An acid-resistant sulfate-reducing bacterium and its application

Abstract

This invention relates to an acid-resistant sulfate-reducing bacterium and its application, belonging to the field of microbial technology. The acid-resistant sulfate-reducing bacterium described in this invention is *Desulfovibrio desulfuricans* QY411, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on March 14, 2024, with accession number GDMCC No: 64412. This invention provides an acid-resistant sulfate-reducing bacterium that can survive in an environment with a pH of 4, which can be applied to the ecological reconstruction of acidic tailings and spoil heap soils. After ecological reconstruction, the pH of the soil and surface water in mining waste sites increases significantly, and the SO4 content in the soil decreases. 2‑ The concentration decreased significantly, effectively inhibiting the acidification process in the soil of mining waste sites, thus achieving effective control over the acidification process in mining waste sites. Simultaneously, it reduced soil heavy metal pollution and restored vegetation growth.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to an acid-tolerant sulfate-reducing bacterium and its application. Background Art

[0002] Due to the production and consumption of non-ferrous metals, large areas of mining waste lands have been accumulated during the long-term mineral development process. The sulfur-containing waste residues stored in these areas will continuously produce acid to form mine acid wastewater after contacting with air and moisture under oxidation conditions, which has characteristics such as high salinity and high heavy metal concentration. Through surface runoff and groundwater infiltration, the pollutants in the mine acid wastewater can diffuse and migrate to the surrounding ecological systems, triggering a series of ecological effects such as soil compaction and degradation, water eutrophication, and sharp reduction of biodiversity, posing a serious threat to the regional human settlement environmental safety and sustainable development.

[0003] In recent years, pollution control and ecological restoration technologies based on functional microorganisms have become research hotspots due to their environmentally friendly characteristics. In response to key issues such as mine acidification regulation, heavy metal stabilization, and ecological function reconstruction, scholars at home and abroad have carried out a large number of basic research and engineering practices. Among them, sulfate-reducing microorganisms (SRMs) have attracted much attention due to their unique biogeochemical functions. Sulfate-reducing bacteria are a class of strictly anaerobic bacteria or archaea with various morphologies, and they can reduce SO4 2- to S 2- . In this process, when every 1 g of SO4 2- is reduced, 1.042 g of alkalinity can be generated, and these alkalinities can neutralize the environmental acidity and increase the pH. At the same time, the S 2- produced during the reduction process can combine with heavy metal ions in the environment to form metal sulfide precipitates, reducing the bioavailability of heavy metals in mining waste lands. Compared with traditional physical isolation and chemical neutralization methods, the microbial treatment technology using sulfate-reducing bacteria has advantages such as source acid control, long-term stability, and no secondary pollution, showing significant ecological and economic benefits.

[0004] However, the pH range for the growth of most sulfate-reducing bacteria is 7-9, and the extreme acidic environment of mining waste lands will cause problems such as inhibition of metabolic activity and hindrance of biofilm formation, resulting in technical bottlenecks such as unstable acid control effect and unsustainable restoration effect when applying sulfate-reducing bacteria to mine ecological reconstruction technology, and finally phenomena such as soil acid return and vegetation degradation occur. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide an acid-tolerant sulfate-reducing bacterium and its application. The acid-tolerant sulfate-reducing bacterium can grow under the condition that the environmental pH is as low as 4, effectively inhibit the acidification in mining wasteland, and reduce SO4 2- , and increase the pH of soil and surface water.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention provides an acid-tolerant sulfate-reducing bacterium, which is characterized in that the acid-tolerant sulfate-reducing bacterium is Desulfovibrio desulfuricans QY411, which was deposited with the Guangdong Provincial Microbial Culture Collection Center on March 14, 2024, and its deposit number is: GDMCC No: 64412.

[0008] The acid-tolerant sulfate-reducing bacterium Desulfovibrio desulfuricans QY411 of the present invention can grow under the condition that the environmental pH is as low as 4, effectively inhibit soil acidification, increase the pH of soil and surface water, and reduce SO4 2- , reduce soil heavy metal pollution, and restore vegetation growth.

[0009] In the second aspect, the present invention provides the application of the acid-tolerant sulfate-reducing bacterium and / or its fermentation product in soil ecological reconstruction.

[0010] Further, the soil ecological reconstruction includes at least one of reducing soil heavy metal pollution, reducing SO4 2- , neutralizing the pH of soil and / or surface water, and restoring vegetation growth.

[0011] Further, the heavy metals include at least one of Cu, Cd, Zn and Pb.

[0012] Further, it is characterized in that the soil includes tailings and soil of waste dump sites.

[0013] Further, the vegetation includes at least one of alfalfa, ryegrass, poa annua, tall fescue and Elymus dahuricus.

[0014] In the third aspect, the present invention provides a biological preparation, and the biological preparation contains the acid-tolerant sulfate-reducing bacterium and / or its fermentation product.

[0015] In the specific embodiment of the present invention, the biological preparation contains the bacterial liquid of the acid-tolerant sulfate-reducing bacterium.

[0016] Further, the OD of the acid-tolerant sulfate-reducing bacterium in the bacterial liquid 600 is 0.7 to 1.0, preferably 1.0.

[0017] Furthermore, the bacterial solution also contains a sulfate-reducing bacteria enrichment medium.

[0018] Furthermore, the sulfate-reducing bacteria enrichment medium contains at least one of NH2SO4, KCl, MgSO4·7H2O, KH2PO4, Ca(NO3)2·4H2O, yeast extract, glycerol, FeSO4·7H2O, ascorbic acid, and CH3C(S)NH4.

[0019] Preferably, the sulfate-reducing bacteria enrichment medium contains 0.44–0.46 g / L NH₂SO₄, 0.04–0.06 g / L KCl, 0.4–0.5 g / L MgSO₄·7H₂O, 0.04–0.06 g / L KH₂PO₄, 0.014–0.015 g / L Ca(NO₃)₂·4H₂O, 0.19–0.21 g / L yeast extract, 0.9–0.95 g / L glycerol, 0.49–0.51 g / L FeSO₄·7H₂O, 0.019–0.021 g / L ascorbic acid, and 0.09–0.11 g / L CH₃C(S)NH₄.

[0020] More preferably, the sulfate-reducing bacteria enrichment medium contains 0.45 g / L NH2SO4, 0.05 g / L KCl, 0.5 g / L MgSO4·7H2O, 0.05 g / L KH2PO4, 0.014 g / L Ca(NO3)2·4H2O, 0.2 g / L yeast extract, and 0.92 g / L glycerol, 0.5 g / L FeSO4·7H2O, 0.02 g / L ascorbic acid, and 0.1 g / L CH3C(S)NH4.

[0021] Further, the method for preparing the sulfate-reducing bacteria enrichment medium is as follows: NH2SO4, KCl, MgSO4·7H2O, KH2PO4, Ca(NO3)2·4H2O, yeast extract and glycerol are mixed, the pH is adjusted to 4-7 with H2SO4, diluted with water, nitrogen is purged for 10-30 min, sterilized at 121℃ or above for 20 min or more, ascorbic acid and CH3C(S)NH4 are added to obtain the sulfate-reducing bacteria enrichment medium.

[0022] More preferably, the pH is adjusted to 4 with H2SO4, nitrogen is passed through for 30 minutes, and sterilization is carried out at 121°C for 20 minutes.

[0023] Fourthly, the present invention provides the application of the biological agent in soil ecological reconstruction.

[0024] Furthermore, the soil ecological reconstruction includes reducing soil heavy metal pollution and reducing SO4.2- Neutralize at least one of the pH of soil and / or surface water and restore vegetation growth.

[0025] Furthermore, the heavy metals include at least one of Cu, Cd, Zn, and Pb.

[0026] Furthermore, the soil includes tailings and waste dump soil.

[0027] Furthermore, the vegetation includes at least one of alfalfa, ryegrass, Kentucky bluegrass, tall fescue, and Elymus dahuricus.

[0028] In a fifth aspect, the present invention provides a method for soil ecological reconstruction, adding the acid-tolerant sulfate-reducing bacteria to the soil for ecological reconstruction.

[0029] [[ID=十六]]The addition method of the acid-tolerant sulfate-reducing bacteria includes at least one of irrigation, spraying, and burial.

[0030] Furthermore, the inoculation amount of the acid-tolerant sulfate-reducing bacteria is 0.1 - 5% v / v. The preferred inoculation amount is 5% v / v.

[0031] Furthermore, the soil is preliminarily improved soil, and the method for preliminary improvement includes the following steps: mixing an alkaline improvement material, an organic improvement material with the soil, stirring evenly, and carrying out an equilibration reaction for 7 - 14 days. The alkaline improvement material includes at least one of caustic soda, plant ash, quicklime, and slaked lime; the organic improvement material includes at least one of livestock and poultry manure, river silt, pond silt, and sewer silt. The alkaline improvement material can neutralize a large amount of acid already generated in the soil of mining waste lands, creating good conditions for the growth and reproduction of the acid-tolerant sulfate-reducing bacteria strains. The organic improvement material contains rich nutrient elements for the growth and reproduction of the acid-tolerant sulfate-reducing bacteria strains.

[0032] Furthermore, when the redox potential of the preliminarily improved soil is lower than 300 mV, dig trenches on the soil surface, with the trench depth being 8 - 12 cm, evenly sprinkle the acid-tolerant sulfate-reducing bacteria in the form of a bacterial solution in the trenches, backfill the trenches, and gently compact the surface to obtain soil inoculated with the acid-tolerant sulfate-reducing bacteria.

[0033] Furthermore, the soil includes tailings and waste dump soil. [[ID=2?]]

[0034] Furthermore, plant seeds can be sown in the soil inoculated with the acid-tolerant sulfate-reducing bacteria. After sowing, cover the surface with rice straw and water for maintenance.

[0035] Furthermore, the livestock and poultry manure includes at least one of chicken manure, pig manure, cow manure, and sheep manure.

[0036] Furthermore, carry out an equilibration reaction for 14 days.

[0037] Furthermore, the sowing rate of plant seeds is 15–30 g / m². 2 Preferred 18g / m 2 .

[0038] Furthermore, the plant includes at least one of alfalfa, ryegrass, Kentucky bluegrass, tall fescue, and crested wheatgrass.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] This invention provides an acid-resistant sulfate-reducing bacterium strain, *Desulfovibrio desulfuricans* QY411, which can survive in an environment with a pH of 4. This strain can be applied to the ecological reconstruction of acidic tailings and spoil heap soils. After ecological reconstruction, the pH of the soil and surface water in mining waste sites significantly increases, and the SO4 content in the soil decreases. 2- The significant decrease in concentration indicates that the acidification process in the soil of mining waste sites has been effectively suppressed, thus achieving effective control over the acidification process in mining waste sites. Simultaneously, it reduces soil heavy metal pollution and restores vegetation growth. Attached Figure Description

[0041] Figure 1 This section shows the enrichment and isolation of the acid-resistant sulfate-reducing bacterium *Desulfovibrio desulfuricans* QY411. In the diagram, A represents the enrichment process, and B represents the isolation process.

[0042] Figure 2 Scanning electron microscope image of the colonization of the acid-tolerant sulfate-reducing bacterium Desulfovibrio desulfuricans QY411 on the surface of its fermentation products.

[0043] Figure 3 Scanning electron microscope image of the morphological characteristics of an individual strain of acid-resistant sulfate-reducing bacterium *Desulfovibrio desulfuricans* QY411.

[0044] Figure 4 Phylogenetic tree of the acid-tolerant sulfate-reducing bacterium Desulfovibrio desulfuricans QY411.

[0045] Figure 5 The growth curves of acid-tolerant sulfate-reducing bacterium *Desulfovibrio desulfuricans* QY411 and acid-intolerant strain *Desulfovibrio desulfuricans* REO-01 at pH 4 are shown.

[0046] Figure 6 This describes the process of ecological reconstruction using the acid-tolerant sulfate-reducing bacterium *Desulfovibrio desulfuricans* QY411. A represents the initial improvement of tailings soil; B represents the sowing of plant seeds and covering with straw after inoculation with the sulfate-reducing bacteria; and C represents the plant growth status three months later.

[0047] Figure 7 pH values ​​of tailings surface water and tailings pH and SO4 levels in tailings before and after ecological reconstruction using the acid-resistant sulfate-reducing bacterium *Desulfovibrio desulfuricans* QY411 were measured. 2- Cu 2+ Cd 2+ Zn 2+ The content of SO4 in the tailings. Where A represents the change in surface water pH; B represents the change in tailings pH; and C represents the content of SO4 in the tailings. 2- Changes in content; D represents Cu in tailings 2+ Changes in content; E represents the Cd content in tailings. 2+ Changes in content; F represents Zn in tailings 2+ Changes in content. The p-values ​​in the figure are the t-test results of the corresponding indicators before and after ecological reconstruction. Detailed Implementation

[0048] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0049] Example 1: Isolation of acid- and sulfate-reducing bacteria strains

[0050] (1) Preparation of enrichment medium: Weigh 0.45g NH2SO4, 0.05g KCl, 0.5g MgSO4·7H2O, 0.05g KH2PO4, 0.014g Ca(NO3)2·4H2O, 0.2g yeast extract and 0.92g glycerol, mix them, adjust the pH to 4 with H2SO4, and then make up the volume to 1L with distilled water. Purge with nitrogen for 30min, sterilize at 121℃ for 20min, and then add 0.5g FeSO4·7H2O, 0.02g ascorbic acid and 0.1g CH3C(S)NH4 to obtain the enrichment medium.

[0051] (2) Preparation of solid culture medium: Weigh 0.45g NH2SO4, 0.05g KCl, 0.5g MgSO4·7H2O, 0.05g KH2PO4, 0.014g Ca(NO3)2·4H2O, 0.2g yeast extract and 0.92g glycerol, dissolve in 700mL distilled water, adjust pH to 4 with H2SO4 to obtain culture medium a; separately weigh 15g agar and dissolve in 300mL distilled water to obtain agar solution; purge culture medium a and agar solution with nitrogen for 30min, sterilize at 121℃ for 20min, and after cooling to about 50℃, mix culture medium a and agar solution evenly, then add 0.5g FeSO4·7H2O, 0.02g ascorbic acid and 0.1g CH3C(S)NH4 was used to obtain a solid culture medium. 15 mL of solid culture medium was poured into each plate. After the plates solidified, they were sealed with sealing film and stored under anaerobic conditions until use.

[0052] (3) Enrichment culture: Take 0.5g of tailings sample from Shuilongwei lead-zinc mine in Fogang County, Qingyuan City, Guangdong Province into an anaerobic tube, add 2 / 3 volume of enrichment culture medium to the anaerobic tube, and incubate in the dark at 34℃ in an incubator until the culture solution changes from clear to ink color.

[0053] (4) Strain isolation: such as Figure 1 A and Figure 1 As shown in B, the bacterial culture obtained from the enrichment culture in step (3) is serially diluted to 10. -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 Then, 100 μL of the culture medium was spread onto a plate containing solid culture medium and incubated in an anaerobic bag at 34°C until single colonies grew. Single colonies were picked and streaked onto a plate, and purified for 3-4 generations until stable single colonies with a single morphology grew.

[0054] Example 2: Identification of acid- and sulfate-reducing bacterial strains

[0055] (1) Morphological characteristics: such as Figure 2 and Figure 3 As shown, the morphology and structure of the isolated colonies were identified and observed using scanning electron microscopy. The scanning electron microscope revealed that the cell surface of the strain was relatively smooth, and the cells mainly grew by attachment on metabolically produced sulfides, with the longest cell reaching 50 μm.

[0056] (2) Species identification: Genomic DNA was extracted from the isolated strain. The 16S rRNA gene sequence (nucleotide sequence as shown in SEQ ID NO: 1) was amplified using 27F (5'-3': AGAGTTTGATCMTGGCTCAG, nucleotide sequence as shown in SEQ ID NO: 1) and 1492R (5'-3': GGTTACCTTGTTACGACTT, nucleotide sequence as shown in SEQ ID NO: 2). The PCR amplification system was 25 μL, specifically containing 12.5 μL Taq mix, 0.5 μL 27F primer, 0.5 μL 1492R primer, 1 μL strain DNA, and 10.5 μL ddH2O. The PCR amplification program was as follows: pre-denaturation temperature 94℃, 5 min; denaturation temperature 94℃, 30 s; annealing temperature 55℃, 30 s; extension temperature 72℃, 90 s; 30 cycles; supplementary extension temperature 72℃, 10 min. The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain sequence information. The 16S rRNA gene sequence of the obtained strain was compared with database sequences using NCBI BLAST. The results showed that the strain had 99% homology with the sequence of *Desulfovibrio desulfuricans* under the genus *Desulfovibrio*. Sixteen 16 16S rRNA gene sequences of the same species, genus, or family of microorganisms were downloaded from the Silva database (version 138.2) as reference sequences and used together with the sequence of this strain to construct a phylogenetic tree, confirming that this strain is *Desulfovibrio desulfuricans*. Figure 4 ).

[0057] The obtained strain was named Desulfovibrio desulfuricans QY411 and deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 14, 2024, with accession number GDMCC No: 64412. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0058] Example 3: Evaluation of the acid resistance of the acid-resistant sulfate-reducing bacterium *Desulfovibrio desulfuricans* QY411

[0059] The acid-resistant sulfate-reducing bacterium *Desulfovibrio desulfuricans* QY411, isolated in Example 2, was inoculated at a ratio of 2% into the enrichment medium prepared in Example 1. Three replicates were set up, and the culture was anaerobic at 30°C to obtain bacterial suspensions. The absorbance of the bacterial suspensions was measured every 24 hours using a spectrophotometer at a wavelength of 600 nm. Simultaneously, the sulfate-reducing bacterium *REO-01*, a strain deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 24267, was used as a control for comparison.

[0060] like Figure 5 As shown, after 13 days of cultivation, the OD of the acid-resistant sulfate-reducing bacterium *Desulfovibriodesulfuricans* QY411 isolated in this invention was... 600 The OD value reached 0.662, while that of the control strain REO-01 was... 600 The value of only 0.007 indicates that the strain isolated in this invention has stronger acid resistance than other strains of the same species and can grow under conditions of pH 4.

[0061] Example 4: Acid-resistant sulfate-reducing bacterium *Desulfovibrio desulfuricans* QY411 used for ecological reconstruction of acidic tailings.

[0062] (1) Acidic tailings sample loading: Test basins with a diameter of 16cm, a height of 17.5cm, and a bottom diameter of 13cm were selected, and about 5kg of tailings sample was loaded into each basin. The tailings sample used in the test came from the Dabao Mountain tailings dam in Shaoguan City, Guangdong Province.

[0063] (2) Preliminary improvement of acidic tailings: Add quicklime and mix evenly with the tailings at a depth of 12cm to neutralize the H in the acidic tailings. + The tailings pH was brought to above 4, then fermented chicken manure was added and mixed evenly with the top 12cm of tailings again. The reaction was allowed to equilibrate for 14 days, during which the oxidation-reduction potential of the tailings was measured periodically. Figure 6 A).

[0064] (3) Functional microbial inoculation: When the redox potential of the tailings is below 300mV, trenches are dug on the surface of the tailings, with a depth of about 8-12cm. Based on the volume of the improved tailings, Desulfovibriodesulfuricans QY411 is inoculated at a rate of 0.5% (v / v) in bacterial suspension (OD). 600 =1) Spray evenly in the trench, backfill the trench, gently compact the surface, use a thin film to seal the test basin, and track and test the physicochemical indicators of the tailings.

[0065] (4) Planting: according to 18g / m2 The amount of ryegrass seeds used was sown, and after sowing, the surface was covered with rice straw. Regular watering and maintenance were carried out, and the germination rate and plant height were measured and compared with untreated tailings. Figure 6 B).

[0066] like Figure 6 As shown in Table C and Table 2, the ryegrass grew well after inoculating the tailings with Desulfovibrio desulfuricans QY411.

[0067] Table 1. Changes in physicochemical properties of tailings after 12 weeks of treatment with Desulfovibrio desulfuricans QY411.

[0068]

[0069]

[0070] *Note: ND indicates that the concentration was below the detection limit (<0.001 mg / kg) and was not detected.

[0071] Table 2. Plant growth after 12 weeks of treatment with Desulfovibrio desulfuricans QY411

[0072]

[0073] The physicochemical index data results are shown in Table 1 and Figure 7 A to Figure 7 As shown in Figure F. The acidic tailings ecological reconstruction experiment lasted for 12 weeks. After ecological reconstruction, the pH of the tailings increased from 2.19 to 7.85, the pH of the surface water on the tailings surface increased from 2.15 to 7.74, and the SO4 content in the tailings decreased. 2- The content decreased from 22.37 g / kg to 8.422 g / kg, indicating that the SO4 content in the acidic tailings decreased under the action of Desulfovibrio desulfuricans QY411. 2- Reduced to S 2- The oxidation process of sulfur in the environment is inhibited, and the acidification of tailings is effectively controlled. Cu in the tailings 2+ The leaching toxicity decreased from 136.4 mg / kg to 0.480 mg / kg, Cd 2+ Zn 2+ The leaching toxicity decreased from 0.320 mg / kg and 48.65 mg / kg to below the instrument detection limit, indicating a significant reduction in the migration risk of pollutants in the tailings. This demonstrates that the present invention, Desulfovibrio desulfuricans QY411, possesses the functions of sulfur inhibition, oxidation control, and acid control, and is applicable to the ecological reconstruction of mining waste sites.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An acid-resistant sulfate-reducing bacterium, characterized in that, The acid- and sulfate-reducing bacteria are Desulfovibrio desulfuricans QY411 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 14, 2024, with accession number GDMCC No: 64412.

2. The application of the acid-resistant sulfate-reducing bacteria described in claim 1 in soil ecological reconstruction; The soil ecological reconstruction includes reducing heavy metal pollution in the soil and reducing SO4. 2- At least one of the following: neutralizing soil and / or surface water pH, or restoring vegetation growth; The heavy metals include at least one of Cu, Cd, and Zn.

3. The application according to claim 2, characterized in that, The soil includes tailings and spoil heap soil.

4. The application according to claim 2, characterized in that, The vegetation includes at least one of alfalfa, ryegrass, Kentucky bluegrass, tall fescue, and crested wheatgrass.

5. A method for soil ecological reconstruction, characterized in that, The acid-resistant sulfate-reducing bacteria described in claim 1 are added to the soil for ecological reconstruction. The soil ecological reconstruction includes reducing heavy metal pollution in the soil and reducing SO4. 2- At least one of the following: neutralizing soil and / or surface water pH, or restoring vegetation growth; The heavy metals include at least one of Cu, Cd, and Zn.

6. The method according to claim 5, characterized in that, The inoculation amount of the acid-resistant sulfate-reducing bacteria is 0.1–5% v / v.

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