Sulfate-reducing bacterium and application thereof

Abstract

The application belongs to the technical field of environmental microorganisms, and particularly relates to a strain of sulfate-reducing bacteria and application thereof. Desulfomicrobium The sulfate-reducing bacteria is Desulfomicrobium sp. SRB-12 IV, which was preserved in the China General Microbiological Culture Collection Center on November 28, 2025, and the preservation number is CGMCC NO. 46907. The application aims to effectively reduce the content of sulfate in the soil by applying the Desulfomicrobium SRB-12 IV to the coal gangue ecological backfilling technology, reduce the content of heavy metal ions separated out due to coordination, balance the pH of the soil, and reduce the environmental pollution caused by soil acidification. The Desulfomicrobium SRB-12 IV significantly improves the economy and efficiency of the whole application through domestication.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a sulfate-reducing bacterium and its applications. Background Technology

[0002] Sulfate-reducing bacteria are a class of heterotrophic bacteria that can use sulfate as an electron acceptor to reduce it to sulfides through dissimilatory processes. They belong to the domain Prokaryotes and, taxonomically, encompass multiple phyla and genera, such as Proteobacteria (…). Proteobacteria Desulfuric Vibrio spp. in ) Desulfovibrio ), Desulfurized Enterobacteriaceae ( Desulfotomaculum Firmicutes ( Firmicutes Some members of the genera are sulfate-reducing bacteria. Different genera of sulfate-reducing bacteria exhibit certain differences in cell morphology, physiological characteristics, and ecological distribution.

[0003] Currently, sulfate-reducing bacteria used in coal gangue treatment include desulfurizing Vibrio, acidophilic Thiobacillus, and methanogen-desulfurizing enterobacteria composite agents. While these sulfate-reducing bacteria can function under neutral pH, strict anaerobic conditions, and stable temperatures, the complex composition of actual coal gangue dumps prevents these bacteria from maintaining stable and continuous growth in harsh environments. For example, in environments where coal gangue comes into contact with excessively acidified soil or high levels of heavy metals. Summary of the Invention

[0004] To address the problem that sulfate-reducing bacteria cannot sustain stable growth under harsh environments in actual coal gangue dumps due to their complex composition, this invention provides a sulfate-reducing bacterium and its applications. To achieve the above objectives, this invention adopts the following technical solution.

[0005] This invention provides a sulfate-reducing bacterium, wherein the sulfate-reducing bacterium is a desulfurizing microorganism (… Desulfomicrobium SRB-12 IV (sp.) was deposited on November 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46907.

[0006] The desulfurizing microorganism SRB-12 IV provided by this invention was screened from deep-sea sediments in the western Pacific Ocean and domesticated in a coal gangue acidification wastewater environment containing high concentrations of heavy metal ions. After domestication, it exhibits characteristics such as acid resistance and heavy metal resistance. This desulfurizing microorganism SRB-12 IV is a chemoautotrophic microorganism, capable of obtaining energy through ferrous oxide or reducing sulfides to complete chemoautotrophic metabolism. It can grow under strictly anaerobic conditions, pH 7.6-8.0, and high concentrations of heavy metals. Ferrous oxide is also known as Fe... 2+ .

[0007] The desulfurizing microbacterium SRB-12 IV provided by this invention can grow and effectively degrade sulfate in acidic wastewater in coal gangue mining areas. It can grow in a low-temperature environment of 15°C and in environments with high sulfate and heavy metal ions. It can alleviate soil acidification caused by coal gangue contact in harsh environments such as coal gangue mining areas and inhibit the precipitation of heavy metal ions. In this way, it can solve the problem in the prior art that the sulfate-reducing bacteria cannot grow stably and continuously in harsh environments due to the complex composition of actual coal gangue stockpiles.

[0008] Preferably, the 16S rRNA sequence of the desulfurizing microorganism SRB-12 IV is shown in SEQ ID NO.1.

[0009] The present invention also provides a method for screening and acclimatizing the sulfate-reducing bacteria, comprising the following steps: Using collected deep-sea sediments from the western Pacific Ocean as an inoculum, the microbial enrichment culture was obtained after culturing at a salinity of 5% and a temperature of 15℃.

[0010] The microbial enrichment culture medium was mixed with a sulfate-reducing bacteria screening medium, and the optimal sulfate-reducing bacteria enrichment culture medium was obtained.

[0011] The optimal sulfate-reducing bacteria enrichment culture medium was mixed with the sulfate-reducing bacteria acclimatization culture medium, and after acclimatization culture, the sulfate-reducing bacteria were obtained.

[0012] Preferably, the sulfate-reducing bacteria screening medium is prepared from a material with the following final concentration: 0.1 g / L calcium chloride dihydrate, 2 g / L magnesium sulfate heptahydrate, 1 g / L ammonium chloride, 2 g / L sodium lactate, 5 g / L yeast extract, 10 g / L tryptone, 1 g / L sodium sulfate, 0.3 g / L potassium dihydrogen phosphate, 0.1 g / L ascorbic acid, and 0.5 g / L ferrous sulfate heptahydrate, with water as the solvent.

[0013] Preferably, the sulfate-reducing bacteria acclimatization culture medium is prepared from a material with the following final concentration: The solvent consisted of 0.1 g / L calcium chloride dihydrate, 2 g / L magnesium chloride hexahydrate, 1 g / L ammonium chloride, 2 g / L sodium lactate, 5 g / L yeast extract, and 10 g / L tryptone, with coal gangue acidification wastewater containing 1000 mg / L heavy metal ions.

[0014] The pH value of the sulfate-reducing bacteria acclimatization medium is 7.6~8.0.

[0015] Preferably, the domestication culture refers to four consecutive subcultures.

[0016] The culture temperature for four consecutive subcultures was 25°C to 30°C.

[0017] The present invention also provides a microbial inoculant, comprising the sulfate-reducing bacteria or a culture thereof.

[0018] Preferably, the microbial agent is a liquid agent.

[0019] The present invention also provides the application of the sulfate-reducing bacteria or the microbial agent in the ecological backfilling of coal gangue.

[0020] Preferably, the sulfate-reducing bacteria or microbial agents are used to reduce sulfate in the soil of coal gangue mines, balance the pH value of the soil, slow down the acidification rate of the soil, thereby reducing ecological pollution and realizing ecological backfilling of coal gangue.

[0021] Preferably, during the ecological backfilling process of coal gangue, the sulfate-reducing bacteria or microbial agents are mixed with the soil in the coal gangue mine pit before application.

[0022] Preferably, the sulfate-reducing bacteria or microbial agent is applied by spraying.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a sulfate-reducing bacterium, namely the desulfurizing microbacterium SRB-12 IV. The desulfurizing microbacterium SRB-12 IV provided by this invention can grow in a low-temperature environment of 15°C and in environments with high sulfate and heavy metal ion concentrations. It can alleviate soil acidification caused by coal gangue contact in harsh environments such as coal gangue mining areas and inhibit the precipitation of heavy metal ions. This solves the problem in existing technologies where the composition of actual coal gangue stockpiles is complex, and these sulfate-reducing bacteria cannot grow stably and continuously under harsh environments.

[0024] 2. The desulfurizing microbacterium SRB-12 IV provided by this invention can grow stably under high concentration of heavy metals and remove sulfates. When applied to the ecological backfilling technology of coal gangue, it significantly improves the ability of microbial methods to inhibit soil acidification and improve the soil environment. At the same time, its efficient reduction of sulfates effectively inhibits the content of heavy metal ions released due to synergistic effects, greatly reduces environmental pollution, and improves ecological value. Attached Figure Description

[0025] Figure 1 Multiple strains of anaerobic bacteria with different morphologies were obtained by isothermal culture in anaerobic 1 / 2 YTSS solid medium.

[0026] Figure 2 The figures show the growth curves and sulfate reduction efficiency of sulfate-reducing bacteria during four subcultures in this invention; wherein: A represents the growth curves of the first to fourth generation strains of sulfate-reducing bacteria SRB-6. B represents the sulfate reduction efficiency of the first to fourth generation strains of sulfate-reducing bacteria SRB-6. C represents the growth curves of the first to fourth generation strains of sulfate-reducing bacteria SRB-12; D represents the sulfate reduction efficiency of sulfate-reducing bacteria SRB-12 from the first generation to the fourth generation. The fourth generation strain of sulfate-reducing bacteria SRB-12, which is the strain obtained by domestication of sulfate-reducing bacteria SRB-12 after four generations, is identified as desulfurizing microbacterium SRB-12 IV.

[0027] Figure 3 This invention describes the change in arsenic content in the soil exposed to coal gangue from landfills after 60 days of treatment with desulfurizing microorganism SRB-12 IV and T5 bioprotective agent.

[0028] Figure 4 The change in mercury content in the soil in contact with coal gangue in landfills treated with T5 bioprotectant was observed after 60 days of treatment with desulfurizing microorganism SRB-12 IV in this invention.

[0029] Figure 5 This invention describes the change in cadmium content in soil exposed to coal gangue from landfills after 60 days of treatment with desulfurizing microorganism SRB-12 IV and T5 bioprotective agent.

[0030] Figure 6 The change in chromium content in the soil in contact with coal gangue in landfills treated with T5 bioprotectant was observed after 60 days of treatment with desulfurizing microorganism SRB-12 IV.

[0031] Figure 7 This invention describes the change in lead content in the soil in contact with coal gangue from landfills after 60 days of treatment with desulfurizing microorganism SRB-12 IV and T5 bioprotective agent.

[0032] Figure 8 This invention describes the change in zinc content in the soil in contact with coal gangue in landfills after 60 days of treatment with desulfurizing microorganism SRB-12 IV and T5 bioprotective agent.

[0033] Figure 9 This invention describes the pH changes in soil contact with coal gangue in landfills after 60 days of treatment with desulfurizing microorganism SRB-12 IV and T5 bioprotective agent.

[0034] Figure 10 This study analyzes the horizontal microbial community in the soil in contact with coal gangue in landfills treated with T5 bioprotective agent after 60 days of treatment with desulfurizing microorganism SRB-12 IV.

[0035] Figure 11This invention compares the abundance of sulfate reduction genes in the contact soil of coal gangue from landfills in T4 and T5 after 60 days of treatment with the desulfurizing microorganism SRB-12 IV.

[0036] Figure 12 This invention compares the abundance of heterolytic sulfate reduction genes in the contact soil of coal gangue from landfills in T4 and T5 after 60 days of treatment with the desulfurizing microorganism SRB-12 IV. Detailed Implementation

[0037] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0038] Example 1: Screening and domestication culture of desulfurizing microorganism SRB-12 IV The screening and acclimatization method for desulfurizing microorganism SRB-12 IV includes the following steps: S1. Using collected deep-sea sediments from the western Pacific Ocean as an inoculum, the microbial enrichment culture was added to an enrichment medium and cultured at a salinity of 5% and a temperature of 15°C to obtain the microbial enrichment culture solution. Details are as follows: 1) Collect deep-sea sediments from the western Pacific Ocean as inoculum, use 5% salinity and 15℃ as core selection pressure, add them to enrichment culture medium, and culture at a constant temperature with shaking for 7 days.

[0039] The source of the deep-sea sediments in the western Pacific Ocean is the first phase of the Jiaolong submersible's 80th scientific expedition to the Chinese Ocean.

[0040] The enrichment medium was prepared from the following final concentrations of materials: 5 g / L beef extract, 10 g / L peptone and 5 g / L sodium chloride, with water as the solvent.

[0041] 2) Mix the bacterial suspension obtained in step 1) at a ratio of 10... -2 10 -4 10 -6 After being diluted proportionally, the bacteria were evenly spread on anaerobic 1 / 2 YTSS solid culture medium and cultured at a constant temperature to obtain multiple strains of anaerobic bacteria with different morphologies.

[0042] The anaerobic 1 / 2 YTSS solid culture medium was prepared from the following final concentrations of materials: 20 g / L sea salt, 4 g / L yeast extract, 2.5 g / L tryptone and 20 g / L agar powder, with water as the solvent, and deoxygenated by blowing argon gas for 10 min.

[0043] Multiple strains of anaerobic bacteria with different morphologies, such as Figure 1 As shown.

[0044] 3) The multiple anaerobic bacteria with different morphologies obtained in step 2) were inoculated into anaerobic 1 / 2 YTSS liquid medium and purified and cultured at constant temperature to obtain the purified bacterial suspensions of each strain.

[0045] The anaerobic 1 / 2 YTSS liquid culture medium was prepared from the following final concentrations of materials: 20 g / L sea salt, 4 g / L yeast extract and 2.5 g / L tryptone, with water as the solvent, and deoxygenated by blowing argon gas for 10 min.

[0046] Each purified bacterial suspension is a microbial enrichment culture medium.

[0047] S2. Mix the microbial enrichment culture medium with the sulfate-reducing bacteria selection medium, and culture to obtain the optimal sulfate-reducing bacteria enrichment culture medium. Details are as follows: The purified bacterial suspensions obtained above were inoculated into sulfate-reducing bacteria selection medium and cultured for 7 days. Their respective sulfate-reducing abilities were then measured to obtain the bacterial suspension with the best sulfate-reducing ability.

[0048] The sulfate-reducing bacteria screening medium was prepared from the following materials at the following final concentrations: 0.1 g / L calcium chloride dihydrate, 2 g / L magnesium sulfate heptahydrate, 1 g / L ammonium chloride, 2 g / L sodium lactate, 5 g / L yeast extract, 10 g / L tryptone, 1 g / L sodium sulfate, 0.3 g / L potassium dihydrogen phosphate, 0.1 g / L ascorbic acid, and 0.5 g / L ferrous sulfate heptahydrate, with water as the solvent.

[0049] The bacterial culture of bacteria with optimal sulfate-reducing capacity is called the optimal sulfate-reducing bacteria enrichment culture medium. Bacteria with optimal sulfate-reducing capacity include sulfate-reducing bacteria SRB-12 and SRB-6. The optimal sulfate-reducing bacteria enrichment culture medium includes bacterial cultures of sulfate-reducing bacteria SRB-12 and SRB-6.

[0050] S3. Mix the optimal sulfate-reducing bacteria enrichment culture medium with the sulfate-reducing bacteria acclimatization culture medium, and after acclimatization culture, obtain the acclimatized sulfate-reducing bacteria. Details are as follows: 1) First acclimatization: 200 μL of each of the sulfate-reducing bacteria SRB-12 and SRB-6 were inoculated into 100 mL of sulfate-reducing bacteria acclimatization medium and cultured at 30°C and 200 rpm for 36 h to obtain first-generation cultures of sulfate-reducing bacteria SRB-12 and SRB-6. The strains obtained in this stage are called first-generation strains, and these strains are the result of one passage acclimatization.

[0051] The sulfate-reducing bacteria acclimatization medium was prepared from the following components at the following final concentrations: 0.1 g / L calcium chloride dihydrate, 2 g / L magnesium chloride hexahydrate, 1 g / L ammonium chloride, 2 g / L sodium lactate, 5 g / L yeast extract, and 10 g / L tryptone were diluted to 1 L with coal gangue acidification wastewater containing 1000 mg / L heavy metal ions. The solutions were then dispensed and purged with argon gas for 10 min to create an anaerobic environment. The coal gangue acidification wastewater containing high concentrations of heavy metal ions was obtained from a coal mining subsidence area in Changzhi City, Shanxi Province.

[0052] The first-generation bacterial suspensions of sulfate-reducing bacteria SRB-12 and SRB-6 are referred to as first-generation bacterial suspensions.

[0053] 2) Second acclimatization: Take 200 μL of first-generation culture of sulfate-reducing bacteria SRB-12 and SRB-6 respectively, and subculture them again in 100 mL of sulfate-reducing bacteria acclimatization medium. Continue to culture at 30°C and 200 rpm on a shaker for 36 h to obtain second-generation culture of sulfate-reducing bacteria SRB-12 and SRB-6. The strains obtained in this stage are called second-generation strains, and the strains obtained in this stage are the strains obtained through the second subculture acclimatization.

[0054] 3) Third acclimatization: 200 μL of second-generation sulfate-reducing bacteria SRB-12 and SRB-6 were respectively subcultured and inoculated into 100 mL of sulfate-reducing bacteria acclimatization medium. The cultures were then incubated at 30°C and 200 rpm for 36 h on a shaker to obtain third-generation sulfate-reducing bacteria SRB-12 and SRB-6. The strains obtained in this stage are called third-generation strains, and these strains represent the result of three subcultures for acclimatization.

[0055] 4) Fourth acclimatization: Take 200 μL of the third-generation culture of sulfate-reducing bacteria SRB-12 and SRB-6 respectively, and subculture them again in 100 mL of sulfate-reducing bacteria acclimatization medium. Continue to culture at 30°C and 200 rpm on a shaker for 36 h to obtain the fourth-generation culture of sulfate-reducing bacteria SRB-12 and SRB-6. The strains obtained in this stage are called fourth-generation strains, and the strains obtained in this stage are the strains obtained through four subcultures of acclimatization.

[0056] The above steps involved four consecutive subcultures of sulfate-reducing bacteria SRB-12 and SRB-6, resulting in four generations of strains. The growth curves of the first to fourth generations of SRB-12 and SRB-6 strains, as well as their sulfate reduction efficiencies, were then measured. The results are as follows: Figure 2 As shown.

[0057] The method for detecting the growth curve is as follows: Starting from 0h, 1 mL of first-generation to fourth-generation bacterial suspensions of sulfate-reducing bacteria SRB-12 and first-generation to fourth-generation bacterial suspensions of sulfate-reducing bacteria SRB-6 were collected every 6 hours, for a total of 36 hours. The collected bacterial suspensions were placed in a spectrophotometer, and the absorbance at 600 nm was measured. The OD values ​​were plotted with growth time on the x-axis. 600nm The absorbance value was used as the ordinate to plot the growth curves. Based on the growth curves, the growth status of the first to fourth generation strains of sulfate-reducing bacteria SRB-12 and the first to fourth generation strains of sulfate-reducing bacteria SRB-6 was determined.

[0058] The method for detecting sulfate reduction efficiency is as follows: (1) Sample preparation for ion chromatography: The first-generation to fourth-generation bacterial cultures of sulfate-reducing bacteria SRB-12 and the first-generation to fourth-generation bacterial cultures of sulfate-reducing bacteria SRB-6 were centrifuged at 4°C and 17,000×g for 3 min. Then, 800 µL of the supernatant was added to 8 µL of 4M KOH and centrifuged again at 17,000×g for 2 min. After filtering the supernatant through a 0.22 µm filter membrane, 600 µL of the supernatant was collected into a sample tube for ion chromatography detection.

[0059] (2) Conditions for ion chromatography detection: Anion detection mode was used, with an Ion Pac AS19 column and a temperature of 30°C. An automatic eluent generator and an ASRS_4mm suppressor were used for gradient elution with KOH at a flow rate of 1 mL / min. The KOH concentration was 25 mM for the first 14 min, gradually increasing to 35 mM after 2.5 min and continuing for 18.5 min. Under these conditions, the elution time for sulfate was 12.5 min.

[0060] (3) By comparing the growth of sulfate-reducing bacteria SRB-12 from generation 1 to generation 4 and sulfate-reducing bacteria SRB-6 from generation 1 to generation 4 after 36 hours, and the sulfate reduction efficiency of sulfate-reducing bacteria SRB-12 from generation 1 to generation 4 and sulfate-reducing bacteria SRB-6 from generation 1 to generation 4, it was found that the OD of sulfate-reducing bacteria SRB-6 from generation 1 to generation 4 after 36 hours of growth was... 600nm All could only reach 0.4. The OD of sulfate-reducing bacteria SRB-12 from the first generation to the fourth generation strain after 36 hours of growth was... 600nmThe sulfate reduction efficiency gradually increased from 0.3 to 0.6. The second to fourth generation strains of sulfate-reducing bacteria SRB-6 achieved a sulfate reduction efficiency of 25%, slightly higher than the 20% of its first generation. The sulfate reduction efficiency of the first to fourth generation strains of sulfate-reducing bacteria SRB-12 increased from 29% to 40%. Therefore, the fourth generation strain of sulfate-reducing bacteria SRB-12 was selected as the best-performing sulfate-reducing bacterium through domestication and screening, and it was preserved. The sulfate-reducing bacterium with the highest sulfate reduction efficiency is the optimal sulfate-reducing bacterium. The fourth generation strain of sulfate-reducing bacteria SRB-12 is the strain obtained from four passages of sulfate-reducing bacteria SRB-12.

[0061] The above results indicate that the fourth generation strain of sulfate-reducing bacteria SRB-12 has stronger sulfate-reducing ability and environmental tolerance.

[0062] Furthermore, identification revealed that the fourth-generation strain of sulfate-reducing bacteria SRB-12 belongs to the genus *Desulfurobacterium*, and it was named *Desulfurobacterium SRB-12 IV*. It was deposited on November 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46907.

[0063] The 16S rRNA sequence of the desulfurizing microorganism SRB-12 IV is shown in SEQ ID NO.1:

[0064] Example 2: Application of desulfurizing microorganism SRB-12 IV in ecological backfilling technology for coal gangue The steps for applying desulfurizing microorganism SRB-12 IV to the ecological backfilling technology of coal gangue are as follows: 1) Activate the desulfurizing microorganism SRB-12 IV, which has been stored at low temperature, in a test tube containing 5 mL of sulfate-reducing bacteria anaerobic medium. After activation, transfer the strain to 100 mL of the above sulfate-reducing bacteria anaerobic medium and culture for 15 h. Then, expand the culture to 500 mL with an inoculum of 10 mL as the seed culture.

[0065] The sulfate-reducing bacteria anaerobic culture medium is prepared from materials with the following final concentrations: 0.1 g / L calcium chloride dihydrate, 2 g / L magnesium chloride hexahydrate, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L ammonium chloride, 2 g / L sodium lactate, 5 g / L yeast extract and 10 g / L tryptone, with distilled water as solvent.

[0066] 2) The above seed culture was inoculated into 30L of purified water at a volume ratio of 2%, and 20g / L glucose, 2g / L sodium lactate, 5g / L yeast extract, 10g / L tryptone, 0.1g / L calcium chloride dihydrate, 2g / L magnesium chloride hexahydrate, 0.5g / L dipotassium hydrogen phosphate, and 1g / L ammonium chloride were added for expansion culture. After the bacterial culture reached the logarithmic growth phase, the bioprotective agent prepared by the desulfurizing microorganism SRB-12IV was obtained and transported to the mine. The bioprotective agent prepared by the desulfurizing microorganism SRB-12IV is a microbial inoculum, or simply a bioprotective agent.

[0067] 3) Three adjacent pits were opened in the coal mining subsidence area of ​​Changzhi City, Shanxi Province, as control unit T4, biological protective agent treatment unit T5 and chemical protective agent treatment unit T6, respectively.

[0068] Each pit is 10 meters long, 2 meters wide, and 5 meters deep. After compaction and leveling, the walls and bottom of all pits are first laid with a high-density polyethylene geomembrane, and then a 75-centimeter-thick compacted clay lining is laid on top, forming the main seepage prevention layer. In control unit T4, the pit filling material only uses 2.5 meters of artificially pre-acidified coal gangue, without laying an intermediate barrier soil layer, and the surface is directly covered with 1 meter of topsoil. In biological protection agent treatment unit T5, the pit filling material uses 2.5 meters of artificially pre-acidified coal gangue. After each layer of coal gangue is compacted, 10L of biological protection agent prepared by desulfurizing microorganism SRB-12 IV is sprayed. In chemical protection agent treatment unit T6, before filling the pit with artificially pre-acidified coal gangue, a 0.1-meter-high solid chelating agent is evenly laid to ensure that heavy metals are stabilized in the filling soil layer.

[0069] Soil samples in direct contact with coal gangue were collected from pits at a depth of 4.4 meters in the control unit T4, the biological protection agent treatment unit T5, and the chemical protection agent treatment unit T6 after treatment with desulfurizing microorganism SRB-12 IV and heavy metal chelating agent for 10, 30, and 60 days, respectively.

[0070] During the filling process of artificially pre-acidified coal gangue, dump trucks are used to transport the gangue to the edge of the pit, and small bulldozers are used for layered filling, with each layer controlled to be less than 50cm thick. After filling, a vibratory roller is used for compaction to ensure that the compaction degree of each layer is not less than 85%. In addition, after each layer of artificially pre-acidified coal gangue is compacted, activated desulfurization microorganisms SRB-12 IV liquid are sprayed onto the artificially pre-acidified coal gangue in the T5 biological protective agent treatment unit, with the spraying amount controlled at 1L / m³. 3 This ensures uniform distribution and activity. Simultaneously, for the chemical protective agent treatment unit T6, a 0.1m high layer of solid chelating agent is evenly laid before filling with artificially pre-acidified coal gangue to ensure that heavy metals are stabilized in the backfill soil layer.

[0071] Artificial pre-acidified coal gangue was obtained by soaking natural coal gangue from the coal mining subsidence area in Changzhi City, Shanxi Province, in dilute sulfuric acid for 6 hours.

[0072] The solid chelating agent is quicklime.

[0073] 4) Using a soil sampler, 100g of soil samples were collected from the three sets of unit pits at 10d, 30d, and 60d in direct contact with coal gangue. The pH value was measured using a pH meter. Arsenic, zinc, cadmium, and mercury ions in the soil samples from the three sets of unit pits in direct contact with coal gangue were detected using microwave digestion atomic fluorescence spectrometry. Chromium and lead ions were detected using flame atomic absorption spectrophotometry.

[0074] The changes in heavy metal ion content over time in soil samples from pits in direct contact with coal gangue in control unit T4, biological protective agent treatment unit T5, and chemical protective agent treatment unit T6 are shown below. Figures 3-8 .

[0075] The concentrations of arsenic, mercury, cadmium, chromium, lead, and zinc in soil samples taken from pits in biological protective agent treatment unit T5 that were in direct contact with coal gangue were all lower than those in soil samples taken from pits in control unit T4 that were in direct contact with coal gangue, and the concentrations of most metals did not increase over time.

[0076] The above results indicate that the bioprotective agent can inhibit the release of metal ions from coal gangue and prevent them from seeping into the soil. The bioprotective agent is the activated desulfurizing microorganism SRB-12 IV liquid.

[0077] The pH changes over time of soil samples in direct contact with coal gangue in pits of control unit T4, biological protective agent treatment unit T5, and chemical protective agent treatment unit T6 are shown below. Figure 9 .

[0078] The pH value of soil samples taken from pits in biological protection unit T5, which were in direct contact with coal gangue, was significantly higher than that of soil samples taken from pits in control unit T4, indicating that the biological protection agent has an anti-acidification effect. Therefore, it is inferred that long-term addition of the desulfurizing microorganism SRB-12 IV can effectively mitigate the acidification impact of mine coal gangue on the surrounding soil, reducing the environmental harm caused by excessive acidification.

[0079] 5) Using a soil sampler, collect 50g of soil samples from the above three groups of pits at 10, 30 and 60 days of direct contact with coal gangue, and perform microbial community analysis, metagenomic library construction and sequencing and data analysis.

[0080] The genus-level microbial community analysis results of soil samples in direct contact with coal gangue in pits of control unit T4, biological protective agent treatment unit T5, and chemical protective agent treatment unit T6 are as follows: Figure 10 Genus-level microbial community analysis was performed on soil samples from the T5 bioprotective agent treatment unit, which was in direct contact with coal gangue, 60 days after landfilling. The analysis revealed that desulfurizing bacteria accounted for approximately 1.45%. Combined with metagenomic analysis, the abundance comparisons of assimilation sulfate reduction genes and dissimilation sulfate reduction genes showed... Figure 11 and Figure 12 As shown, the abundance of dissimilatory sulfate reduction genes was significantly higher in the bioprotective agent treatment unit T5 than in the control unit T4. The content of the bioprotective agent remained stable at 1.5% of the microbial community, and the abundance of related genes showed an increasing trend over time, indicating that the bioprotective agent can exist stably.

[0081] In summary, the desulfurizing microorganism SRB-12 IV provided by this invention can be applied to the ecological backfilling technology of coal gangue. The domesticated strain significantly improves the acid and heavy metal resistance characteristics, enhances the sulfate reduction capacity, and inhibits the coordinated precipitation of acid-soluble heavy metals. This greatly reduces the environmental hazards caused by excessive soil acidification, improves the bioeconomic value and soil utilization rate, and reduces the damage of environmental pollution to biodiversity.

[0082] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0083] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. The application of a sulfate-reducing bacterium or microbial agent in the ecological backfilling of coal gangue, characterized in that, The sulfate-reducing bacteria are desulfurizing microorganisms ( Desulfomicrobium SRB-12 IV (sp.) was deposited at the China General Microbiological Culture Collection Center on November 28, 2025, with accession number CGMCC NO.46907; The microbial agent includes the sulfate-reducing bacteria; The sulfate-reducing bacteria or microbial agents are used to reduce sulfate in the soil of coal gangue mines, balance the pH value of the soil, slow down the acidification rate of the soil, thereby reducing ecological pollution and realizing ecological backfilling of coal gangue.

2. The application according to claim 1, characterized in that, The 16S rRNA sequence of the desulfurizing microorganism SRB-12 IV is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The microbial agent is a liquid agent.

4. The application according to claim 1, characterized in that, During the ecological backfilling process of coal gangue, the sulfate-reducing bacteria or microbial agents are mixed with the soil in the coal gangue mine pit and then applied.

5. The application according to claim 4, characterized in that, The sulfate-reducing bacteria or microbial agents are applied by spraying.

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