Heavy metal tolerant sphingomonas 1PPS-Zn1 and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to a heavy metal tolerant sphingomonas strain 1PPS-Zn1 and application thereof, the heavy metal tolerant sphingomonas strain 1PPS-Zn1 is preserved in China Center for Type Culture Collection on March 20, 2025, the preservation number is CCTCC NO: M 2025544, the sphingomonas strain 1PPS-Zn1 can adsorb and tolerate high-concentration zinc, the highest tolerable concentration reaches 80 mmol / L, and the sphingomonas strain 1PPS-Zn1 also has the capabilities of producing IAA, fixing nitrogen and promoting plant growth, and can be applied to the field of heavy metal tolerant plants. And the absorption, enrichment and transfer capabilities of the black nightshade to heavy metal zinc in the soil can be improved, finally, the remediation efficiency of the zinc-polluted soil is improved, a new way is provided for soil remediation and ecological environment remediation, and the method has wide development and application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a heavy metal-tolerant Sphingomonas 1PPS-Zn1 strain and application thereof. Background Art

[0002] Heavy metal contamination of soil is a serious problem currently facing countries around the world. Heavy metals not only affect soil properties but also accumulate in plants and animals, threatening ecosystems and human health. Zinc is an essential trace element for plant growth and development, but excessive zinc can also lead to reduced crop yields and quality. Furthermore, zinc in the soil can migrate and be transferred through the food chain, posing a serious threat to human health. Currently, methods for remediating zinc contamination primarily include physicochemical and bioremediation technologies. Physicochemical remediation technologies include chemical precipitation, adsorption, and electrokinetic remediation. While these technologies have diverse application scenarios, they are generally costly and complex to operate, and can also lead to a decline in soil fertility and secondary environmental pollution.

[0003] Bioremediation is a relatively popular remediation technology in recent years. It refers to the use of plants, microorganisms or their combined systems to remove or fix zinc in the environment. Compared with traditional physical remediation and chemical remediation technologies, bioremediation has the advantages of simple operation, low cost, and environmental protection. For example, invention patent CN103642702B discloses a fungal strain LP-16 and its use in the treatment of zinc-containing wastewater. By contacting the fungal strain LP-16 with zinc-containing wastewater, its strong adsorption capacity is utilized to remove or recycle zinc ions, solving the problems of low efficiency and waste of resources in the treatment of zinc-containing wastewater in the prior art, and achieving low-cost and efficient wastewater treatment. Invention patent CN118185838B discloses an oligotrophic bacterium with zinc adsorption capacity and the application of its extracellular polymers, solving the problems of high cost and low efficiency of zinc pollution treatment in the prior art, and providing an environmentally friendly zinc remover and pollution mitigation method.

[0004] Sphingomonas is a type of Gram-negative bacteria with unique biological characteristics and a wide range of environmental adaptability. Researchers have found that Sphingomonas can be used to prevent and treat tobacco black shank (see invention patent CN109182213B), effectively reduce the cadmium content in rice, and increase the selenium content in rice (see invention patent CN114317373B). Regarding Sphingomonas' tolerance to zinc, invention patent CN101974471A discloses that it can tolerate 4mmol / L zinc, but its potential to promote plant growth in zinc-contaminated environments has not yet been reported. During the research process, the inventors screened and obtained a strain of Sphingomonas sp. 1PPS-Zn1, which can tolerate high concentrations of zinc and can grow at zinc concentrations of up to 80mmol / L, which is 19 times higher than the existing technology. In addition, the fungus also has the functions of producing IAA (19.94 mg / L), fixing nitrogen, promoting plant growth, and enhancing the restoration effect, providing a new way for soil management and ecological environment restoration, and has broad development and application prospects. Summary of the Invention

[0005] The primary purpose of the present invention is to provide a heavy metal-tolerant Sphingomonas sp. 1PPS-Zn1, wherein the Sphingomonas sp. 1PPS-Zn1 was deposited in the China Center for Type Culture Collection on March 20, 2025, with a deposit number of CCTCC NO: M 2025544.

[0006] The second object of the present invention is to provide a bacterial agent, which contains the Sphingomonas 1PPS-Zn1 or its strain fermentation liquid.

[0007] The third object of the present invention is to provide the use of the Sphingomonas 1PPS-Zn1 or the bacterial agent in removing heavy metals.

[0008] The fourth object of the present invention is to provide the use of the Sphingomonas 1PPS-Zn1 or the bacterial agent in the remediation of heavy metal contaminated soil.

[0009] The fifth object of the present invention is to provide the use of the Sphingomonas 1PPS-Zn1 or the bacterial agent in promoting plant growth under heavy metal stress environment.

[0010] Preferably, the heavy metal is zinc.

[0011] The sixth object of the present invention is to provide a bioremediation preparation containing the Sphingomonas 1PPS-Zn1 or the bacterial agent.

[0012] The seventh object of the present invention is to provide a plant growth-promoting bacterial agent, which contains the Sphingomonas 1PPS-Zn1 or the bacterial agent.

[0013] The beneficial effects of the present invention are as follows: the present invention provides a heavy metal-tolerant Sphingomonas sp. 1PPS-Zn1, which was deposited in the China Center for Type Culture Collection on March 20, 2025, with a deposit number of CCTCC NO: M2025544. The Sphingomonas sp. 1PPS-Zn1 can tolerate and adsorb high concentrations of zinc, and can grow at a zinc concentration of up to 80 mmol / L, which is 19 times higher than the existing technology. It also has the functions of producing IAA (19.94 mg / L), fixing nitrogen, promoting plant growth, and enhancing the repair effect, providing a new way for soil management and ecological environment restoration, and has broad development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Colony morphology of strain 1PPS-Zn1

[0015] Figure 2 Identification results and phylogenetic tree of strain 1PPS-Zn1

[0016] Figure 3 Growth-promoting properties of Sphingomonas 1PPS-Zn1

[0017] Note: a: IAA color reaction; b: nitrogen fixation characteristics

[0018] Figure 4 Growth curve of Sphingomonas 1PPS-Zn1 Note: a: Growth curve of Sphingomonas 1PPS-Zn1 in R2A medium; b: Growth curve of Sphingomonas 1PPS-Zn1 in heavy metal (Zn 2+ ) Growth curve in culture medium Figure 5 Adsorption rate of zinc by Sphingomonas 1PPS-Zn1 at different growth stages

[0019] Note: Different lowercase letters indicate significant differences (p<0.05)

[0020] Figure 6 Growth-promoting effect of Sphingomonas 1PPS-Zn1 on Solanum nigrum under zinc stress

[0021] Note: a: plant height; b: aboveground dry weight; c: root dry weight

[0022] Figure 7 Enhanced effect of Sphingomonas 1PPS-Zn1 on remediation of zinc-contaminated soil by Solanum nigrum

[0023] Note: a: stems and leaves; b: roots; c: soil; d: enrichment coefficient DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0025] It should be noted that, in the following examples, unless otherwise specified, the methods described are conventional methods, and the reagents described can be purchased from the market.

[0026] The following culture medium is used in the embodiments of the present invention:

[0027] R2A medium: yeast extract 0.5 g / L, peptone 0.5 g / L, acid hydrolyzed casein 0.5 g / L, soluble starch 0.5 g / L, sodium pyruvate 0.3 g / L, K2HPO4 0.3 g / L, MgSO4 0.024 g / L (solid medium plus agar);

[0028] Heavy metal culture medium: R2A medium containing ZnSO4 aqueous solution;

[0029] King liquid medium: K2HPO4 1.5 g, MgSO4·7H2O 1.5 g, peptone 20 g, tryptophan 0.1 g, glycerol 15 mL, H2O 1 L, pH 7.2;

[0030] Ashby nitrogen-free medium: mannitol 10 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaSO4·2H2O 0.2 g, CaCO3 5 g, H2O 1 L, Agar 15 g, pH 7.2.

[0031] Example 1: Isolation, screening and identification of strains

[0032] 1. Strain Isolation and Culture

[0033] The rhizosphere soil of a grass plant growing in an abandoned mine was collected, 10 g of soil sample was weighed and placed in a conical flask containing 90 mL of sterile water and shaken for 20 minutes. The soil suspension was diluted in a gradient manner and applied to the Zn 2+ Culture the strain on 2 mmol / L R2A solid medium in an incubator at 28°C upside down for 3-5 days. Observe the growth and morphology of the strain.

[0034] The isolated and purified strain was inoculated with Zn 2+ The concentration of 5mmol / L R2A liquid medium was added, and when the medium reached the logarithmic phase, 200μL was taken and transferred to a higher Zn2+ Concentration (10, 15, 20, 30, 40, 50, 60, 70, 80, 90 mmol / L) 的 culture medium until the strain no longer grows.

[0035] After separation, screening and domestication, we finally obtained a strain that could tolerate 80mmol / L Zn 2+ The strain 1PPS-Zn1 has a yellow, round, smooth colony on R2A solid medium. Figure 1 ).

[0036] 2. Strain Identification

[0037] Genomic DNA from strain 1PPS-Zn1 was extracted using a bacterial DNA extraction kit. The bacterial 16S rRNA gene was amplified using universal primers 27F / 1492R. The PCR product with distinct bands was purified and sequenced. The strain sequence was then compared using BLAST in the NCBI database, and a phylogenetic tree was constructed using MEGA11 software.

[0038] The strain 1PPS-Zn1 was identified to have a similarity of 98.93% with the previously published strain Sphingomonas parapaucimobilis (AB680768.1), belonging to the genus Sphingomonas, with the Latin name Sphingomonas sp., so it was named Sphingomonas sp. 1PPS-Zn1. The phylogenetic tree is shown in Figure 2. Figure 2 shown.

[0039] The Sphingomonas sp. 1PPS-Zn1 was deposited in the China Center for Type Culture Collection on March 20, 2025, with a deposit number of CCTCC NO: M 2025544. The deposit address is Wuhan University, Wuhan, China, telephone: 027-68754052, fax: 027-68754833.

[0040] 3. Determination of growth-promoting properties of strains

[0041] IAA production characteristics: The strain was inoculated into King liquid medium and cultured with shaking at 180 rpm and 28°C for 8 days. The bacterial solution was then centrifuged at 10,000 rpm for 2 minutes. The supernatant was collected and an equal volume of Salkowski colorimetric solution was added. The solution was reacted in the dark for 20-30 minutes. The strain producing IAA would cause the colorimetric solution to change from colorless to pink. Analytical grade IAA was used to prepare a gradient standard solution and draw an IAA standard curve. Finally, the OD value of each reaction solution was measured on a spectrophotometer. 530 The value was read and the IAA content was calculated according to the standard curve.

[0042] Nitrogen-fixing properties: Add 2 μL of bacterial solution to Ashby nitrogen-free medium, culture at 28°C for 5-7 days, and observe. If the strain can grow well on the medium, it is determined that the strain has the ability to fix nitrogen.

[0043] The results are as follows Figure 3 As shown, the IAA production level of Sphingomonas 1PPS-Zn1 was as high as 19.94 mg / L (there is no corresponding chart for the value), and the colorimetric solution turned dark red after inoculation, indicating that Sphingomonas 1PPS-Zn1 has a strong ability to produce IAA; at the same time, Sphingomonas 1PPS-Zn1 grew well on Ashby nitrogen-free medium, indicating that it also has the ability to fix nitrogen.

[0044] Example 2: Growth curve of Sphingomonas 1PPS-Zn1 in culture medium and its zinc adsorption rate

[0045] 1. Growth curve of Sphingomonas 1PPS-Zn1 in culture medium

[0046] Sphingomonas 1PPS-Zn1 was inoculated into R2A liquid medium and Zn 2+ The cells were cultured in a heavy metal medium with a concentration of 2000 mg / L at 28°C and 220 rpm. Samples were taken every 2 hours and the OD of the bacterial solution was measured using a spectrophotometer. 600 The latter was sampled every 4 hours to measure the OD of the bacterial solution. 600 , and 3 replicates were set for each sampling.

[0047] The results are as follows Figure 4 As shown, under normal conditions, Sphingomonas 1PPS-Zn1 is in the logarithmic growth phase from 6 to 12 hours, and the strain is in the stationary phase from 12 to 28 hours ( Figure 4 a). In Zn 2+ In the liquid culture medium with a concentration of 2000 mg / L, Sphingomonas 1PPS-Zn1 can also grow normally, but the growth is slow, and the time to enter each growth stage is doubled ( Figure 4 b), which indicates that heavy metals have a certain effect on the growth of strains.

[0048] 2. Adsorption rate of zinc by Sphingomonas 1PPS-Zn1

[0049] The strain was inoculated with Zn 2+ The bacterial cultures were cultured in a heavy metal medium with a concentration of 2000 mg / L. The bacterial cultures in the delayed phase, logarithmic phase, stable phase, and decay phase were centrifuged (6000 r / min, 10 min). The supernatants were collected and the Zn content was determined using an inductively coupled plasma emission spectrometer (ICP). 2+ Concentration, and calculate the strain's zinc adsorption rate. Calculation formula:

[0050] Adsorption rate = (C0-C e ) / C0×100%.

[0051] Where: C0 is the supernatant Zn before inoculation 2+ Concentration (mg / L); C e Zn in the supernatant after inoculation 2+ Concentration (mg / L).

[0052] The results showed that the adsorption capacity of Sphingomonas 1PPS-Zn1 on zinc varied in different growth periods, and the adsorption capacity of zinc on zinc in the stable period was the highest. 2+ The adsorption rate is the strongest. 2+ When the concentration is 2000 mg / L, 1PPS-Zn1 has a significant effect on Zn 2+ The adsorption rate is as high as 85.94% ( Figure 5 ).

[0053] Example 3: Growth-promoting effect of Sphingomonas 1PPS-Zn1 on Solanum nigrum under zinc stress

[0054] Transplant the Solanum nigrum that has grown two cotyledons and has the same growth pattern into a soil containing 2000mg / kg Zn 2+ In the soil, after 3 cotyledons grow, root irrigation is carried out every week for 5 consecutive weeks. Before root irrigation, the bacterial solution is diluted with sterile water and then added to the soil to make each gram of soil contain 1×10 8 CFU (1PPS-Zn1), and the control group (CK) was irrigated with the same amount of sterile water. Each treatment had three replicates, each containing 9 Solanum nigrum plants.

[0055] On the 45th day after root irrigation, the above-ground parts (stems and leaves) and underground parts (roots) of Solanum nigrum were separated, cleaned with distilled water, and the surface moisture was absorbed. After that, they were placed in an oven at 60°C and dried to constant weight, and their dry weight was calculated.

[0056] The results showed that the height, dry weight of above-ground and underground parts of Solanum nigrum inoculated with Sphingomonas 1PPS-Zn1 increased by 25.35%, 56.14% and 120% respectively compared with the control group. Figure 6 ). These results indicate that inoculation with 1PPS-Zn1 can significantly promote the growth of Solanum nigrum under zinc stress and increase its biomass.

[0057] Example 4: Remediation Effect of Sphingomonas 1PPS-Zn1 on Zinc-Contaminated Soil

[0058] The experimental method is the same as that of Example 3. The stems, leaves, roots and rhizosphere soil of Solanum nigrum after drying were ground and sieved (100 mesh sieve). Weigh 0.1g of sample and put it in a digestion tube, add 6mL of aqua regia and soak overnight, then put it in a digestion furnace, keep the aqua regia in a slightly boiling state, and cool it to room temperature after the digestion is completed. Then wash it all with deionized water into a 50mL volumetric flask, adjust the volume, shake well, filter, and finally use inductively coupled plasma emission spectrometer (ICP) to determine the Zn content in the sample. 2+ The enrichment coefficient was calculated, and 3 replicates were performed for each treatment.

[0059] BCF = Zn in Solanum nigrum 2+ Content (mg / kg) / Zn in soil 2+ Content (mg / kg)

[0060] The results are as follows Figure 7 As shown in the figure, compared with the control, the Zn content in the stems, leaves and roots of Solanum nigrum after inoculation with Sphingomonas 1PPS-Zn1 was significantly increased. 2+ The contents of Zn in Solanum nigrum roots increased to varying degrees. 2+ The content is much higher than that of stems and leaves ( Figure 7 a, 7b), indicating that Solanum nigrum is enriched in Zn 2+ It is mainly concentrated in the roots. It is also found that the Zn enriched in Solanum nigrum after the strain treatment 2+ The content of Zn in the soil was 1.34 times that of the uninoculated control group. This indicates that Sphingomonas 1PPS-Zn1 can enrich Zn in soil with Solanum nigrum. 2+ It has an obvious promoting effect.

[0061] At the same time, the Zn content in the rhizosphere soil of Solanum nigrum was significantly increased in the group inoculated with Sphingomonas 1PPS-Zn1. 2+ The content (355.42 mg / kg) was significantly lower than that of the control group (555.41 mg / kg) ( Figure 7 c), and the zinc enrichment coefficient of Solanum nigrum was as high as 3.14, which was significantly higher than that of the control group without inoculation (1.5) ( Figure 7 d). This indicates that Sphingomonas 1PPS-Zn1 can improve the absorption, accumulation and transport capacity of Solanum nigrum for heavy metal zinc in soil, thereby improving the remediation efficiency of zinc-contaminated soil.

[0062] In summary, the present invention provides a heavy metal-tolerant Sphingomonas sp. 1PPS-Zn1, which was deposited in the China Center for Type Culture Collection on March 20, 2025, with a deposit number of CCTCC NO: M2025544. The Sphingomonas sp. 1PPS-Zn1 can tolerate and adsorb high concentrations of zinc, with the maximum tolerance concentration reaching 80 mmol / L, which is 19 times higher than the existing technology. It also has the functions of producing IAA (19.94 mg / L), fixing nitrogen, promoting plant growth, and enhancing the repair effect. It has the advantages of high efficiency, safety, green environmental protection, and no damage to soil structure. It provides a new way for soil management and ecological environment restoration, and has broad development and application prospects.

Claims

1. A heavy metal-tolerant Sphingomonas sp. 1PPS-Zn1, characterized in that: The Sphingomonas 1PPS-Zn1 was deposited in the China Center for Type Culture Collection on March 20, 2025, with the deposit number being CCTCCNO: M 2025544.

2. A bacterial agent, characterized in that The bacterial agent contains the Sphingomonas 1PPS-Zn1 according to claim 1 or its strain fermentation liquid.

3. Use of the Sphingomonas 1PPS-Zn1 according to claim 1 or the bacterial agent according to claim 2 in removing heavy metals.

4. Use of the Sphingomonas 1PPS-Zn1 according to claim 1 or the bacterial agent according to claim 2 in the remediation of heavy metal contaminated soil.

5. Use of the Sphingomonas 1PPS-Zn1 according to claim 1 or the bacterial agent according to claim 2 in promoting plant growth under heavy metal stress environment.

6. The use according to any one of claims 3 to 5, characterized in that The heavy metal is zinc.

7. A bioremediation preparation, characterized in that The bioremediation preparation contains the Sphingomonas 1PPS-Zn1 according to claim 1 or the bacterial agent according to claim 2.

8. A plant growth promoter, characterized in that The plant growth promoter contains the Sphingomonas 1PPS-Zn1 according to claim 1 or the bacterial agent according to claim 2.

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