A new sulfate-reducing bacterial strain and application thereof

Abstract

The application relates to the field of agricultural heavy metal microbial remediation, and discloses a new sulfate-reducing strain, a new sulfate-reducing strain Desulfovibrio sp.GLDM01 which has been preserved in the China Center for Type Culture Collection on March 9, 2026, and the preservation number is CCTCC NO:M 2026395. The new sulfate-reducing strain has the functions of adsorbing cadmium elements in soil and improving the ability of rice to enrich methylmercury, thereby providing new strain resources for farmland heavy metal remediation.

Description

Technical Field

[0001] This invention relates to the field of microbial remediation of heavy metals in agriculture, specifically to a novel sulfate-reducing strain and its application. Background Technology

[0002] Globally, 14% to 17% of farmland is contaminated with toxic metals, impacting agricultural production and food security. Calculations show that my country loses over 10 million tons of grain annually due to heavy metal pollution, resulting in cumulative losses of up to 20 billion yuan. Approximately 900 million to 1.4 billion people worldwide live in areas with excessive heavy metal pollution in their soil, posing potential risks to human health. Therefore, the remediation of heavy metal pollution in farmland is a crucial foundation for ensuring safe crop production and a healthy diet for residents.

[0003] Compared to physical and chemical remediation, bioremediation relies on animals, plants, and microorganisms in the soil environment to reduce the bioavailability of heavy metals. This technology is low-cost and widely applicable. Microbial remediation mechanisms are mainly divided into intracellular and extracellular processes. Extracellular removal primarily involves adsorption-fixation and ion exchange, while intracellular removal mainly utilizes peptidoglycans and enzymes on the bacterial surface to enhance the solubility of heavy metals, thereby accumulating them within the bacteria. Sulfate-reducing bacteria (SRBs) possess a unique advantage, not only passivating heavy metals both extracellularly and intracellularly but also fixing heavy metals in the soil through sulfide ions generated by sulfate reduction.

[0004] Currently, known sulfate-reducing bacteria, including Desulfricans MB and Desulfovibrio legalliidsv3, have been found in anaerobic environments, but their application in agricultural heavy metal remediation is limited. Therefore, it is necessary to explore more sulfate-reducing bacteria and their application in farmland heavy metal remediation. Summary of the Invention

[0005] The present invention aims to provide a novel sulfate-reducing strain and study its application in passivating cadmium in farmland and enhancing the ability of rice to accumulate methylmercury, thereby providing a new strain resource for the microbial remediation of heavy metals in farmland.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a novel sulfate-reducing strain, Desulfovibrio sp. GLDM01, was deposited at the China Center for Type Culture Collection on March 9, 2026, with accession number CCTCC NO: M 2026395.

[0007] The novel sulfate-reducing strain provided by this invention has been experimentally verified to passivate cadmium in farmland and enhance the ability of rice to accumulate methylmercury, thereby providing a new strain resource for the microbial remediation of heavy metals in farmland.

[0008] Preferably, the sulfate-reducing strain is a Gram-negative bacterium, and the dissolved oxygen in the culture environment of the strain is 0.23-0.42 mg·L⁻¹. -1 The temperature is 35 degrees Celsius. o C.

[0009] The present invention also provides a sulfate-reducing bacterial agent, comprising a live bacterial preparation of the aforementioned sulfate-reducing new strain, bacterial lysate, or fermentation supernatant.

[0010] This invention also provides a method for obtaining novel sulfate-reducing strains, comprising the following steps: Enrichment culture: Soil samples were taken to prepare supernatant, and the supernatant was inoculated into liquid culture medium and cultured at constant temperature until sulfurization characteristics appeared. The enriched bacterial solution was obtained by continuous transfer. Separate and purify the bacteria by adding agar to the liquid culture medium to form a plate, streak the diluted enriched bacterial solution on the surface of the plate, cover the bottom of the streak with solid culture medium, seal and anaerobic culture until a single colony grows. To obtain the target sulfate-reducing strain, single colonies were picked under anaerobic conditions and inoculated into sterile liquid culture medium for culture.

[0011] The beneficial effects of this scheme are as follows: This scheme combines the sandwich method and the dilution shaker method. This combined improvement retains the advantages of the sandwich method, such as good anaerobic sealing, clear colony stratification, and easy observation and picking, while incorporating the characteristics of the dilution shaker method, such as gradient dilution and dispersion of bacteria, deep oxygen-limited shaping, and inhibition of spread and adhesion. Compared with using either traditional method alone, it can efficiently remove impurities and enrich bacteria, significantly reduce the risk of trace oxygen contamination, and quickly obtain dispersed, independent, and highly pure single colonies. It has high operational tolerance, strong repeatability, and short screening cycle, and is more suitable for the precise isolation and preservation of new strains of strict anaerobic sulfate reduction.

[0012] Preferably, in the enrichment culture step, the soil sample is purple soil, and the purple soil is paddy soil; the liquid culture medium includes NaCl, NH4Cl, MgSO4·7H2O, Na2SO4, K2HPO4, and the pH is 7.5~8.0.

[0013] Preferably, in the separation and purification step, the solid culture medium is obtained by mixing the liquid culture medium with agar, and the concentration of agar is 1.5-2%.

[0014] The present invention also provides an application of a novel sulfate-reducing strain, which is used to passivate cadmium in farmland.

[0015] Preferably, the sulfate-reducing strain can reduce the content of exchangeable and carbonate-bound heavy metals in cadmium-containing soils, and increase the content of iron-manganese-bound, organic matter and sulfide-bound, and residual heavy metals.

[0016] The beneficial effects of this scheme are: using a new sulfate-reducing strain to efficiently passivate cadmium pollution in farmland, specifically reducing the highly active, easily migratable exchangeable and carbonate-bound cadmium in the soil, and increasing the proportion of insoluble and stable iron-manganese oxide-bound, organosulfur compound-bound, and residual cadmium. The passivation is long-lasting, the in-situ remediation cost is low, and the ecological compatibility is good.

[0017] Preferred sulfate-reducing strains are used to reduce the content of methylmercury in soil.

[0018] Preferably, the sulfate-reducing strain can enhance the ability of rice to accumulate methylmercury, thereby reducing the methylmercury content in rice-grown soil.

[0019] The beneficial effects of this scheme are as follows: by applying sulfate-reducing new strains, it can not only effectively reduce the inherent methylmercury stock in the soil, but also directionally enhance the enrichment and translocation capacity of rice roots, efficiently migrate the soil-reduced methylmercury to the aboveground parts of rice for centralized removal, and achieve low-cost, sustainable detoxification and remediation of polluted soil, which takes into account both the symptoms and the root causes, minimizes ecological disturbance, and meets the urgent need for in-situ remediation of farmland. Attached Figure Description

[0020] Figure 1 The diagram shows the extraction and identification steps of sulfate-reducing bacteria in Example 1; where: (A)-(F) are diagrams of the extraction process of single colonies of sulfate-reducing bacteria; (G) is a Gram staining diagram of sulfate-reducing bacteria; (H) is a gel electrophoresis diagram of sulfate-reducing bacteria; (I) is a phylogenetic tree diagram constructed from the 16S rRNA gene sequence of sulfate-reducing bacteria; Figure 2 This is a growth curve of sulfate-reducing bacteria plotted based on sampling time points in Example 1; Figure 3 This is a growth curve of sulfate-reducing bacteria in culture media with different cadmium concentrations in Example 2; Figure 4 This is an X-ray diffraction analysis diagram of the yellow precipitate obtained by reacting sulfate-reducing bacteria with cadmium-containing liquid culture medium in Example 2; Figure 5 This is a graph showing the changes in cadmium levels before and after the reaction of sulfate-reducing bacteria with different clay samples in Example 3. Figure 6 This is the experimental design diagram for the ability of sulfate-reducing bacteria to accumulate methylmercury in rice seedlings in Example 4; Figure 7 This is a comparison chart of the sterilization effect in the serial dilution experiment in Example 4; Figure 8 This is a diagram showing the growth of rice seedlings two months after transplanting in Example 4; Figure 9In Example 4, rice seedlings were exposed to a mercury concentration of 30 mg / kg. -1 Figure showing the concentration of methylmercury in rhizosphere and non-rhizosphere soils two months later; Figure 10 In Example 4, rice seedlings were exposed to a mercury concentration of 30 mg / kg. -1 Differential upregulated and downregulated expression genes in soil after 2 months; Figure 11 This is the experimental design diagram for the ability of sulfate-reducing bacteria to accumulate methylmercury in rice plants in Example 5. Figure 12 This is a graph showing the concentrations of methylmercury in the rhizosphere and non-rhizosphere soils of mature rice in a greenhouse pot experiment in Example 5. Figure 13 The graph shows the MeHg content and mass in the roots, stems and leaves of rice during the tillering, flowering and maturity stages in the greenhouse pot experiment of Example 5. Detailed Implementation

[0021] The main contents of the present invention are further illustrated below with reference to specific embodiments, but the contents of the present invention are not limited to the following embodiments. Unless otherwise specified, the technical solutions involved in the embodiments of the present invention are conventional solutions in the art; the reagents or materials mentioned, unless otherwise specified, are all from commercial sources.

[0022] The sulfate-reducing strain Desulfovibrio sp.GLDM01 in this invention is a facultative anaerobic bacterium screened from paddy soil. It belongs to the genus Desulfovibrio and is a Gram-negative bacterium.

[0023] Example 1: Method for extraction and identification of the sulfate-reducing strain Desulfovibrio sp. GLDM01.

[0024] S1, enrichment culture of the sulfate-reducing strain Desulfovibrio sp. GLDM01. The specific steps are as follows: Using purple soil as the main research object, this embodiment uses paddy soil (106°24′E, 29°48′N) from the Chongqing Purple Soil Ecological Environment Key Field Scientific Observation and Experiment Station to extract new sulfate-reducing strains.

[0025] like Figure 1As shown, 10g of soil sample was weighed and placed in a conical flask containing 50mL of sterile water and sealed. The flask was then shaken on a shaker at an ambient temperature of 30℃ and a rotation speed of 120 r / min for 1 hour. The supernatant was collected after settling. Postgate liquid culture medium (a dedicated medium for sulfate-reducing bacteria) suitable for the growth of sulfate-reducing bacteria was selected. The composition of the Postgate liquid culture medium in this example is detailed in Table 1. The pH of the prepared liquid culture medium was then adjusted to 7.0-7.5 using sodium hydroxide or hydrochloric acid. The liquid culture medium, distilled water, pipettes, and rubber stoppers were subjected to high-temperature sterilization at 121℃, a sterilization pressure of 0.1MPa, and a sterilization time of 30 minutes. After sterilization, the sterilized sample was transferred to an anaerobic clean bench for cooling. Next, weigh 0.01 g of ascorbic acid and 0.05 g of L-cysteine, and place them together in a 2 mL centrifuge tube for UV sterilization for 30 min. After sterilization, dissolve the mixture of ascorbic acid and L-cysteine ​​in sterile water, filter the solution through a 0.45 μm filter membrane, and then add it to the above liquid culture medium. Finally, use a pipette to transfer 1 mL of the supernatant from the above soil sample to 100 mL of liquid culture medium, and place it in a constant temperature incubator at 35°C for anaerobic culture until the liquid culture medium turns black (a characteristic of sulfidation), i.e., when the liquid culture medium turns black as detected by lead acetate test paper, then transfer.

[0026] Repeat the above inoculation and culture steps several times, specifically twice a week in this example, for one and a half months. Once the inoculation and culture are complete, a bacterial suspension of the sulfate-reducing strain Desulfovibrio sp. GLDM01 will be obtained.

[0027] Table 1: Composition of Postgate Liquid Culture Medium S2, the isolation and purification of the sulfate-reducing strain Desulfovibrio sp. GLDM01. The specific procedures are as follows: The isolation and purification of sulfate-reducing bacteria strains is a complex process requiring stringent operating conditions. Therefore, this embodiment employs a combination of the sandwich plate method and the dilution shaker method. After adding 1.5-2% agar to the liquid culture medium, the culture is sterilized at high temperature. Once the liquid culture medium has cooled to 50°C, it is poured into petri dishes, ensuring the thickness reaches one-quarter of the dish's height. After the bottom layer of liquid culture medium cools and forms a plate, the bacterial suspension is diluted 10... 3Double the amount of culture medium and streak it diagonally across the surface of the plate using an inoculation loop. Finally, pour in the sterilized and cooled liquid culture medium again, ensuring it completely covers the bottom of the streaks. After the upper layer of liquid culture medium has completely solidified, cover the plate with the lid, seal it with sealing film, and place it in a 35°C incubator for constant temperature incubation until single colonies grow.

[0028] S3, Obtaining the new sulfate-reducing strain Desulfovibrio sp. GLDM01. The specific steps are as follows: The strain was cultured and genomic DNA extracted under anaerobic conditions. The liquid culture medium was purged with nitrogen for 30 min, and the oxygen content was controlled at 0.23–0.42 mg / L. -1 Single colonies of sulfate-reducing bacteria were picked from the anaerobic incubator and placed into the sterilized liquid culture medium. The liquid culture medium was then incubated on a shaker for 8 hours. In this embodiment, the incubation temperature was set at 35°C and the shaker speed was 125 r / min. Finally, a concentrated bacterial solution of the new sulfate-reducing strain with high bacterial activity and sufficient quantity was obtained.

[0029] Next, high-quality genomic DNA was prepared from bacteria using the M5 Bacteria Genomic DNA Kit. The components of the M5 Bacteria Genomic DNA Kit are detailed in Table 2 for this embodiment.

[0030] Table 2. M5 bacterial genomic DNA kit The specific steps for genomic DNA extraction are as follows: Take 0.5-3 mL of the enriched bacterial culture into a centrifuge tube and centrifuge at 10,000 rpm for 1 min at room temperature, discarding the supernatant. Add 200 μL of GTL buffer, thoroughly pipette-pipette, then add 20 μL of proteinase K solution. Mix well in the centrifuge tube and centrifuge briefly, discarding the supernatant. Add another 200 μL of GL buffer, and centrifuge at 56°C. oIncubate the centrifuge tube in a C2 water bath for 10 min, inverting the tube periodically to disperse the sample evenly. Once the solution becomes clear, centrifuge and discard the supernatant. Next, add 200 μL of anhydrous ethanol, shake thoroughly, and briefly centrifuge after the formation of flocculent precipitate, discarding the supernatant. Then, add 500 μL of GW1 buffer to the adsorption column, centrifuge at 12,000 rpm for 30 s at room temperature, discarding the filtrate in the collection tube. Add 600 μL of GW2 buffer, centrifuge at 12,000 rpm for 30 s at room temperature, discarding the waste liquid. Next, add 500 μL of GW2 buffer, centrifuge at 12,000 rpm for 2 min at room temperature, discarding the waste liquid. Finally, place the centrifuge column in a new 1.5 mL plastic centrifuge tube, add 50–100 μL of GE buffer, incubate at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min. The solution at the bottom of the tube is the crude extract of sulfate-reduced genomic DNA from the new strain.

[0031] The universal primers 27F and 1492R for 16S rDNA were selected, and polymerase chain reaction (PCR) was performed using a PCR instrument. The specific PCR steps were as follows: initial preheating at 98℃ for 2 min, denaturation at 98℃ for 10 s, annealing at 57℃ for 10 s, and extension at 72℃ for 10 s. This process was repeated for 30 cycles. Finally, the extension was performed at 72℃ for 2 min to obtain the complete DNA fragment of the sulfate-reduced strain.

[0032] Next, the quality of the extracted DNA was detected by electrophoresis. The detection steps are as follows: First, TAE buffer (containing 193.6 g·L⁻¹) was added... -1 Tris base, 45.7 μL·L -1 Acetic acid and 14.9 g·L -1 The 1.2% w / v agarose gel in EDTA was heated to melt; after cooling, 2-3 μL of ethidium bromide was added to prepare the transfer plate and wells, and a comb was vertically inserted above the transfer plate; after the gel solidified, the comb was removed and the gel was placed in the electrophoresis tank, and buffer was added; then 1-2 μL of loading buffer was added to the gel wells, followed by the above DNA sample and a blank sample, and electrophoresis was performed. After electrophoresis, the sample was removed and observed under UV light, and the image was taken as shown. Figure 1 As shown, the DNA fragment is intact, and the target amplified fragment is approximately 1360 bp.

[0033] Furthermore, the novel sulfate-reducing strain Desulfovibrio sp. GLDM01 obtained above was identified, and the specific steps are as follows: Combination Figure 1As shown in Figures (G), (H), and (I), Gram staining of the novel sulfate-reducing strain revealed it to be a Gram-negative bacterium. The amplified products were sent to Beijing Qingke Xinyue Biotechnology Co., Ltd. for species identification. The obtained DNA gene sequences were compared for homology in the NCBI database, and a phylogenetic tree was constructed using MEGA6. Given that this strain shares 75% kinship with *Desulfricans MB* and *Desulfovibrio legallii dsv3*, indicating they are homologous strains of sulfate-reducing bacteria, and that a 75% 16S rDNA similarity is sufficient to distinguish between the two species, based on the 16S rDNA similarity, strain *Desulfovibrio sp. GLDM01* is taxonomically distinct from existing species in the *Desulfovibrio* genus, and is thus identified as a novel species within the *Desulfovibrio* genus.

[0034] The 16S rDNA nucleotide sequence number of the specific sulfate-reducing strain Desulfovibrio sp. GLDM01 is detailed in Sequence Listing 1: The selected sulfate-reducing strains were inoculated into the above-mentioned liquid culture medium to obtain the initial bacterial suspension. The initial bacterial suspension was shaken at 180 rpm for 14 hours. Then, sterile glycerol was added to the shaken initial bacterial suspension at a ratio of 30% glycerol to bacterial agent of 1:1 (v / v). After mixing, the mixture was dispensed into sterile bacterial culture tubes (1-2 mL / tube) and stored at -80℃ or in liquid nitrogen. For subsequent experiments, the bacterial suspension was transferred to Postgate liquid medium for rejuvenation before use.

[0035] The novel sulfate-reducing strain Desulfovibrio sp. GLDM01 was deposited at the China Center for Type Culture Collection on March 9, 2026, with accession number CCTCC NO: M 2026395.

[0036] Example 2: Application of the novel sulfate-reducing strain Desulfovibrio sp.GLDM01 in the adsorption of cadmium.

[0037] First, the growth of the novel sulfate-reducing strain Desulfovibrio sp. GLDM01 obtained in Example 1 was investigated. Since Desulfovibrio sp. GLDM01 is a facultative anaerobe, the dissolved oxygen level in the culture environment was set at 0.23-0.42 mg·L⁻¹. -1 The culture temperature was 35℃. The enriched bacterial solution obtained in Example 1 was added to the liquid culture medium at a volume ratio of 1:25 and then added to the glass tube. The tube was then placed on a shaker and shaken at 125 r / min and 35℃ for 24 h. Samples were taken every 3 h to measure the pH and OD600 of the sulfate-reducing bacteria and generate a bacterial growth curve.

[0038] The results are as follows Figure 2 As shown, the sampling point at 3 hours represents the lag phase of the sulfate-reducing bacteria's growth, 6 hours the logarithmic phase, 12 hours the stationary phase, and 21 hours the decline phase. The pH change from 7.03±0.05 immediately after inoculation to 6.78±0.03 during the lag phase is mainly due to the microorganism's regulation of the environment. The slightly alkaline environment created by the sulfate-reducing bacteria's growth and metabolism during the logarithmic phase (6.81±0.05) to the stationary phase (7.21±0.02) is conducive to microbial growth. Simultaneously, the sulfate-reducing bacteria generate H2S during their growth, which is toxic to the bacteria, leading to reduced bacterial activity and decreased sulfate reduction. This, in turn, causes the pH to drop to 6.46±0.09 during the decline phase, indicating that the growth of the sulfate-reducing bacteria influences pH changes.

[0039] Next, the sulfate-reducing bacteria obtained in Example 1 were inoculated at a volume ratio of 1:25 into cadmium concentrations of 0 and 2 mg·L⁻¹, respectively. -1 5 mg·L -1 8 mg·L -1 10 mg·L -1 The cadmium was added to the liquid culture medium (with a cadmium concentration of 0 serving as a blank control), and then shaken at 125 r / min for 32 h. Similarly, samples were taken every 4 h to measure OD600. The supernatant obtained from shaking was filtered, centrifuged, and the cadmium removal rate in the above liquid culture medium was measured again. The results are detailed in Table 3 and [Table data missing]. Figure 3 As shown.

[0040] Table 3. Removal rate of cadmium at sampling time points (%) As shown in Table 3, the concentration of each cadmium-containing culture medium was 2 mg·L⁻¹ 8 h after inoculation with sulfate-reducing bacteria. -1 The cadmium removal rate in the culture medium was 69.79%, with a concentration of 8 mg·L⁻¹. -1 The cadmium removal rate in the culture medium was 19.10%, with a concentration of 10 mg·L⁻¹. -1 The cadmium removal rate in the culture medium was 12.60%. After inoculating each cadmium-containing culture medium with sulfate-reducing bacteria for 32 hours, the concentration was 2 mg·L⁻¹. -1 The cadmium removal rate in the culture medium was as high as 99.42%, with a concentration of 10 mg·L⁻¹. -1 The cadmium removal rate in the culture medium reached 96.63%, indicating that this sulfate-reducing bacterium can effectively adsorb high cadmium substances.

[0041] The yellow precipitate (specifically cadmium sulfide, CdS) obtained by reacting sulfate-reducing bacteria with cadmium-containing liquid culture medium was collected by centrifugation, naturally dried, and then subjected to X-ray diffraction analysis. The results are as follows: Figure 4 As shown, three distinct diffraction peaks are observed at 26.5°, 43.7°, and 51.8°, pointing to the 111, 220, and 311 crystal planes of CdS, respectively, corresponding to the typical cadmium sulfide peaks in the PDF card (10-0454). This further indicates that after the sulfate-reducing bacteria reduce sulfate, sulfide ions and cadmium ions form an insoluble precipitate.

[0042] In other words, the sulfate-reducing bacteria extracted in Example 1 have good cadmium passivation ability and can be applied to cadmium removal in soil microbial remediation to improve soil quality and soil safety.

[0043] Example 3: Application of the sulfate-reducing strain Desulfovibrio sp. GLDM01 in the passivation of cadmium in soil.

[0044] Clay minerals formed from rock weathering are soil components. The heavy metal content in the parent rock affects the background element values ​​in the soil. Therefore, this embodiment further selects farmland near the mining area as the research object to analyze the solidification effect of heavy metal cadmium on iron-bearing minerals and microorganisms in clay minerals of different strata of farmland from a natural geological perspective.

[0045] This embodiment selected soil samples from different stratigraphic sections, and the cadmium content in the soil samples was tested to be 0.41-1.83 mg·kg⁻¹. -1 After extraction from clay particles, the cadmium content was found to be 4.0812-14.8426 mg·kg⁻¹. -1 Clay samples were collected. Strata from the Nanhua, Sinian, Cambrian, Ordovician, Silurian, Permian, and Triassic systems all showed excessive levels of three or more heavy metals. Based on the chemical speciation of iron, the sulfate-reducing strain from Example 1 was reacted with the aforementioned clay samples. The exchangeable, carbonate-bound, iron-manganese oxide-bound, organic and sulfide-bound, and residual forms of iron were extracted using the Tessier method for further analysis. The extraction methods are as follows: Exchangeable state: Weigh 1g of soil clay mineral into a 50 mL centrifuge tube, add 16 mL of 1mol·L⁻¹ -1 The MgCl2 solution (adjusted to pH 7.0) was continuously shaken at 220 r / min for 2 h, centrifuged at 4000 r / min for 5 min, filtered, and diluted to volume.

[0046] Carbonate-bound state: Add 16 mL of 1 mol·L⁻¹ to the residue from the previous step. -1 CH3COONa was used to adjust the pH to 5.0 using CH3COOH. After continuous shaking at 220 r / min for 5 h at room temperature, it was centrifuged for 5 min, filtered, and the supernatant was diluted to 50 mL.

[0047] Iron-manganese oxide bound state: Add 30 mL of 0.4 mol·L⁻¹ to the residue from the previous step. -1 NH₂OH·HCl solution (pH adjusted to 2.0 with HNO₃), 96±3 o Extraction by heating in a water bath for 6 hours, replenishing with NH2OH·HCl-HAc solution 1-2 times during the process, shaking intermittently, and after cooling, centrifuging at 3500 r / min for 5 min, filtering and making up to volume.

[0048] Organic matter and sulfide bound states: Add 4 mL of 0.02 mol·L⁻¹ solution to the residue from the previous step. -1 HNO3 and 10 mL of 30% H2O2 (adjust pH to 2.0 with HNO3, (85±2) oOxidation was carried out by heating in a water bath for 3 hours, followed by the addition of 30% H2O2 (with pH adjusted to 2.0 using HNO3), and the mixture was kept in the water bath for 2 hours with intermittent shaking. After cooling, 5 mL of 0.02 mol·L⁻¹ solution was added. -1 HNO3 and 8 mL of 3.2 mol·L⁻¹ -1 CH3COONH4 solution, constant temperature and continuous shaking at 220 r / min for 30 min, centrifuged at 4000 r / min for 5 min, filtered and diluted to volume.

[0049] Residual state: Add 3 mL of concentrated hydrochloric acid and 5 mL of concentrated nitric acid to the remaining soil sample in the centrifuge tube, and incubate at 120°C in a sealed reaction vessel. o Heat at 120°C for 3 hours. Then evaporate the aqua regia to dryness, add 3 mL of concentrated hydrochloric acid, and heat in a sealed reaction vessel at 120°C. o Continue heating until the sample is completely digested. Evaporate the hydrofluoric acid to dryness, then add 1 mL of concentrated hydrochloric acid to remove the hydrogen fluoride. Centrifuge at 5000 r / min for 5 min, then bring the volume to 50 mL with 2% nitric acid solution.

[0050] The results are as follows Figure 5As shown in the figure, F1 represents exchangeable state, F2 represents carbonate-bound state, F3 represents iron-manganese oxide-bound state, F4 represents organic matter and sulfide-bound state, F5 represents residual state, and +SRB represents the addition of sulfate-reducing bacteria extracted in Example 1. In Silurian clay sample LY45, after the addition of bacteria, the exchangeable state decreased by 27.16%, the iron-manganese bound state increased by 37.93%, and the residual state increased by 13.32%. In Triassic clay sample LY7, after reaction with sulfate-reducing bacteria, the exchangeable state decreased from 54.86% to 16.15%, the carbonate-bound state decreased from 22.91% to 7.28%, the iron-manganese bound state increased from 2.92% to 56.80%, and the residual state increased from 8.17% to 18.31%. After reacting with sulfate-reducing bacteria, the exchangeable content of Sinian clay sample LY23 decreased from 50.94% to 16.22%, the carbonate-bound content decreased from 23.58% to 7.62%, the iron-manganese-bound content increased from 2.77% to 52.08%, and the residual content increased from 8.84% to 20.90%. Similarly, after reacting with sulfate-reducing bacteria, the exchangeable content of Ordovician clay sample LY15 decreased from 58.63% to 12.47%, the carbonate-bound content decreased from 20.64% to 6.34%, the iron-manganese-bound content increased from 2.84% to 55.73%, and the residual content increased from 7.64% to 24.72%. After reacting with sulfate-reducing bacteria, the exchangeable content of Nanhua clay sample LY15 decreased from 49.80% to 16.44%, the carbonate-bound content decreased from 23.26% to 6.31%, the iron-manganese-bound content increased from 3.37% to 50.44%, and the residual content increased from 14.87% to 25.73%. After reacting with sulfate-reducing bacteria, the exchangeable content of Cambrian clay sample LY59 decreased from 53.52% to 11.24%, the carbonate-bound content decreased from 20.62% to 7.87%, the iron-manganese-bound content increased from 2.60% to 48.33%, the organic matter and sulfide-bound content decreased from 7.93% to 5.03%, and the residual content increased from 15.33% to 27.53%. After reacting with sulfate-reducing bacteria, the exchangeable content of Sinian clay sample LY23 decreased by 34.72%, the iron-manganese-bound content increased by 49.31%, and the residual content increased by 12.06%. After reacting with sulfate-reducing bacteria, the exchangeable state of Ordovician clay sample LY15 decreased by 46.16%, while the iron-manganese bound state increased by 52.89%. Similarly, after reacting with sulfate-reducing bacteria, the exchangeable state of Cambrian clay sample LY59 decreased by 42.28%, while the iron-manganese bound state increased by 45.73%.

[0051] The decrease in exchangeable and carbonate-bound content indicates a significant passivation effect, while the increase in iron-manganese-bound, organic matter and sulfide-bound, and residual content also indicates a significant passivation effect. All clay samples after inoculation showed a significant negative correlation between exchangeable and iron-manganese-bound content (r = -0.650, P < 0.05), and a significant negative correlation between carbonate-bound and iron-manganese-bound content (r = -0.533, P < 0.05). In other words, after the sulfate-reducing bacteria reduce the iron in clay minerals and regenerate secondary iron minerals, cadmium will be redistributed to the stable phase, thus enhancing the solidification effect of cadmium in the secondary minerals through bacterial inoculation.

[0052] Example 4: Application of the sulfate-reducing strain Desulfovibrio sp. GLDM01 in the enrichment of methylmercury in rice seedlings.

[0053] The soil used in this example was collected from a paddy field in Beibei suburbs of Chongqing in 2021 (106.420016E, 29.818452N). The soil was air-dried, ground, and sieved before use. The sulfate-reducing bacteria obtained in Example 1 were inoculated into unsterilized soil and gamma-ray (60 kGy) sterilized soil to further investigate the effect of the addition of this new strain on the formation of methylmercury in the soil and the accumulation of inorganic mercury and methylmercury in rice. First, the feasibility of sterilizing the sample soil with 60 kGy gamma radiation was verified through a serial dilution experiment. Figure 7 As shown, 60 kGy gamma radiation is sufficient for soil sterilization, and no live microorganisms were observed after gamma radiation sterilization treatment.

[0054] Considering the factors of sulfate-reducing bacteria and rice addition, multiple treatments were designed, including soil sterilization (whether sterilization was performed), rice planting (whether rice seedlings were transplanted), and inoculation with sulfate-reducing bacteria (whether the bacterial strain was inoculated). A control group was also designed, consisting of soil without added mercury (Hg) solution for rice seedling transplantation and treatment with added sulfate-reducing bacteria. Figure 6 As shown, a total of 10 treatments were generated, with each treatment having three replicates.

[0055] The specific procedure is as follows: Weigh out 6 portions of 0.25 kg sterilized soil and 4 portions of 0.25 kg unsterilized soil and place them separately into 125 mL glass bottles. Then, add 10 g·L⁻¹ to each glass bottle. -1 HgCl2 solution, and further control the Hg concentration in the sterilized soil to 30 mg·kg⁻¹. -1 The Hg concentrations in the unsterilized soil were 0 and 30 mg·kg⁻¹, respectively. -1 Next, add sterile water and place in a sterile incubator for two weeks to age. Then proceed as follows... Figure 6As shown, rice seedlings with similar growth and cultured in sterile nutrient solution were transplanted into the above-mentioned soil samples, with half of the soil remaining unplanted. The rice growth conditions in the sterile incubator were set as follows: 16 hours of light (light temperature 28°C, light intensity 5000 Lux) and 8 hours of darkness (dark temperature 20°C). The soil with transplanted rice seedlings was considered rhizosphere soil, and the soil without rice seedlings was considered non-rhizosphere soil. Immediately after transplanting the rice seedlings, they were inoculated with the sulfate-reducing bacteria described in Example 1. Details of the inoculated soil are provided below. Figure 6 The remaining uninoculated soil was treated with 0.9% physiological saline.

[0056] The specific procedure for inoculating sulfate-reducing bacteria is as follows: After thawing the sulfate-reducing bacteria enrichment solution preserved in Example 1, inoculate it into Postgate liquid medium for rejuvenation culture. Pour the cultured bacterial agent into a 50mL sterile centrifuge tube, centrifuge at 7000rpm for 5min, rinse once with sterile physiological saline, and then suspend the bacterial cells in physiological saline. Adjust the absorbance OD600 to 1.0 using a UV spectrophotometer. Finally, inoculate the bacterial suspension onto the roots of rice plants using a syringe, with an inoculation volume of 5mL to 10mL.

[0057] like Figure 6 As shown, five groups of soil experimental samples were finally obtained (Hg0, Hg30, Hg30, Hg30). NSRB , γ Hg30 and γ Hg30 NSRB (); where Hg0 is unsterilized soil with a Hg concentration of 0 (i.e., control group), and Hg30 is Hg concentration of 30 mg·kg⁻¹. -1 Unsterilized soil, Hg30 NSRB Hg concentration 30 mg·kg -1 And unsterilized soil inoculated with sulfate-reducing bacteria, γ Hg30 refers to an Hg concentration of 30 mg·kg⁻¹. -1 Sterilized soil, γ Hg30 NSRB Hg concentration 30 mg·kg -1 The soil was sterilized and inoculated with sulfate-reducing bacteria. Each soil group included both non-rhizosphere and rhizosphere soils, meaning each group included soils with and without rice seedlings.

[0058] Two months after rice seedling transplantation, soil samples and rice tissue samples (roots, stems, and leaves) were collected and stored at -80°C for later use. Figure 8As shown, superficially, Hg stress inhibited rice growth in both unsterilized and sterilized soils, while the introduction of sulfate-reducing bacteria promoted rice growth under mercury stress in sterilized soil to some extent, but inhibited rice growth in unsterilized soil. This indicates that sulfate-reducing bacteria can alleviate mercury toxicity and promote rice growth; moreover, the application effect of sulfate-reducing bacteria is influenced by native soil microorganisms.

[0059] Figure 9 In this embodiment, the rice seedlings were treated with 30 mg·kg⁻¹ -1 The methylmercury content in rhizosphere and non-rhizosphere soils after two months of exposure to soil mercury concentrations includes MeHg concentration, MeHg / THg concentration, Hg_w concentration, and Hg_s concentration, as well as MeHg concentration in roots and shoots and IHg concentration in shoots. In the figure, the data bars represent the mean, the error bars represent the standard error of the mean, different lowercase letters indicate significant differences, Hg_w is water-soluble mercury, and Hg_s is sulfide-bound mercury. Figure 10 In this embodiment, the rice seedlings were exposed to a mercury concentration of 30 mg·kg⁻¹. -1 Soil gene expression diagrams after 2 months, where Figure A is... γ Hg30 NSRB and γ Figure 1 shows differentially upregulated and downregulated gene expression by Hg30; Figure 2 shows GO terms for enrichment of differentially upregulated genes; Figure 3 shows the KEGG enrichment pathway for differentially upregulated genes; Figure 4 shows a heatmap of log2 fold differences in upregulated genes in calcium ion binding, sequence-specific DNA binding, and glutathione metabolism; CML in the figure refers to calcium-binding protein CML, PLD1. 2 refers to phospholipase D1 / 2, psbQ refers to photosystem II evolutionary oxygen-promoting protein 3, CALM refers to calmodulin, AMY refers to alpha amylase, RBOH refers to respiratory burst oxidase, CPK refers to calcium-dependent protein kinase, EHD1 refers to EH domain-containing protein 1, KIC refers to calcium-binding protein KIC and related proteins, SMAP refers to matrix membrane-associated protein, WRKY22 refers to WRKY transcription factor 22, WRKY33 refers to WRKY transcription factor 33, TGA refers to transcription factor TGA, GST refers to glutathione S-transferase, CARP refers to leucine aminopeptidase, GSR refers to glutathione reductase, and IDH1 refers to isocitrate dehydrogenase.

[0060] Although Hg_w and Hg_s showed no obvious changing patterns, in non-rhizosphere soils, the addition of sulfate-reducing bacteria had no effect on methylmercury production regardless of soil sterilization treatment. Conversely, in rhizosphere soils, the addition of sulfate-reducing bacteria reduced methylmercury production by 22.86% in unsterilized soil and 17.11% in sterilized soil. For rice plants, the addition of sulfate-reducing bacteria significantly increased the accumulation of methylmercury in rice seedlings, with methylmercury concentrations in rice seedling roots and shoots increasing by 54.09% and 12.10%, respectively, in unsterilized soil; and by 67.68% and 52.33%, respectively, in sterilized soil. Figure 10 As shown, the addition of sulfate-reducing bacteria led to a significant enrichment of differentially expressed genes upregulated in rice roots in the G0 term molecular function category, particularly calcium ion binding and sequence-specific DNA binding. Furthermore, metabolic pathway enrichment analysis revealed that the upregulated differentially expressed genes primarily participated in glutathione metabolism, and their related genes mainly regulated glutathione S-transferase (GST). This indicates that sulfate-reducing bacteria can reduce methylmercury content in rhizosphere soil while significantly promoting the absorption and accumulation of methylmercury in rice seedlings.

[0061] Example 5: Application of the sulfate-reducing strain Desulfovibrio sp. GLDM01 in enhancing the accumulation of methylmercury in rice plants.

[0062] To further verify whether the accumulation capacity of these rice seedlings with differential gene expression induced by sulfate-reducing bacteria exists throughout the entire growth period of rice, this example conducted a pot experiment on the effect of sulfate-reducing bacteria inoculation on the accumulation of methylmercury in rice plants.

[0063] The soil used in this embodiment is the same as that in Example 4. After air-drying, grinding, and sieving, the soil is placed in a plastic basin with a length, width, and height of 90cm, 36cm, and 25cm respectively (the amount of soil is 40 kg per basin). -1 Then add 100 mg / kg of the solution to the soil in the pot. -1 Urea, 150 mg / kg -1 superphosphate and 85 mg·kg -1 Potassium chloride was used as the base fertilizer and thoroughly mixed. Then 10g L was added. -1 The HgCl2 solution was used to make the THg concentration in the soil 30 mg·kg⁻¹. -1 During the 3-week soil aging process, the water level above the soil was maintained at 3-5 cm. Figure 11 As shown in Example 4, this example also includes a sterilized soil control group and an unsterilized soil control group.

[0064] In the incubator, hydroponically grown rice seedlings were transplanted to unsterilized and sterilized soils, and then inoculated with sulfate-reducing bacteria. This example included four groups of seedlings, with controls at Hg30 and Hg30, respectively. NSRB , γ Hg30 and γ Hg30 NSRB Each group had three replicates. Details regarding transplantation and vaccination are available in the [link to relevant documentation]. Figure 11 .like Figure 11 As shown, rice seedlings with similar growth, cultured in sterile broth for two months, were transferred to the aforementioned soils and cultivated in a greenhouse. 77 days after transplanting into the aged soil (i.e., the rice's maturity period), non-rhizosphere soil and rhizosphere soil, as well as rice grains, were collected. It should be noted that during sample collection, the entire rice plant was dug out of the soil and gently shaken; the soil that fell off was considered non-rhizosphere soil, while the soil attached to the roots was considered rhizosphere soil; if no soil fell off, the remaining soil in the pot was considered non-rhizosphere soil. Additionally, during the rice seedling... γ Hg30 and γ Hg30 NSRB Roots, stems, and leaves of rice plants were collected as plant samples at 27, 47, and 77 days after transplanting (corresponding to the tillering, flowering, and maturity stages of rice seedlings, respectively). The collected soil and plant samples were stored at -80℃ for later use.

[0065] Figure 12 This data represents the pH, NH4+, SO42-, MeHg, and MeHg / THg content in the non-rhizosphere and rhizosphere soils of mature rice in a greenhouse pot experiment, as well as the MeHg content in rice grains. Data bars represent the mean, error bars represent the standard error of the mean, and different lowercase letters indicate significant differences. Figure 13 This graph shows the MeHg content and mass in rice roots, stems, and leaves during the tillering, flowering, and maturity stages in a greenhouse pot experiment. Similarly, the data bars represent the mean, the error bars represent the standard error of the mean, and different lowercase letters indicate significant differences.

[0066] Combination Figure 12 and Figure 13 As shown, rice seedlings inoculated with unsterilized soil exhibited a 55.66% increase in methylmercury content in mature grains, a result that can be partially explained by the significant increase in methylmercury content in the rhizosphere soil. However, rice seedlings inoculated with sterilized soil showed a 98.94% increase in methylmercury content in mature grains, while the rhizosphere methylmercury content showed no significant change. This is consistent with the results of Example 4, indicating that sulfate-reducing bacteria increased the accumulation of methylmercury in rice seedlings. Meanwhile, as... Figure 13As shown, rice plants inoculated with sterilized soil exhibited enhanced accumulation of methylmercury in roots and leaves during tillering, flowering, and maturity stages, further demonstrating that this sulfate-reducing bacterium can improve the accumulation of methylmercury in rice plants. After inoculation with sulfate-reducing bacteria, rice's ability to accumulate methylmercury increases, enabling it to absorb large amounts of methylmercury from the soil during its growth, gradually reducing soil mercury content and achieving soil heavy metal remediation.

[0067] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A novel sulfate-reducing bacterial strain, characterized in that, The novel sulfate-reducing strain Desulfovibrio sp. GLDM01 was deposited at the China Center for Type Culture Collection on March 9, 2026, with accession number CCTCC NO: M 2026395.

2. The sulfate-reducing strain according to claim 1, characterized in that, The new sulfate-reducing strain was a Gram-negative bacterium, and the dissolved oxygen in its culture environment was 0.23-0.42 mg·L⁻¹. -1 The temperature is 35 degrees Celsius. o C.

3. A sulfate-reducing bacteria agent, characterized in that, A live bacterial preparation, bacterial lysate, or fermentation supernatant containing the sulfate-reducing novel strain as described in claim 1 or 2.

4. A method for obtaining novel sulfate-reducing strains, characterized in that, Includes the following steps: Enrichment culture: Soil samples were taken to prepare supernatant, and the supernatant was inoculated into liquid culture medium and cultured at constant temperature until sulfurization characteristics appeared. The enriched bacterial solution was obtained by continuous transfer. Separate and purify the bacteria by adding agar to the liquid culture medium to form a plate, streak the diluted enriched bacterial solution on the surface of the plate, cover the bottom of the streak with solid culture medium, seal and anaerobic culture until a single colony grows. To obtain the target sulfate-reducing strain, single colonies were picked under anaerobic conditions and inoculated into sterile liquid culture medium for culture.

5. The method for obtaining a new sulfate-reducing strain according to claim 4, characterized in that, In the enrichment culture step, the soil sample was purple soil, and the purple soil was paddy soil; the liquid culture medium included NaCl, NH4Cl, MgSO4·7H2O, Na2SO4, K2HPO4, and the pH was 7.5~8.

0.

6. The method for obtaining a new sulfate-reducing strain according to claim 4, characterized in that, In the separation and purification step, the solid culture medium is obtained by mixing the liquid culture medium with agar, and the concentration of agar is 1.5-2%.

7. The application of a novel sulfate-reducing strain, characterized in that, A new sulfate-reducing strain was used to passivate cadmium in farmland.

8. The application of the sulfate-reducing strain according to claim 7, characterized in that, The new sulfate-reducing strain can reduce the content of exchangeable and carbonate-bound heavy metals in cadmium-containing soils, and increase the content of iron-manganese-bound, organic matter and sulfide-bound, and residual heavy metals.

9. The application of a novel sulfate-reducing strain, characterized in that, A new sulfate-reducing strain was used to reduce the content of methylmercury in soil.

10. The application of the sulfate-reducing strain according to claim 9, characterized in that: The new sulfate-reducing strain can enhance the ability of rice to accumulate methylmercury, thereby reducing the methylmercury content in rice-grown soil.

Images