Alkaliphilic sulfur-oxidizing bacterium and its application in water pollution treatment and bioleaching

Abstract

The present application relates to the technical field of health, environmental management and resource development, and discloses an alkaline-sulfur oxidizing bacteria and its application in water pollution treatment and bioleaching. The alkaline-sulfur oxidizing bacteria is named 381-2, which has been preserved in the China General Microbiological Culture Collection Center on December 23, 2024, and the preservation number is CGMCC No. 33017; the microbial classification name is Stutzerimonas sp. The present application isolates a new species of Stizolobium strain from a deep-sea hydrothermal area Stutzerimonas sp. . The strain has strong and rapid oxidation ability of thiosulfate ions, and also has the characteristics of producing alkali, so it can be applied to the fields of water pollution treatment and bioleaching.

Description

Technical Field

[0001] This invention relates to the fields of microbiology, environmental remediation, and resource development, and in particular to an alkali-producing sulfur-oxidizing bacterium and its application in water pollution control and bioleaching. Background Technology

[0002] Sulfur-oxidizing bacteria (SOBs) are drivers of the biological oxidation of elemental sulfur or reduced sulfides, playing a crucial role in the process. SOBs are essential for the treatment of pollutants such as wastewater, waste gas, and solid waste, as well as for the recovery of sulfur resources. Therefore, SOBs have been widely applied in agriculture, environmental engineering, and the metallurgical industry.

[0003] Sulfur-oxidizing bacteria are widely distributed and diverse, with a wide variety of sulfur oxidation-related genes, enzymes, and pathways. SOBs have been found in various environments, including marine environments, hydrothermal vents, cold seeps, soils, rivers, and lakes. Discovered SOBs include green sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, and colorless sulfur bacteria. Other SOB groups include those in Firmicutes. Alicyclobacillus spp., green non-sulfur bacteria Chloroflexus aurantiacus and in Aquificae Sulfurihydrogenibium Spp., etc. Sulfur-oxidizing microorganisms also have different oxidation pathways. For the oxidation of thiosulfate, the two most common pathways are: one is the acid-producing pathway, in which thiosulfate is completely oxidized to sulfate by the Sox enzyme system without producing intermediate products; the other is the alkali-producing pathway, in which tetrathionate is produced as an intermediate metabolite by the action of thiosulfate dehydrogenase.

[0004] Deep-sea hydrothermal environments contain abundant sulfur oxides (SOBs), exhibiting characteristics such as high pressure resistance, high temperature resistance, and strong chemical reactivity. The large amounts of H2S gas and reducing sulfides produced by deep-sea hydrothermal eruptions allow sulfur-oxidizing microorganisms in the hydrothermal zone to generate energy through the oxidation of reduced sulfur compounds. On one hand, deep-sea alkali-producing sulfur-oxidizing bacteria raise the pH value of the environment during the oxidation of sulfides, which helps precipitate heavy metal ions, thereby reducing their mobility and biohazard in the environment. On the other hand, deep-sea neutrophilic sulfur-oxidizing bacteria can be used to oxidize deep-sea sulfide minerals, promoting mineral leaching (e.g., patent CN1955279B). Therefore, deep-sea alkali-producing and neutrophilic sulfur-oxidizing bacteria show promising industrial applications in multiple fields such as environmental remediation and bioleaching.

[0005] Therefore, the exploration of SOB resources in deep-sea hydrothermal environments and the acquisition of more SOBs will help discover new sulfur oxidation genes, enzymes, or pathways, providing a guarantee of microbial resources for pollution control and resource recovery. Summary of the Invention

[0006] This invention provides an alkali-producing sulfur-oxidizing bacterium. A novel strain of the genus *Stizemona* was isolated from a deep-sea hydrothermal vent area. Stutzerimonas sp. This strain has a strong ability to rapidly oxidize thiosulfate ions and also exhibits alkali-producing properties, thus it can be applied in water pollution control and bioleaching.

[0007] The specific technical solution of the present invention includes:

[0008] In a first aspect, this invention provides an alkali-producing sulfur-oxidizing bacterium, named 381-2, which was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33017; its microbiological classification is *Stizemona*. Stutzerimonas sp. ).

[0009] The strain of this invention was isolated from a deep-sea hydrothermal sample (collected from the Carlsberg Ridge hydrothermal vent area in the northwestern Indian Ocean, at a water depth greater than 3000 m). Testing revealed that this strain possesses a strong and rapid ability to oxidize thiosulfate ions, and also exhibits alkali-producing characteristics. The 16S rRNA nucleotide sequence of this strain is shown in SEQ ID No. 1, and its genomic sequence NCBI accession number is JBPVWS000000000. Genomic sequence identification showed an ANI value of less than 95% and a DDH value of less than 70%, indicating that this strain belongs to the genus *Stizemona* (*Stizemona*). Stutzerimonas A new species of the genus ( ).

[0010] After cultivation, the colonies of this strain are white, round, and relatively flat. Transmission electron microscopy reveals that the bacterial cells are rod-shaped. This strain can utilize glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, adipic acid, malic acid, citric acid, and phenylacetic acid as carbon sources, but cannot utilize decanoic acid. This strain produces β-glucosidase but does not produce tryptophan hydrolase, arginine hydrolase, urease, or protease. The optimal growth range for this strain is 0–5% NaCl concentration (optimum 0%), 20–40 °C temperature (optimum 28 °C), and 6–11 pH (optimum 7).

[0011] In a second aspect, the present invention provides a microbial inoculant containing one or more of the above-mentioned live alkali-producing sulfur-oxidizing bacteria, fermentation broth, exosomes, and their metabolites.

[0012] Thirdly, the present invention provides the application of the above-mentioned alkali-producing sulfur-oxidizing bacteria or microbial agents in water pollution treatment. Preferably, the water pollution is water containing metal sulfides; more preferably, the water is acidic.

[0013] Because the strain of this invention has a strong ability to rapidly oxidize thiosulfate ions and also exhibits alkali-producing properties, it can, on the one hand, promote the release of metal ions by oxidizing metal sulfides, and on the other hand, raise the pH value of acidic water bodies by producing alkali, thereby causing metal ions to precipitate, thus reducing the metal content in the water body and decreasing its mobility and biological hazards in the environment.

[0014] Fourthly, the present invention provides the application of the aforementioned alkali-producing sulfur-oxidizing bacteria or microbial agents in bioleaching. Preferably, the mineral targeted by the bioleaching is a sulfide mineral.

[0015] The alkali-producing sulfur-oxidizing bacteria of this invention possess highly efficient oxidation capabilities for elemental sulfur and inorganic sulfur compounds. They can oxidize sulfur compounds in sulfide minerals, promoting ore leaching and thus increasing metal extraction rates. For example, the strain can oxidize sulfides (such as pyrite FeS2) in ores, releasing metal ions for the recovery of low-grade ores.

[0016] Further preferably, the sulfide mineral is a deep-sea sulfide mineral.

[0017] Deep-sea polymetallic sulfide deposits are rich in copper, zinc, lead, gold, silver, and other rare metals, making them more valuable for mining. The polymetallic sulfide resources in submarine hydrothermal vent areas are estimated to be hundreds of millions of tons. As terrestrial mineral resources gradually deplete, developing deep-sea resources has become an important way to meet future needs.

[0018] Furthermore, the alkali-producing sulfur-oxidizing bacteria or microbial agents promote the leaching of sulfide minerals by oxidizing them, thereby releasing metal ions.

[0019] Fifthly, the present invention provides the application of the above-mentioned alkali-producing sulfur-oxidizing bacteria or microbial agents in sulfides.

[0020] In a sixth aspect, the present invention provides the application of the above-mentioned alkali-producing sulfur-oxidizing bacteria or the above-mentioned microbial agents in the metabolism of sodium thiosulfate to generate tetrathionate.

[0021] The product of sodium thiosulfate metabolized by the strain of this invention is tetrathionate.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] (1) The strain 381-2 of this invention is a new species of strain of the genus Stizemonas. Stutzerimonas sp. Obtaining more SOBs will help in the discovery of new sulfur oxidation genes, enzymes, or pathways, providing a guarantee of microbial resources for pollution control and resource recycling.

[0024] (2) The strain of the present invention has an ideal ability to rapidly oxidize thiosulfate ions in MMT medium. After 24 hours of cultivation, the removal rate of sodium thiosulfate by strain 381-2 is 95%. At the same time, the strain also has alkali-producing characteristics. Therefore, when it is applied to bioleaching, it not only promotes the leaching of sulfide minerals, but also maintains the pH of the environment in a neutral or slightly alkaline state, which can control the generation of acidic wastewater caused by mineral oxidation.

[0025] (3) The strain of the present invention can be applied to environmental remediation and treatment such as water pollution. Attached Figure Description

[0026] Figure 1 This is a schematic diagram (phylum level) of the community structure of culturable microorganisms in the hydrothermal vent area of ​​the Northwest Indian Ocean.

[0027] Figure 2 A schematic diagram of the community structure of culturable microorganisms in the hydrothermal vent area of ​​the Northwest Indian Ocean (horizontal).

[0028] Figure 3 This is a phylogenetic diagram of strains based on the 16S rRNA gene.

[0029] Figure 4 This is a schematic diagram of a genome phylogenetic tree based on homologous protein sequences.

[0030] Figure 5 This is a transmission electron microscope image of the strain after culturing in 2216 medium at 28°C for 48 hours.

[0031] Figure 6 This is an HPLC peak diagram of the sulfur metabolites of the strain. Detailed Implementation

[0032] The present invention will be further described below with reference to embodiments.

[0033] General Implementation Examples

[0034] Firstly, an alkali-producing sulfur-oxidizing bacterium, named 381-2, was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33017; its microbiological classification is *Stizemonas*. Stutzerimonas sp. ).

[0035]

[0036] Its genomic sequence has an NCBI accession number of JBPVWS000000000.

[0037] Secondly, a microbial inoculant containing one or more of the above-mentioned alkali-producing sulfur-oxidizing bacteria, fermentation broth, exosomes, and their metabolites.

[0038] Thirdly, the application of the aforementioned alkali-producing sulfur-oxidizing bacteria or microbial agents in water pollution control.

[0039] Preferably, the water pollution is water containing metal sulfides; more preferably, the water is acidic.

[0040] Furthermore, the alkali-producing sulfur-oxidizing bacteria or microbial agents promote the release of metal ions by oxidizing metal sulfides, and increase the pH value of acidic water by producing alkali, thereby causing metal ions to precipitate and reducing the metal content in the water.

[0041] Fourthly, the application of the aforementioned alkali-producing sulfur-oxidizing bacteria or microbial agents in bioleaching.

[0042] Preferably, the mineral targeted by the bioleaching is a sulfide mineral.

[0043] Further preferably, the sulfide mineral is a deep-sea sulfide mineral.

[0044] Further preferred, the sulfide mineral is pyrite.

[0045] Furthermore, the alkali-producing sulfur-oxidizing bacteria or microbial agents promote the leaching of sulfide minerals by oxidizing them, thereby releasing metal ions.

[0046] Fifthly, the application of the aforementioned alkali-producing sulfur-oxidizing bacteria or microbial agents in the oxidation of sulfides.

[0047] The sixth aspect concerns the application of the aforementioned alkali-producing sulfur-oxidizing bacteria or the aforementioned microbial agents in the metabolism of sodium thiosulfate to produce tetrathionate. Specific Implementation

[0049] Example 1: Enrichment of sulfur-oxidizing strains from deep-sea hydrothermal vents

[0050] The deep-sea hydrothermal samples used in this invention were collected in the Carlsberg Ridge hydrothermal vent area of ​​the Northwest Indian Ocean by a box sampler carried on the 72nd Chinese Ocean Expedition, at a water depth greater than 3000 m.

[0051] SOB enrichment medium is an inorganic culture medium formulated with sodium thiosulfate as the main energy source. Since the metabolic pathway of sodium thiosulfate is similar to that of sulfides, microorganisms that grow ideally when using sodium thiosulfate as their metabolic energy source often also have a good ability to oxidize sulfides. The main energy source for SOB enrichment medium is Na₂S₂O₃, with CO₂ and NH₄Cl as the carbon and nitrogen sources, respectively.

[0052] Specifically, the culture formula for SOB enrichment culture is as follows: NH4Cl 0.4 g, NaHCO3 0.2016 g, FeSO4·7H2O 0.001 g, phenol red 0.004 g, KH2PO4 0.1 g, Na2S2O3·5H2O 3 g, trace element solution (1000×) 1 ml, microbial solution (1000×) 1 ml, VB 12 0.015 mg was prepared with 1 L of aged seawater, and the pH was adjusted to 7.5. The trace element solution (mmol / L) consisted of: EDTA-Na₂·2H₂O 14, FeSO₄·7H₂O 7.5, ZnCl₂ 0.5, MnCl₂·4H₂O 0.5, H₃BO₃ 1.0, CoCl₂·6H₂O 0.8, CuCl₂·2H₂O 0.1, NiCl₂·6H₂O 0.1, NaMoO₄·2H₂O 0.15, with a pH of 3.4, and was used after sterilization by filtration through a 0.22 μm filter. The vitamin solution (mg / L) consisted of: VB7 2, VB9 2, VB5 5, VB1 5, VB6 5, VB3 5, p-Aminobenzoic Acid 5, VB2 5, and was filtered through a 0.22 μm filter membrane. Na₂S₂O₃ was dissolved in sterile ultrapure water and filtered through a 0.22 μm filter membrane. Other components were sterilized at 121 °C for 20 min. MgSO₄·7H₂O and CaCl₂·2H₂O were sterilized separately and then added to the culture medium. 1.5% agar powder was added to the SOB solid culture medium.

[0053] Take 1g of the collected deep-sea hydrothermal sediment sample, suspend it in sterile seawater, and then serially dilute it. Take 200 μL of the diluted solution and spread it on SOB agar plates or transfer it to SOB liquid medium. Incubate at room temperature. After multiple transfers, the SOB liquid medium sample is spread on SOB agar plates.

[0054] Example 2: High-throughput screening and identification of sulfur-oxidizing strains from deep-sea hydrothermal vents

[0055] Colonies with different morphological characteristics were randomly selected from agar plates and inoculated into SOB liquid medium. The medium was incubated at 30 °C with shaking at 180 rpm. Changes in the medium color were used as the basis for initial screening of sulfur-oxidizing microorganisms. The triple-line streak plate method was used for purification until single colonies were obtained. The bacterial culture was then stored at -80 °C with 30% glycerol added.

[0056] The strain underwent 16S rRNA gene sequencing. 2 μL of bacterial culture was placed in a 1.5 mL centrifuge tube, 30 μL of sterile water was added, and the mixture was quickly centrifuged to prevent liquid adhesion to the tube walls. The centrifuge tube was then incubated in a 100 ℃ metal bath for 10 min. After centrifugation at 12000 rpm for 1 min, 2 μL of the supernatant was collected as template DNA for amplification and sequencing of the 16S rRNA of the test strain. The 16S rDNA sequence of the purified strain was amplified by PCR using universal bacterial primers: 27f (5'-AGAGTTTGATCMTGCCTCAG-3') and 1492r (5'-TACGGYTACCTTGTTACGACTT-3'). The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the obtained 16S rDNA sequence was submitted to EzBioCloud (http: / / www.ezbiocloud.net / ) for comparison and analysis.

[0057] The 16S rRNA gene sequences of 121 bacterial strains were isolated, purified, amplified, and sequenced. Molecular identification results showed that the 121 isolated strains belonged to the class Alpha-Proteobacteria (α-Proteobacteria). Alphaproteobacteria ), Gamma-Proteobacteria ( Gammaproteobacteria ) and Flavobacteria ( Flavobacteriia ) 3 taxa. Belonging to 19 genera, including *Stizemonas* ( Stutzerimonas 29), *Pseudomonas* spp. Pseudoalteromonas 21), genus Halomonas ( Halomonas 19) and Alternating Monotamos ( Alteromonas (9) are the dominant genera. Stutzerimonas stutzeri (21 strains) and Pseudoalteromonas lipolytica (20 plants) are the dominant species (e.g.) Figure 1-2 (As shown).

[0058] Example 3: Oxidizing power test of sodium thiosulfate strain

[0059] The sulfur oxidation capacity of the strain was tested. The strain was then transferred at a 1% inoculum to MMT medium containing 20 mM sodium thiosulfate (added after sterilization) and cultured at 30°C and 160 rpm for 24 h. The MMT medium formulation was: NaCl 23 g, CaCl2·2H2O 0.1 g, K2HPO4 0.5 g, MgCl2 0.4 g, Na2S2O3·5H2O 2.48 g, CH3COONa 0.8 g, yeast extract 0.2 g, and trace element solution (1000×) 1 ml, pH adjusted to 7.0. The trace element solution formulation was the same as in Example 1.

[0060] Centrifuge the cultured bacterial solution at 10,000 rpm for 10 min at 4℃ and collect the supernatant. Determine the consumption of sodium thiosulfate in the cultured system using indirect iodine titration. Each sample group is titrated twice. The indirect iodine titration method is as follows: Add 1 ml of potassium dichromate standard solution, 0.1 g of potassium iodide, and 4 ml of acidic buffer (1 M HCl) to an iodine flask. Shake in the dark for 10 min, then dilute with 30 ml of boiled distilled water to titrate the remaining thiosulfate ions in the solution. Add the cultured supernatant to a burette, record the initial position of the solution in the burette, titrate to near the endpoint, and add 200 μl of 1% activated soluble starch. The color in the iodine flask changes from dark blue to light blue, and the blue color disappears at the titration endpoint. Record the titration endpoint position and calculate the volume of the test solution consumed during the titration, ΔV. Titrate each sample twice to reduce error. The method for calculating thiosulfate ions in the sample is: C Na2S2O3 =6×(cV) K2CR2O2 / V Na2S2O3 .

[0061] Comparing the sodium thiosulfate oxidation efficiency of all strains, strain 381-2 was found to have the highest sulfur oxidation efficiency, with a sodium thiosulfate removal rate of 95%. Simultaneously, the pH changes of the system before and after cultivation were measured. The pH of the system increased significantly after cultivation, with strain 381-2 reaching a pH of 1.84, indicating that strain 381-2 is an alkali-producing sulfur-oxidizing strain.

[0062] Example 4: Determination of the taxonomic position of the strain

[0063] A draft genome sequence of strain 381-2 was performed. After rapid freezing of the bacterial cells, high-throughput paired-end sequencing was performed using an Illumina NovaSeq6000 PE150 platform, yielding approximately 1 Gb of bacterial genome sequencing data. Using the clean reads obtained from the sequencing, genome assembly was performed using SPAdes 3.10.1 and Megahit 1.2.9 software, and its quality was assessed using checkm v1.0.7. The assembled genome of strain 381-2 showed 100.00% integrity and a contamination rate of 1.01%, indicating reliable assembly results suitable for subsequent gene function prediction.

[0064] The closest phylogenetic relationship between strain 381-2 and the type strain is Stutzerimonas zhaodongensis The 16S rDNA of strain 381-2 showed 99.52% homology. Based on the gene sequence of the 16S rRNA of strain 381-2 and closely related type strains, a phylogenetic tree was constructed using the neighbor-linked method. Figure 3 This indicates that strain 381-2 belongs to the genus *Stizemonas* (…). Stutzerimonas ) genus. Download Stizemonas genus ( Stutzerimonas The reference genome sequences of all eligible published species were used to construct genome trees based on homologous protein sequences in the genome sequences using the maximum likelihood method. Figure 4 This indicates that strain 381-2 formed an independent branch. Average Nucleotide Identity (ANI) was calculated using FastANI software installed on the local server (Jain et al., 2018). The in silico DNA-DNA hybridization (DDH) value between strains was calculated using the online Genome-to-Genome Distance Calculator (GGDC, https: / / ggdc.dsmz.de / home.php). The ANI values ​​of strain 381-2 and closely related strains were all less than 88.2%. in silico DDH values ​​were all less than 34% (Table 1). Based on the minimum standards recommended for prokaryotic classification based on genomic data (Chun et al., 2018), ANI values ​​less than 95% and... in silico A DDH value less than 70% indicates a new species. Therefore, strain 381-2 belongs to the genus *Stizemonas*. StutzerimonasThis is a new species of the genus *Prokaryotes*. The basis for this discovery is the authoritative paper published by the International Committee on Systematics of Prokaryotes (ICSP) (Jongsik Chun, Aharon Oren, Antonio Ventosa, Henrik Christensen, David Ruiz Arahal, Milton S. da Costa, Alejandro P. Rooney, Hana Yi, Xue-Wei Xu, Sofie De Meyer and Martha E. Trujillo. 2018. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 2018;68:461–466. DOI10.1099 / ijsem.0.002516). With the popularization of sequencing technology, classification based solely on 16S sequences has been considered one-sided. Therefore, the aforementioned genome similarity is now the gold standard for microbial taxonomy in the genome era, including the two most important indicators, namely ANI and DDH values, with species thresholds of 95% ANI and 70% DDH, respectively.

[0065] Strain 381-2 was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC NO. 33017.

[0066] Table 1. Strains 381-1 and *Stizemonas* spp. ( Stutzerimonas ANI and DDH values ​​of genus species

[0067]

[0068] Example 5: Phenotypic characteristics of deep-sea sulfur-oxidizing strain 381-2

[0069] Strain 381-2 grew well after being cultured on marine broth agar (BD Difco™, 279110) medium at 30°C for 48 hours, with clear, white, round, and relatively flat colonies. Transmission electron microscopy revealed that the bacterial cells were rod-shaped, approximately 1.5-2.5 μm in length. Figure 5The optimal NaCl concentration for this strain is 0-5% (optimal is 0%), the optimal temperature range is 20-40℃ (optimal is 28℃), and the optimal pH range is 6-11 (optimal is 7).

[0070] Among the 12 carbon sources in API 20NE (Biomerieux, 20050), strain 381-2 can utilize glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, adipic acid, malic acid, citric acid, and phenylacetic acid, but cannot utilize decanoic acid. It can produce β-glucosidase but does not produce tryptophan hydrolase, arginine hydrolase, urease, or protease (Table 2).

[0071] Table 2. Physiological and biochemical indicators of strain 381-2

[0072]

[0073] Example 6: Identification of bacterial sulfur oxidation genes and oxidation products

[0074] Gene annotation was performed using the online annotation tool RAST (https: / / rast.nmpdr.org / ), the Prokka software package on a local server, and the eggNOG online website. The annotated amino acid sequences were uploaded to the KEGG database for comparison and analysis. The thiosulfate dehydrogenase gene (tsdA) was annotated in the genome of strain 381-2, indicating its potential for thiosulfate oxidation via the SI4 pathway.

[0075] To detect the sodium thiosulfate oxidation products of the strain, the above-mentioned strain was transferred at a 1% inoculum to MMT medium containing 20 mM sodium thiosulfate (added after sterilization) and cultured at 30°C and 160 rpm for 24 h. The MMT medium formulation is shown in Example 4. The cultured bacterial solution was centrifuged at 10,000 rpm and 4°C for 10 min, and the supernatant was collected. The sulfur oxidation products of the alkali-producing strain 381-2 were further determined by high-performance liquid chromatography (HPLC). The method for determining the sodium thiosulfate products metabolized by the strain is as follows: Potassium tetrathionate of known concentration was used as a standard, and the concentration of tetrathionate in the sample was determined by high-performance liquid chromatography (HPLC). Instrument model: Arc HPLC (Waters); chromatographic column: Syncoris C18, Dim 250 mm 4.6 mm × 5 μm; HPLC conditions: injection volume 10 μl, column temperature 30 ℃, flow rate 0.5 ml / min, mobile phase: 0.05 M potassium dihydrogen phosphate (pH adjusted to 2.68 with phosphoric acid); detection wavelength: 215 nm. The peak chromatogram results showed that the products of sodium thiosulfate metabolism by the strain were all tetrathionate (…). Figure 6 ).

[0076] Example 7: Detection of the ability of strains to oxidize pyrite

[0077] Pyrite was freeze-dried to produce powder. Artificial seawater was used as the basal culture medium, supplemented with trace elements and vitamins (see Example 1), along with micronutrients. The artificial seawater (ASW) consisted of: 28.13 g NaCl, 0.77 g KCl, 1.60 g CaCl2·2H2O, 4.80 g MgCl2·6H2O, 0.11 g NaHCO3, 3.50 g MgSO4·7H2O, 1000 mL sterile water, and a pH of 7.0. After inoculating strain 381-2 at 28°C for one month, the pH of the solution remained stable at neutral to slightly alkaline. Electron microscopy revealed that the mineral surface became rough, with attached surface-mineralized rod-shaped microorganisms and EPS-like filamentous structures, demonstrating that strain 381-2 could oxidize pyrite and promote the leaching of sulfide minerals. Meanwhile, the oxidation of pyrite mediated by alkali-producing sulfur-oxidizing microorganisms maintains the pH of the environment at a neutral or slightly alkaline level, and the alkali-producing characteristics of the strains control the generation of acidic wastewater caused by mineral oxidation. Microbial treatment methods have the advantages of being economical, efficient, and environmentally friendly.

[0078] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An alkaliphilic sulfur-oxidizing bacterium, characterized in that: The strain named 381-2 has been preserved in China General Microbiological Culture Collection Center on December 23, 2024, and the preservation number is CGMCC No. 33017; the microbial classification name is Stutzerimonas sp. .

2. A microbial inoculant, characterized by: The bacteria contain live bacteria of the alkali-producing sulfur-oxidizing bacteria described in claim 1.

3. The application of the alkali-producing sulfur-oxidizing bacteria of claim 1 or the microbial agent of claim 2 in water pollution treatment.

4. Use according to claim 3, characterized in that: The alkali-producing sulfur-oxidizing bacteria or microbial agents promote the release of metal ions by oxidizing metal sulfides, and increase the pH value of acidic water by producing alkali, thereby causing metal ions to precipitate and reducing the metal content in the water.

5. The application of the alkali-producing sulfur-oxidizing bacteria of claim 1 or the microbial agent of claim 2 in bioleaching.

6. Use according to claim 5, characterized in that: The minerals targeted by the bioleaching process are sulfide minerals.

7. Use according to claim 6, characterized in that: The sulfide minerals mentioned are deep-sea sulfide minerals.

8. The use according to claim 6 or 7, characterized in that: The alkali-producing sulfur-oxidizing bacteria or the microbial agent according to claim 2 promote the leaching of sulfide minerals by oxidizing them, thereby releasing metal ions.

9. The application of the alkali-producing sulfur-oxidizing bacteria of claim 1 or the microbial agent of claim 2 in the oxidation of sulfides.

10. The use of the alkali-producing sulfur-oxidizing bacteria of claim 1 or the microbial agent of claim 2 in the metabolism of sodium thiosulfate to produce tetrathionate.

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