Acid-resistant sulfate reducing bacteria and application thereof

Abstract

The invention relates to acid-resistant sulfate reducing bacteria and application thereof, and belongs to the technical field of microorganisms. The acid-resistant sulfate reducing bacterium disclosed by the invention is Desulfovibrio desulfuricans QY411, and is preserved in the Guangdong Microbial Culture Collection Center on March 14, 2024, and the preservation number of the acid-resistant sulfate reducing bacterium is GDMCC No: 64412. The acid-resistant sulfate reducing bacterium provided by the invention can survive in an environment with the pH value of 4, and can be applied to ecological reconstruction of acid tailings and waste dump soil, the pH values of mining wasteland soil and surface water after ecological reconstruction are remarkably increased, the concentration of SO4 < 2-> in the soil is remarkably reduced, the acidification process in the mining wasteland soil is effectively inhibited, and the acid-resistant sulfate reducing bacterium has the advantages of being safe and reliable. Therefore, the acidification process in the mining wasteland is effectively controlled. Meanwhile, soil heavy metal pollution is reduced, and vegetation growth is recovered.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to acid-resistant sulfate-reducing bacteria and applications thereof. Background Art

[0002] Due to the production and consumption of non-ferrous metals, large areas of abandoned mining land have accumulated over the long-term process of mineral development. Sulfur-containing waste residues in these areas, upon contact with air and water under oxidizing conditions, continuously produce acid, forming acid mine drainage (AMD), characterized by high salinity and heavy metal concentrations. Through surface runoff and groundwater infiltration, AMD can cause pollutants to migrate into surrounding ecosystems, triggering chain reactions such as soil compaction and degradation, eutrophication of water bodies, and a sharp decline in biodiversity, posing a serious threat to the safety and sustainable development of the regional human settlement environment.

[0003] In recent years, pollution control and ecological restoration technologies based on functional microorganisms have become a research hotspot due to their environmentally friendly characteristics. In response to key issues such as mining area acidification control, heavy metal stabilization and ecological function reconstruction, domestic and foreign scholars have carried out a lot of basic research and engineering practice. Among them, sulfate-reducing microorganisms (SRMs) have attracted much attention due to their unique biogeochemical functions. Sulfate-reducing bacteria are a type of strictly anaerobic bacteria or archaea with different morphologies that can reduce SO4 2- Restore to S 2- In this process, every 1g of SO4 is reduced 2- 1.042g of alkalinity can be generated when the alkalinity is 1.042g, which can neutralize the acidity of the environment and increase the pH. 2- Sulfate-reducing bacteria can combine with heavy metal ions in the environment to form metal sulfide precipitates, reducing the bioavailability of heavy metals in mining wastelands. Compared to traditional physical isolation and chemical neutralization methods, microbial treatment technology using sulfate-reducing bacteria offers advantages such as source acid control, long-term stability, and no secondary pollution, demonstrating significant ecological and economic benefits.

[0004] However, the pH range for the growth of most sulfate-reducing bacteria is 7 to 9. The extremely acidic environment of mining wastelands can inhibit their metabolic activity and hinder biofilm formation. As a result, when applying sulfate-reducing bacteria to mining area ecological reconstruction technology, there are technical bottlenecks such as unstable acid control effects and unsustainable restoration effects, which ultimately lead to soil acidification and vegetation degradation. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an acid-resistant sulfate-reducing bacteria and its application. The acid-resistant sulfate-reducing bacteria can grow under the condition of environmental pH as low as 4, and effectively inhibit the occurrence of acidification in mining wasteland, reduce SO4 2- , raising soil and surface water pH.

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

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

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

[0009] In a second aspect, the present invention provides the use of the acid-resistant sulfate-reducing bacteria and / or their fermentation products in soil ecological reconstruction.

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

[0011] Furthermore, the heavy metal includes at least one of Cu, Cd, Zn and Pb.

[0012] Furthermore, it is characterized in that the soil includes tailings and spoil dump soil.

[0013] Furthermore, the vegetation includes at least one of alfalfa, ryegrass, bluegrass, tall fescue and Elymus ovata.

[0014] In a third aspect, the present invention provides a biological preparation containing the acid-resistant sulfate-reducing bacteria and / or their fermentation products.

[0015] In a specific embodiment of the present invention, the biological preparation contains a bacterial solution of the acid-resistant sulfate-reducing bacteria.

[0016] Furthermore, the OD of the acid-resistant sulfate-reducing bacteria in the bacterial solution is 600 It is 0.7 to 1.0, preferably 1.0.

[0017] Furthermore, the bacterial solution also contains a sulfate-reducing bacteria enrichment culture 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] Furthermore, the preparation method of 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 passed for 10-30 minutes, sterilized at above 121°C for more than 20 minutes, and 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 sterilized at 121°C for 20 minutes.

[0023] In a fourth aspect, the present invention provides the use of the biological agent in soil ecological reconstruction.

[0024] Furthermore, the soil ecological reconstruction includes reducing soil heavy metal pollution, reducing SO42- , neutralizing soil and / or surface water pH, and restoring vegetation growth.

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

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

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

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

[0029] Furthermore, the acid-resistant sulfate-reducing bacteria are added by at least one of watering, spraying and burying.

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

[0031] Furthermore, the soil is preliminarily improved, and the preliminarily improved method includes the following steps: mixing alkaline improvement materials and organic improvement materials with the soil, stirring uniformly, and allowing equilibrium reaction to occur for 7 to 14 days. The alkaline improvement materials include at least one of caustic soda, wood ash, quicklime, and slaked lime; and the organic improvement materials include at least one of livestock and poultry manure, river silt, pond silt, and sewer silt. The alkaline improvement materials can largely neutralize the acid produced in the soil of mining wastelands, creating favorable conditions for the growth and reproduction of the acid-resistant sulfate-reducing bacteria strain. The organic improvement materials contain rich nutrients for the growth and reproduction of the acid-resistant sulfate-reducing bacteria strain.

[0032] Furthermore, when the redox potential of the soil after preliminary improvement is lower than 300 mV, trenches are dug on the soil surface with a depth of 8 to 12 cm, the acid-resistant sulfate-reducing bacteria are evenly spread in the trenches in the form of a bacterial liquid, the trenches are backfilled, and the surface is lightly compacted to obtain soil inoculated with acid-resistant sulfate-reducing bacteria.

[0033] Furthermore, the soil includes tailings and spoil dump soil.

[0034] Furthermore, plant seeds can be sown in the soil inoculated with acid-resistant sulfate-reducing bacteria. After sowing, the surface is covered with rice straw and watered 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, the reaction was equilibrated for 14 days.

[0037] Furthermore, the sowing amount of plant seeds is 15-30g / m 2 , preferably 18g / m 2 .

[0038] Furthermore, the plant includes at least one of alfalfa, ryegrass, bluegrass, tall fescue and Elymus ovata.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention provides an acid-resistant sulfate-reducing bacteria strain Desulfovibrio desulfuricans QY411 that can survive in an environment with a pH of 4. It can be used in the ecological reconstruction of acidic tailings and waste dump soils. After ecological reconstruction, the pH of the soil and surface water in the mining wasteland increases significantly, and the SO4 in the soil decreases. 2- The concentration has dropped significantly, indicating that the acidification process in the mining wasteland soil has been effectively suppressed, thus achieving effective control of the acidification process in the mining wasteland. At the same time, it has reduced soil heavy metal pollution and restored vegetation growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Figure 4 shows the enrichment and isolation of the acid-tolerant sulfate-reducing bacterium Desulfovibrio desulfuricans QY411. Figure A shows the enrichment process, while Figure B shows the isolation process.

[0042] Figure 2 This is a scanning electron micrograph of the colonization of the acid-tolerant sulfate-reducing bacterium Desulfovibrio desulfuricans QY411 on the surface of its fermentation product.

[0043] Figure 3 This is a scanning electron micrograph of the individual morphological characteristics of the acid-tolerant sulfate-reducing bacterium Desulfovibrio desulfuricans QY411 strain.

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

[0045] Figure 5 This is the growth curve of the acid-tolerant sulfate-reducing bacteria Desulfovibrio desulfuricans QY411 and the acid-intolerant strain Desulfovibrio desulfuricans REO-01 at pH 4.

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

[0047] Figure 7 To investigate the pH of surface water, pH of tailings, and SO4 in tailings before and after ecological reconstruction using acid-tolerant sulfate-reducing bacteria Desulfovibrio desulfuricans QY411. 2- 、Cu 2+ 、Cd 2+ 、Zn 2+ Among them, A is the change of pH value of surface water; B is the change of pH value of tailings; C is the content of SO4 in tailings. 2- Content changes; D is the Cu content in the tailings 2+ E is the change of Cd content in tailings 2+ Content changes; F is the Zn content in the tailings 2+ The p values ​​in the figure are the t-test results of the corresponding indicators before and after ecological reconstruction. DETAILED DESCRIPTION

[0048] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

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

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

[0051] (2) Preparation of solid culture medium: Weigh 0.45 g NH2SO4, 0.05 g KCl, 0.5 g MgSO4·7H2O, 0.05 g KH2PO4, 0.014 g Ca(NO3)2·4H2O, 0.2 g yeast extract and 0.92 g glycerol, dissolve in 700 mL distilled water, and adjust the pH to 4 with H2SO4 to obtain culture medium a; weigh 15 g agar and dissolve in 300 mL distilled water to obtain agar solution; pass nitrogen gas through culture medium a and agar solution for 30 min, sterilize at 121°C for 20 min, cool to about 50°C, mix culture medium a and agar solution evenly, and then add 0.5 g FeSO4·7H2O, 0.02 g ascorbic acid and 0.1 g CH3C(S)NH4, to obtain solid culture medium, pour the plate according to the amount of 15mL solid culture medium per plate, seal the plate with sealing film after solidification and store under anaerobic conditions until use.

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

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

[0054] Example 2 Identification of acid-resistant sulfate-reducing bacteria strains

[0055] (1) Morphological characteristics: Figure 2 and Figure 3 As shown, the bacterial morphology and structure of the isolated colonies were identified and observed using scanning electron microscopy. Scanning electron microscopy revealed that the strain had a smooth cell surface and grew primarily in an attached form on sulfides produced by metabolism. The cells could reach a maximum length of 50 μm.

[0056] (2) Species identification: Genomic DNA of the isolated strain was extracted, and 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) were used to amplify the 16S rRNA gene sequence (nucleotide sequence as shown in SEQ ID NO: 3). The PCR amplification system was 25 μL, and the specific components included 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°C, 5 min, denaturation temperature 94°C, 30 s, annealing temperature 55°C, 30 s, extension temperature 72°C, 90 s; 30 cycles, supplementary extension temperature 72°C, 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 strain obtained by sequencing was compared with the database sequence using NCBI BLAST. The results showed that the strain had a sequence homology of up to 99% with Desulfovibrio desulfuricans under the genus Desulfovibrio. 16 16S rRNA gene sequences of microorganisms of the same species, genus or family were downloaded from the Silva database (version 138.2) as reference sequences. Together with the sequence of the strain, a phylogenetic tree was constructed, proving that the strain was Desulfovibrio desulfuricans. Figure 4 ).

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

[0058] Example 3 Evaluation of Acid Resistance of Desulfovibrio desulfuricans QY411

[0059] The acid-tolerant, sulfate-reducing bacterium Desulfovibrio desulfuricans QY411 isolated in Example 2 was inoculated at a 2% ratio into the enrichment medium prepared in Example 1. Three replicates were cultured anaerobically at 30°C to obtain a bacterial suspension. The absorbance of the suspension was measured at a wavelength of 600 nm using a spectrophotometer every 24 hours. The same strain, sulfate-reducing bacterium REO-01, was used as a control for comparison. This strain is deposited at the General Microbiology Center of the China General Culture Collection Administration under the CGMCC No. 24267.

[0060] like Figure 5 As shown, after 13 days of culture, the OD value of the acid-resistant sulfate-reducing bacteria Desulfovibrio desulfuricans QY411 isolated and obtained by the present invention was 600 reached 0.662, while the OD of the control strain REO-01 600 It is only 0.007, indicating that the strain isolated in the present invention has stronger acid resistance than other strains of the same species and can grow at a pH of 4.

[0061] Example 4: Application of Acid-Resistant Sulfate-Reducing Bacteria Desulfovibrio desulfuricans QY411 in Acidic Tailings Ecological Reconstruction

[0062] (1) Acidic tailings basins: Test basins with a diameter of 16 cm, a height of 17.5 cm, and a bottom diameter of 13 cm were selected, and approximately 5 kg of tailings sample was placed in each basin. The tailings samples used in the test were from the Dabaoshan tailings reservoir in Shaoguan City, Guangdong Province.

[0063] (2) Initial improvement of acidic tailings: Add slaked lime and mix evenly with the tailings at a depth of 12 cm to neutralize the H in the acidic tailings. + , so that the pH value of the tailings reaches above 4, then fermented chicken manure is added and evenly stirred with the tailings at a depth of 12 cm on the surface again, and the equilibrium reaction is carried out for 14 days, during which the redox potential of the tailings is regularly measured ( Figure 6 A).

[0064] (3) Inoculation of functional microorganisms: When the redox potential of the tailings is lower than 300 mV, dig a trench on the surface of the tailings with a depth of about 8 to 12 cm, and inoculate Desulfovibrio desulfuricans QY411 with a bacterial solution (OD 600 =1) in the form of spraying evenly in the trench, backfilling the trench, lightly compacting the surface, using a film to seal the test basin, and tracking the physical and chemical indicators of the tailings.

[0065] (4) Planting: according to 18g / m2 Sow ryegrass seeds at a dosage of 500mg / L. After sowing, cover the surface with rice straw, water regularly, and test the germination rate and plant height of the plants and compare them with those of untreated tailings ( Figure 6 B).

[0066] like Figure 6 C and Table 2, after the tailings were inoculated with Desulfovibrio desulfuricans QY411, the ryegrass grew well.

[0067] Table 1 Changes in physical and chemical indicators of tailings after treatment with Desulfovibrio desulfuricans QY411 for 12 weeks

[0068]

[0069]

[0070] *Note: ND means the amount is below the lower limit of detection (<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 results of physical and chemical index data are shown in Table 1 and Figure 7 A to Figure 7 F. The acidic tailings ecological reconstruction experiment lasted for 12 weeks. After the ecological reconstruction, the pH of the tailings increased from 2.19 to 7.85, and the pH of the surface water on the tailings surface increased from 2.15 to 7.74. The SO4 2- The content decreased from 22.37g / kg to 8.422g / kg, indicating that under the action of Desulfovibrio desulfuricans QY411, SO4 2- Restored to S 2- , the oxidation process of sulfur in the environment is inhibited and the acidification of tailings is effectively controlled. 2+ The leaching toxicity of Cd was reduced from 136.4 mg / kg to 0.480 mg / kg, and the 2+ 、Zn 2+ The leaching toxicity of the tailings was reduced from 0.320 mg / kg and 48.65 mg / kg, respectively, to below the instrument detection limit, indicating a significant reduction in the risk of pollutant migration in the tailings. This demonstrates that Desulfovibrio desulfuricans QY411 has the function of inhibiting sulfur oxidation and controlling acidity, and is suitable for ecological reconstruction of mining wastelands.

[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 the technical solutions of the present invention may be modified or replaced by equivalents 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-resistant sulfate-reducing bacteria is Desulfovibriodesulfuricans QY411, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on March 14, 2024, and its preservation number is: GDMCC No: 64412.

2. Use of the acid-resistant sulfate-reducing bacteria and / or their fermentation products according to claim 1 in soil ecological reconstruction.

3. The use according to claim 2, characterized in that The soil ecological reconstruction includes reducing soil heavy metal pollution, reducing SO4 2- , neutralizing soil and / or surface water pH, and restoring vegetation growth.

4. The use according to claim 3, characterized in that The heavy metal includes at least one of Cu, Cd, Zn and Pb.

5. The use according to claim 2, characterized in that The soil includes tailings and spoil dump soil.

6. The use according to claim 2, characterized in that The vegetation includes at least one of alfalfa, ryegrass, bluegrass, tall fescue and Elymus ovata.

7. A biological agent, characterized in that The biological preparation contains the acid-resistant sulfate-reducing bacteria and / or fermentation products thereof according to claim 1.

8. Use of the biological agent according to claim 7 in soil ecological reconstruction.

9. A method for soil ecological reconstruction, characterized in that: The acid-resistant sulfate-reducing bacteria according to claim 1 or the biological agent according to claim 7 are added to the soil for ecological reconstruction.

10. The method according to claim 9, characterized in that The inoculation amount of the acid-resistant sulfate-reducing bacteria is 0.1-5% v / v.

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