Antimony detoxification synergistic autotrophic nitrogen fixation pseudomonas and application thereof

Abstract

The invention discloses a pseudomonas for antimony detoxification and autotrophic nitrogen fixation and application of the pseudomonas. The strain is separated from the rhizosphere of a pioneer plant in antimony-polluted soil of a tin mine in cold water river city, Hunan province, and is identified to be capable of quickly oxidizing high-toxicity antimony (III) into low-toxicity antimony (V) through autotrophic metabolism in a high-antimony environment, so that antimony detoxification is realized, and the detoxification rate is 108.6 (mu mol / (L.day)). The strain has autotrophic nitrogen fixation activity and can achieve autotrophic growth under the oligotrophic condition of a mining area, and the nitrogenase activity is larger than or equal to 223.4 (nmol C2H2 / (g.h)). Particularly, the antimony detoxification and nitrogen fixation capabilities of the strain have a synergistic effect, so that the strain has unique advantages in the field of heavy metal antimony pollution remediation, and dual effects of antimony pollution treatment and soil improvement can be synchronously realized; the development and application prospects are good.

Description

Technical Field

[0001] The present invention belongs to the field of environmental microbial technology, and specifically relates to a Pseudomonas bacterium with the dual functions of antimony detoxification and autotrophic nitrogen fixation and its application, especially its combined application in antimony pollution remediation and barren soil improvement. Background Art

[0002] Antimony mine tailings, a typical example of a highly degraded ecosystem, present two major challenges in restoration: toxic metal contamination and nutrient deficiencies. Traditional ecological restoration techniques are ineffective in such extreme environments. Therefore, exploring efficient, multifunctional antimony detoxification bioremediation strategies is crucial to overcoming these technical bottlenecks.

[0003] At present, in the field of microbial remediation research of antimony-contaminated environments, there are three core issues that urgently need to be addressed. First, the screening process for efficient antimony detoxification microorganisms is slow, and the detoxification rate of existing strains is not high, which cannot meet the requirements of actual remediation. Most of the functional microorganisms that have been discovered have single functions, especially lacking strains that also have the ability to promote growth. For example, the Bacillus mentioned in the patent application with publication number CN117247874A can oxidize Sb(III) to Sb(V) for detoxification, but it does not have the function of nitrogen fixation and growth promotion; the Enterobacter strain disclosed in the patent application with publication number CN111662847A also has only antimony detoxification ability and does not have nitrogen fixation and growth promotion. Secondly, although many nitrogen-fixing and growth-promoting microorganisms have been reported, most of them are not resistant to antimony toxicity and cannot play a growth-promoting role in the oligotrophic environment under high antimony stress. Furthermore, most of the current research on nitrogen-fixing and promoting microorganisms focuses on symbiotic heterotrophic systems, such as the symbiosis between rhizobia and legumes, while people's understanding of autotrophic nitrogen-fixing and promoting microorganisms in extremely barren mining environments is still very limited.

[0004] Given the dual challenges of antimony pollution and nutrient deficiencies common in antimony mining areas, the development of autotrophic microorganisms capable of both antimony detoxification and efficient nitrogen fixation is crucial. This not only holds important theoretical significance for understanding the adaptive mechanisms of microorganisms in extreme environments but also opens new avenues for developing microbial-based tailings ecological remediation technologies. Summary of the Invention

[0005] The main purpose of this invention is to provide a Pseudomonas strain with the dual functions of antimony detoxification and autotrophic nitrogen fixation, and its application, particularly in the combined application of antimony pollution remediation and barren soil improvement. This strain not only efficiently oxidizes Sb(III) in the environment, reducing antimony toxicity, but also autotrophically fixes nitrogen in oligotrophic environments, thereby alleviating plant rhizosphere stress, promoting plant growth, and improving remediation effectiveness.

[0006] The present invention is named as PseudomonasPseudomonas sp. JLB0503, deposited with CCTCC NO: M20251573, was deposited at the China Center for Type Culture Collection, Wuhan University, China, on July 11, 2025.

[0007] The Pseudomonas can catalyze the oxidation of highly toxic trivalent antimony Sb(III) into low-toxic pentavalent antimony Sb(V), thereby achieving antimony detoxification; and can convert nitrogen gas into ammoniacal nitrogen through the nitrogenase system.

[0008] The bacterium can also use the energy released by Sb(III) oxidation to drive autotrophic growth, without the need for an organic carbon source, and only uses CO2 as a carbon source for growth.

[0009] The Pseudomonas can alleviate plant rhizosphere stress and promote plant growth, taking Arabidopsis thaliana as an example.

[0010] Furthermore, the strain was able to grow in the medium with a maximum Sb(III) concentration of 3 mM.

[0011] The present invention also provides the use of the Pseudomonas in antimony detoxification and / or nitrogen fixation.

[0012] Specifically, it is used for detoxification of antimony in soil or water, and for nitrogen fixation in the atmosphere, soil or water. It can also be used for antimony pollution control and soil nitrogen replenishment in antimony-contaminated tailings areas.

[0013] The invention also provides application of the Pseudomonas in promoting plant growth.

[0014] The Pseudomonas strain provided by the present invention is a Gram-negative, straight rod-shaped bacterium with an optimal growth temperature of 28-35°C and an optimal pH of 7.0-8.0. This strain can grow in media containing Sb(III) concentrations up to 3 mM, making it suitable for environments contaminated by the heavy metal antimony. It also exhibits efficient Sb(III) detoxification (detoxification rate of 108.6 (μmol / (L·day)) and nitrogenase activity (≥ 223.4 nmol C2H2 / (g·h)).

[0015] The strain of this invention uses Sb(III) as an electron donor, achieving chemoautotrophic growth. This detoxifies metallic antimony while also synergistically completing the biological nitrogen fixation process. This unique metabolic pathway offers new insights into tailings ecological remediation: biological nitrogen fixation can alleviate nitrogen deficiencies in mining areas, improve soil fertility, and lay a good foundation for subsequent plant colonization. Furthermore, Sb(III) is converted to the less toxic Sb(V) through biological oxidation, significantly reducing environmental risks.

[0016] The advantages and positive effects of the present invention are:

[0017] Pseudomonas ( Pseudomonas sp. The JLB0503 strain exhibits three key advantages, providing a highly effective solution for mine remediation. It can survive stably in the extreme environment of high antimony pollution and oligotrophic conditions, adapting to the unique conditions of mining areas with its strong tolerance. Furthermore, its efficient Sb(III) oxidation ability and nitrogen fixation create a synergistic effect, reducing antimony toxicity while improving soil nutrient status and promoting plant growth. More notably, the strain's simple cultivation conditions make it easy to scale up and apply in practice, and its environmentally friendly process facilitates the implementation of ecological restoration technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Colony diagram of Pseudomonas of the present invention;

[0019] Figure 2 : Antimony resistance curve of Pseudomonas sp. of the present invention;

[0020] Figure 3 : Antimony oxidation ability curve of Pseudomonas sp. of the present invention;

[0021] Figure 4 : Comparison of the antimony oxidation efficiency of the Pseudomonas sp. of the present invention with some other bacteria that have been reported;

[0022] Figure 5 : Plate photo of nitrogen fixation ability of Pseudomonas of the present invention;

[0023] Figure 6 : Autotrophic capacity curve of Pseudomonas of the present invention;

[0024] Figure 7 : qPCR results of autotrophic antimony oxidation and nitrogen fixation by Pseudomonas sp. of the present invention;

[0025] Figure 8 : The growth-promoting effect of the Pseudomonas sp. of the present invention on Arabidopsis thaliana. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0027] Example 1 Antimony detoxification and nitrogen-fixing bacteria Pseudomonas sp. Isolation, screening and identification of:

[0028] The fungus was obtained from the rhizosphere soil sample of Miscanthus collected from the Xikuangshan antimony mine area in Lengshuijiang City, Hunan Province in October 2024.

[0029] 1. Isolation and culture of Miscanthus rhizosphere microorganisms:

[0030] Sample Collection and Pretreatment: Rhizosphere soil samples were collected from the Xikuangshan antimony mining area in Lengshuijiang City, Hunan Province, and stored in sterile bags at 4°C. For strain isolation, 10 g of rhizosphere soil sample was weighed and added to 90 mL of sterile saline solution. The sample was shaken at 30°C for 30 min. After limiting dilution, 160 μL of the dilution was pipetted into bacterial culture plates. For each serial dilution, 45 plates were plated, sealed, and incubated at room temperature. 1 / 10 TSB medium (supplemented with 0.5 mmol / L Sb(III)) was plated on 96-well plates. After incubation at 30°C for 14 days, the gradient with turbidity in 30-40% of the wells was retained for identification. A portion of the cultured bacteria from each well was removed for identification, and the remaining culture was added to 80% (v / v) glycerol at a 1:1 volume ratio and stored at -80°C until further purification.

[0031] 2. Identification and strain comparison of isolated bacteria:

[0032] 6 μL of bacterial culture medium was transferred to a 1.5 mL centrifuge tube, and 10 μL of buffer (containing 20 mM NaOH and 0.2 mM EDTA, pH 12) was added. The mixture was vortexed and heated in a 95°C metal bath for 30 min for crude DNA extraction. The reaction was then terminated by adding 10 μL of neutralization buffer II (0 mM Tris-HCl, pH 7.5). After centrifugation at 12,000 × g for 5 min, the supernatant was used as a PCR template. The full-length 16S rRNA gene was amplified using the bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR product was verified by 1% agarose gel electrophoresis, gel-purified, and sent for Sanger sequencing. The sequencing results were compared with the NCBI database using BLASTn. Pseudomonas sp. ) The 16S rRNA gene sequence of JLB0503 is shown in SEQ ID NO.1 (same as Pseudomonas aeruginosa Similarity > 99%).

[0033] Example 2 strains ( Pseudomonas sp. )Physiological and biochemical characteristics of JLB0503:

[0034] 1. strain ( Pseudomonas sp. ) Species identification of JLB0503:

[0035] The isolated strains were identified by biological characteristics and morphological observation as follows:

[0036] Pseudomonas ( Pseudomonas sp.JLB0503 is a spherical bacterium that is Gram-negative and has flagella. It grows in the temperature range of 5-40°C, with an optimum temperature of 28-35°C and an optimum pH of 7.0-8.0. On LB solid medium, the colonies are orange-yellow, opaque, with neat edges and a smooth, opaque surface (see Figure 1 The bacteria were cultured on TSB solid medium for 24 hours, and the colonies were moist, smooth and opaque, with neat edges.

[0037] 2. Antimony resistance verification:

[0038] By determining the strain ( Pseudomonas sp. ) JLB0503 growth curves in culture medium containing different concentrations of Sb(III) were used to evaluate Sb(III) resistance. The method is as follows:

[0039] The bacterial suspension was added to liquid LB containing different concentrations of Sb(III) at a 1% inoculum volume (v / v) to culture the strain. The Sb(III) concentrations were 0.1, 1, 2, and 3 mmol / L, respectively. Three replicates were used for each group. The culture was continuously incubated at 28°C and 180 rpm in a constant temperature shaker for 72 h. Samples were taken at 12 h, 24 h, 48 h, and 72 h, and the OD was measured using a UV spectrophotometer. 600 Growth curves were drawn to evaluate the Sb(III) resistance.

[0040] 3. Antimony detoxification function determination:

[0041] Sb(V) is less toxic than Sb(III), so the detoxification function of antimony (Sb) is assessed by measuring the oxidation capacity of Sb(III). The method is as follows:

[0042] Add 1 mmol / L Sb(III) to the LB solution and measure oxidation activity every 24 hours. Three parallel groups and a blank control (no inoculation) are set up. For curve analysis, the sample is centrifuged at 5000 rpm for 5 minutes. The supernatant is then collected and the Sb(III) concentration is measured using an atomic fluorescence spectrometer. The Sb(III) oxidation capacity is determined using an atomic fluorescence spectrometer.

[0043] 4. Autotrophic growth ability test:

[0044] Two rounds of continuous cultivation were performed in carbon-free liquid medium. Each round, the bacterial suspension was transferred to a 1% inoculum (v / v) culture. Three replicates were used for each group. Cultures were continued in a shaker at 28°C and 180 rpm for 6 days. Samples were taken on the 1st, 2nd, 4th, and 6th day, and the OD values ​​were measured using a UV spectrophotometer. 600 , and draw growth curves to evaluate the autotrophic ability of the strain.

[0045] Carbon-free liquid medium: manganese sulfate (0.02 g / L), sodium dihydrogen phosphate (1 g / L), potassium dihydrogen phosphate (1.8 g / L), magnesium sulfate (0.4 g / L), calcium chloride (0.05 g / L), sodium chloride (1 g / L), ammonium chloride (0.5 g / L), ferric chloride (0.02 g / L), sodium sulfite (10 g / L).

[0046] 5. Nitrogenase Activity Assay:

[0047] The nitrogen fixation ability of the strain was verified by Ashby nitrogen-free culture, and the nitrogenase activity of the strain was determined by the acetylene reduction method. The specific method is as follows:

[0048] (1) Qualitative analysis: Take 1 μL OD 600 = 0.5 was inoculated into Ashby nitrogen-free medium for 7 days, and its growth and whether a transparent zone appeared around the colony were observed. If a transparent zone appeared, the strain had the ability to fix nitrogen.

[0049] (2) Quantitative analysis: Ashby medium was injected into a bottle with a rubber stopper, leaving 10% of the space in the bottle. The activated strain was inoculated and argon was flushed into the bottle to expel all air. Then, acetylene gas was used to replace 10% (v / v) of the headspace argon in the tube. The reaction was carried out at 28°C for 12 h. Then, 1 mL of gas was withdrawn from the bottle and the ethylene content in the headspace gas was determined by gas chromatography. The nitrogenase activity was quantified by measuring the amount of ethylene produced by acetylene reduction.

[0050] 6. Study on the ability of antimony detoxification and nitrogen fixation:

[0051] The antimony detoxification and nitrogen fixation capabilities were investigated using real-time fluorescence quantitative PCR (qPCR). The specific methods are as follows:

[0052] Microcosm culture experiments were conducted using sterile, nitrogen-free, and organic carbon-free Ashby medium. A 1 mmol / L Sb(III)-supplemented group and a blank control group were set up. After 7 days of culture at 28°C, real-time quantitative PCR (qPCR) was used to measure the abundance of the following functional genes: the antimony oxidation gene anoA, the autotrophic gene cbbL, and the nitrogenase gene nifH. The formula for nitrogen-free and organic carbon-free Ashby medium is as follows.

[0053] ;

[0054] 7. Arabidopsis growth promotion experiment:

[0055] Pour the 1 / 2 MS solid medium (300 ppm Sb(III) added to the experimental group and not added to the control group) to be sterilized into a sterile culture dish and cool it down for later use. When preparing the bacterial suspension, take the Pseudomonas aeruginosa ( Pseudomonas sp . )JLB0503, resuspended with sterile water, and measured OD 600 The value was adjusted to 0.5. Wild-type Arabidopsis thaliana (Col-0) seeds with full grains were surface-sterilized with a 5% NaClO solution for 10 minutes while shaking on a shaker at 150 rpm. The washed seeds were then thoroughly washed six times with sterile water. The washed seeds were immersed in 10 mL of the bacterial suspension and incubated at 28°C with shaking for 12 hours. The seeds were then washed three times with sterile water to remove unadhered bacteria. The seeds were transferred to the 1 / 2 MS medium plates prepared above using sterile forceps. The plates were vernalized at 4°C for 48 hours and then transferred to an upright position in a culture chamber at 25°C, with a 16 h / 8 h light / dark cycle and a relative humidity of 60% for 14 days. Phenotypes were observed and photographed.

[0056] result:

[0057] The Pseudomonas strain of the present invention can tolerate antimony environments. Even in a high concentration of 3 mM Sb(III), it still has a strong Sb(III) tolerance and good adaptability to high antimony environments ( Figure 2 ).

[0058] The Pseudomonas strain of the present invention has the ability to detoxify Sb(III) by oxidizing highly toxic Sb(III) to less toxic Sb(V), and can oxidize 1 mM Sb(III) by 43.95% within 4 days. It has good high antimony oxidation and detoxification rate of 108.6 μmol / L / d ( Figure 3 ). Its antimony oxidation ability is higher than that of most other common antimony-oxidizing microorganisms ( Figure 4 ).

[0059] The Pseudomonas sp. of the present invention can form sticky and translucent colonies on Ashby medium without organic or inorganic nitrogen sources, and also has a strong nitrogen fixation ability ( Figure 5 ), and the nitrogenase activity was measured to be ≥ 223.4 (nmol C2H2 / (g·h)).

[0060] The strain was cultured using inorganic carbon medium, and the results of two rounds showed that it had the ability to grow, proving that it still had the ability to grow autotrophically in the absence of long-term carbon sources ( Figure 6 ).

[0061] The Pseudomonas strain of the present invention was enriched using a medium containing Sb(III) without organic carbon and nitrogen sources. qPCR results showed that the antimony oxidation gene (anoA) and carbon fixation (cbbL) / nitrogen fixation (nifH) increased after the addition of Sb(III), indicating that the strain has the ability to autotrophically oxidize and fix nitrogen through antimony. Figure 7 ).

[0062] The Pseudomonas sp. of the present invention has a significant plant growth-promoting effect. It can alleviate plant stress under the conditions of high antimony (300 ppm) oligotrophy, indicating that Pseudomonas sp. has good application potential in plant growth promotion ( Figure 8 ).

Claims

1. A strain of Pseudomonas that detoxifies antimony and autotrophically fixes nitrogen ( Pseudomonas sp. ) JLB0503, characterized in that, The deposit number is CCTCC NO: M 20251573.

2. The Pseudomonas according to claim 1, characterized in that It can catalyze the oxidation of highly toxic trivalent antimony Sb(III) into low-toxic pentavalent antimony Sb(V), achieving antimony Sb detoxification; and can convert nitrogen gas into ammonia nitrogen through the nitrogenase system.

3. The Pseudomonas according to claim 1, characterized in that The energy released by Sb(III) oxidation is used to drive autotrophic growth, without the need for an organic carbon source, and only using CO2 as a carbon source for growth.

4. The Pseudomonas according to claim 1, characterized in that It can alleviate plant rhizosphere stress and promote plant growth.

5. The Pseudomonas according to claim 1, characterized in that The Pseudomonas sp. can grow in a culture medium with a Sb(III) concentration of 3 mM.

6. Use of the Pseudomonas according to any one of claims 1 to 5 in antimony detoxification and / or nitrogen fixation.

7. The use according to claim 6, characterized in that Used for detoxification of antimony in soil or water.

8. The use according to claim 6, characterized in that Used for nitrogen fixation in the atmosphere, soil or water.

9. The use according to claim 6, characterized in that Used for antimony pollution control and soil nitrogen replenishment in antimony-contaminated tailings areas.

10. Use of the Pseudomonas according to any one of claims 1 to 5 in promoting plant growth.

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