Paenibacillus and application thereof in microbial fertilizer

Abstract

The invention provides a paenibacillus and an application thereof in a microbial fertilizer. The classification name of the paenibacillus is Paenibacillus sp.LXY-3, and the preservation unit is China General Microbiological Culture Collection Center (CGMCC); the address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is October 18, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 1.64949. The invention also provides an application of the bacillus in a microbial fertilizer.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to the application of microbial treatment of pollutants, specifically to a strain of Bacillus subtilis and its application. Background Technology

[0002] With increasingly frequent industrial production activities, heavy metal pollution has become a more severe problem, among which chromium (Cr(VI)) pollution has become a globally significant environmental issue. Chromium is widely used in many industrial fields such as electroplating, metallurgy, leather making, and chemicals. Large amounts of chromium-containing wastewater and waste residue are directly discharged into the environment without effective treatment, causing serious pollution to soil and water bodies. High concentrations of chromium not only cause persistent damage to the ecological environment but also threaten human health through the food chain, causing a series of serious diseases such as cancer, organ damage, and genetic mutations.

[0003] Soil, as the foundation of agricultural production, directly affects the yield and quality of crops. In chromium-contaminated soil environments, crop growth and development are significantly inhibited. Chromium ions interfere with various physiological and biochemical processes within plants, affecting their absorption and transport of water and nutrients, damaging cell structure and function, and consequently leading to stunted growth, yellowing leaves, poor root development, and even death. This not only reduces crop yields but also compromises the safety of agricultural products, causing significant economic losses to agricultural production.

[0004] Traditional methods for remediating chromium-contaminated soil mainly include physical remediation and chemical remediation. Physical remediation methods, such as soil replacement and topsoil replacement, can reduce the chromium content in the soil to some extent, but they have drawbacks such as large engineering scale, high cost, and potential damage to soil structure, and cannot fundamentally eliminate chromium pollution. Chemical remediation methods involve adding chemical reagents to the soil to induce chromium precipitation, adsorption, and redox reactions, thereby reducing its bioavailability. However, chemical remediation may introduce new pollutants, alter the soil's physicochemical properties, and cause secondary damage to the soil ecosystem.

[0005] Microbial remediation, as an emerging soil remediation method, has gradually gained widespread attention due to its advantages such as low cost, environmental friendliness, and no secondary pollution. Microorganisms can reduce the toxicity and bioavailability of heavy metals in soil through mechanisms such as adsorption, precipitation, redox reactions, and methylation, promoting the absorption, transformation, and fixation of heavy metals by plants. Simultaneously, some microorganisms can also secrete plant growth regulators, improving soil fertility, promoting plant growth and development, and enhancing plant stress resistance.

[0006] Currently, although some research reports have been published on the microbial remediation of chromium-contaminated soil, microbial strains that can simultaneously and effectively tolerate high concentrations of chromium and significantly promote plant growth remain relatively scarce. Therefore, finding a microorganism with highly efficient chromium tolerance and growth-promoting capabilities, and applying it to soil remediation and agricultural production in areas with high concentrations of chromium contamination, has significant practical implications and application value. Summary of the Invention

[0007] To address the aforementioned problems, the present invention aims to provide a Bacillus-like bacterium that can promote plant growth under conditions of high Cr(VI) concentration.

[0008] Another object of the present invention is to provide the application of the above-mentioned Bacillus subtilis.

[0009] To achieve the above objectives, this invention provides a strain of Bacillus sp. LXY-3, with the following classification: CGMCC No. 1.64949; deposit date: October 18, 2024; depositing institution: China General Microbiological Culture Collection Center.

[0010] The present invention also provides the application of the above-mentioned Bacillus subtilis in Cr(VI) contaminated areas.

[0011] The present invention relates to the application of the above-mentioned Bacillus subtilis as a microbial fertilizer to promote crop growth in Cr(VI) polluted areas.

[0012] The above-mentioned methods promote plant germination and growth under Cr(VI) pollution conditions, providing a large amount of nutrients for crop production, and have great potential in the production of high-efficiency bio-organic fertilizers.

[0013] The beneficial effects of this invention are as follows:

[0014] This invention provides a Bacillus-like bacterium that shows great promise for applications in the remediation of high-concentration Cr(VI) sites and agricultural production. It can promote plant germination and growth, providing abundant nutrients for crop production. Through the controlled large-scale production of this Bacillus-like bacterium, it can be applied to the preparation of microbial fertilizers. This microbial fertilizer based on Bacillus-like bacteria not only improves tolerance to high concentrations of Cr(VI) but also promotes crop growth and development, thereby increasing crop yield and quality. Furthermore, because its production process is relatively controllable and easily scaled up, it has high economic value and practicality in real-world applications. Attached Figure Description

[0015] Figure 1 This is a diagram showing the growth status of Bacillus LXY-3 on a culture medium, as provided by the present invention.

[0016] Figure 2 The phylogenetic tree of Bacillus LXY-3 based on 16S rDNA provided by the present invention.

[0017] Figure 3 The growth curve of Bacillus LXY-3 provided by this invention.

[0018] Figure 4 The growth curves of Bacillus LXY-3 provided by this invention under different concentrations of Cr(VI). Detailed Implementation

[0019] The embodiments of the present invention will now be described in detail and comprehensively so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0020] Material

[0021] 1. The DNA extraction kit was purchased from Beijing Jinsha Biotechnology Co., Ltd., product number: DE703-50.

[0022] Preparation of culture medium:

[0023] The microbial enrichment medium (1L) formula is as follows: 2g anhydrous magnesium sulfate, 5g sodium citrate dihydrate, 1g calcium sulfate dihydrate, 1g ammonium chloride, 0.5g dipotassium hydrogen phosphate, 3.5g sodium lactate, and 1g yeast extract. The preparation method is as follows: Mix the above ingredients and add water to 1L. Adjust the pH to 7.2 with 1mol / L dilute sulfuric acid. Dispense the mixture into anaerobic tubes in an anaerobic workbench and sterilize in a high-pressure steam autoclave at 121℃ for 20min. Cool before use.

[0024] The formula for solid culture medium (1L) is as follows: 2g anhydrous magnesium sulfate, 5g sodium citrate dihydrate, 1g calcium sulfate dihydrate, 1g ammonium chloride, 0.5g dipotassium hydrogen phosphate, 3.5g sodium lactate, 1g yeast extract, and 15g agar. The preparation method is as follows: Mix the above ingredients and add water to 1L. Adjust the pH to 7.2 with 1mol / L dilute sulfuric acid. After venting the bottle containing the culture medium with 95% high-purity nitrogen in an anaerobic environment, seal the bottle and autoclave at 121℃ for 20 minutes. While still hot, pour the mixture into sterile petri dishes. After cooling, the solid culture medium plates are obtained.

[0025] LB liquid medium (1L): 5g yeast extract, 10g sodium chloride, 10g tryptone. After thorough mixing, adjust the pH to 7 using NaOH or HCl, autoclave at 121℃ for 20 minutes, and then cool for later use.

[0026] LB solid culture medium plates are made by adding 15g of agar powder to LB liquid culture medium, adjusting the pH to 7, autoclaving at 121℃ for 20min, pouring the hot mixture into sterile petri dishes, and cooling to obtain LB solid culture medium plates.

[0027] LB medium is used for the large-scale culture of microorganisms.

[0028] Example 1: Isolation and Identification of Strains

[0029] Soil samples were collected from a heavy metal smelting plant in Guangxi Province. Sterilized shovels or samplers were used to collect samples from a depth of 10-40 cm. The samples were placed in sterile plastic bags or containers and labeled with information. The collected samples were then sent to the laboratory at low temperature for microbial isolation and screening.

[0030] Take 5g of soil sample and add 45mL of sterile physiological saline. Shake thoroughly for 10 minutes to fully suspend the soil particles. Let the soil mixture stand for 10 minutes to allow solid particles to settle. The supernatant is the bacterial suspension used for screening. Add 100μL of the bacterial suspension to 900μL of sterile physiological saline and mix thoroughly to form a 10... -1 Diluent. Take 100 μL of 10 -1 The diluent was added to 900 μL of sterile physiological saline to form a 10... -2 Diluent, and so on, up to 10 -6 Dilution. Take 200 μL of 10... -4 10 -5 and 10 -6 The serially diluted solutions were evenly spread onto different solid culture medium plates using a sterile spreader, and then inverted and placed in a 30°C incubator for 48 hours to observe the growth of the colonies.

[0031] After clear single colonies have grown on the solid medium plate, select representative and well-grown colonies and inoculate them using a sterile inoculation loop. Streak the colonies on a fresh solid medium plate to ensure the isolation of single colonies. Invert the plate and incubate at 30°C for 48 hours. Observe the growth of the colonies and repeat the streaking process until a purified strain is obtained. Figure 1 The image shown depicts the growth status on a solid culture medium.

[0032] Genomic DNA was extracted from the purified strain using a DNA extraction kit and amplified by PCR. The PCR products were then subjected to agarose gel electrophoresis to confirm the amplification effect. The 16S rRNA of this bacterium was amplified by PCR and sequenced, yielding the sequence shown in SeqID No. 1. Comparison with the Ezbiocloud database (https: / / www.ezbiocloud.net / ) showed that the maximum full-length similarity of the 16S rRNA gene sequence of this strain with all standard strains in the genus *Paenibacillus* was 96.66%, which is below the 98.5% threshold for new species classification. Figure 2 As shown, based on physiological and biochemical characteristics, this bacterium was identified as a new species of the genus *Bacillus*, and named...

[0033] Paenibacillus sp. LXY-3, abbreviated as Bacillus LXY-3, has been deposited for preservation with accession number CGMCCNo.1.64949; the deposit date is October 18, 2024, and the depositary institution is the China General Microbiological Culture Collection Center.

[0034] Example 2: Detection of the growth ability of Bacillus subtilis LXY-3 at different concentrations of Cr(VI)

[0035] The *Bacillus* LXY-3 strain obtained in Example 1 was inoculated into liquid LB medium and cultured at 30°C and 180 rpm for 72 h. The OD of the bacterial culture was adjusted. 600 =1, to obtain the seed culture. The environment of a high-Cr(VI) region was simulated by preparing liquid LB medium with different Cr(VI) concentrations: seven concentration gradients of Cr(VI) were adjusted to 0, 50, 100, 150, 200, 250, and 300 mg / L, resulting in liquid LB medium containing different concentrations of Cr(VI). Each concentration was prepared in triplicate. The pH was adjusted to 7, and the medium was autoclaved at 121℃ and then cooled for later use. OD 600 The seed culture of 1 was inoculated into the above LB medium at a volume percentage of 1%, and cultured at 30°C and 160 rpm for 72 h with shaking. The OD of the bacterial suspension was then measured. 600 Value, result as Figure 4 As shown: When the Cr(VI) concentration in the culture environment is 0 mg / L, the OD of Bacillus subtilis LXY-3 is... 600 The value reached 0.8; when the Cr(VI) concentration increased to 50 mg / L, its OD value... 600 The value rose slightly to 0.83; at a Cr(VI) concentration of 100 mg / L, the OD... 600 The value is 0.81; when the Cr(VI) concentration reaches 150 mg / L, the OD 600The value dropped to 0.79; as the Cr(VI) concentration further increased to 200 mg / L, the OD value... 600 The value decreased significantly to 0.6; when the Cr(VI) concentration was 250 mg / L, the OD value... 600 The value dropped sharply to 0.2; while when the Cr(VI) concentration reached 300 mg / L, the OD value... 600 The value is only 0.09.

[0036] At low Cr(VI) concentrations ranging from 0 to 100 mg / L, Bacillus subtilis LXY-3 exhibited good adaptability and stability. When the Cr(VI) concentration was 0 mg / L, OD... 600 A value of 0.8 represents the normal growth level of this strain in an environment free of Cr(VI) contamination. As the Cr(VI) concentration gradually increases to 50 mg / L, the OD... 600 The value not only did not decrease, but actually increased slightly to 0.83, indicating that at this concentration, Cr(VI) did not inhibit the growth of Bacillus subtilis LXY-3, and may even have stimulated the growth and metabolic activities of the strain to some extent, promoting an increase in its biomass. When the Cr(VI) concentration continued to increase to 100 mg / L, the OD... 600 Although the value decreased slightly to 0.81, the overall change was small, indicating that Bacillus subtilis LXY-3 can still adapt well to this concentration of Cr(VI) environment and maintain a relatively stable growth state.

[0037] When the Cr(VI) concentration reaches 150 mg / L, OD 600 The value decreased to 0.79, a slight reduction compared to the value at 100 mg / h. This suggests that as the concentration of Cr(VI) further increases, it begins to inhibit the growth of Bacillus subtilis LXY-3. However, this inhibition is relatively mild, and the strain can still maintain a certain growth capacity at this concentration, indicating that Bacillus subtilis LXY-3 has a certain tolerance to moderate concentrations of Cr(VI).

[0038] At a Cr(VI) concentration of 200 mg / L, OD 600 When the Cr(VI) concentration decreased to 0.6, the inhibitory effect of Cr(VI) on the strain intensified, leading to a slowdown in growth and a reduction in biomass. As the Cr(VI) concentration continued to rise to 250 mg / L, the OD... 600 The value dropped sharply to 0.2, indicating that high concentrations of Cr(VI) exerted a stress effect on the growth of Bacillus subtilis LXY-3. Furthermore, when the Cr(VI) concentration reached 300 mg / L, the OD value... 60 The value is only 0.09, almost close to 0.

[0039] Based on the above data and analysis, it can be concluded that Bacillus subtilis LXY-3 exhibits a certain tolerance to Cr(VI) when the Cr(VI) concentration is less than 200 mg / L. This characteristic makes Bacillus subtilis LXY-3 potentially valuable in treating environments with high Cr(VI) pollution. For example, it can be used for the bioremediation of Cr(VI) contaminated soil or water, reducing the concentration of Cr(VI) in the environment and mitigating pollution hazards through its growth and metabolic activities.

[0040] Example 3: Detection of nitrogen fixation capacity of Bacillus subtilis LXY-3

[0041] To prepare liquid nitrogen-free Ashby medium: Add 10g mannitol, 0.2g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 0.2g sodium chloride, 0.1g calcium sulfate, and 5g calcium carbonate to 1L of water, adjust the pH to 7, autoclave at 121℃ for 20min, and cool for later use.

[0042] Preparation of solid nitrogen-free Ashby medium: Add 15g of agar powder to 1L of liquid Ashby medium, adjust the pH to 7, autoclave at 121℃ for 20min, pour it into sterile petri dishes before it solidifies, and obtain solid culture medium plates after cooling.

[0043] Bacillus subtilis LXY-3 was streaked on nitrogen-free Assumption solid medium. After 3 days of culture, the solid culture plate near the bacteria changed from opaque white to transparent. The substances produced during the culture process dissolved the calcium carbonate solid in the medium, indicating that strain LXY-3 has nitrogen-fixing ability.

[0044] Example 4: The plant growth promoting effect of Bacillus subtilis LXY-3 under high concentration of Cr(VI) environment.

[0045] Bacillus subtilis LXY-3 was cultured in LB liquid medium at 30℃ and 150 rpm until the logarithmic growth phase. The culture was then transferred to sterile centrifuge tubes in a laminar flow hood and centrifuged at 8000 rpm for 5 min. The supernatant was discarded, and the culture was washed with sterile water and the concentration adjusted to 10. 8 CFU / ml. Take 100g of air-dried soil and inoculate the bacterial suspension into the soil at an inoculation rate of 10. 8 CFU / g dry soil; a control group (CK) was set up with no inoculation, and a substitute group was set up with an equal volume of sterile water added to the bacterial suspension. Each group had 5 replicates.

[0046] Rice seeds with uniform germination were selected and inoculated into planting pots. The temperature was 25℃, the light duration was 16 h / d, and the dark duration was 8 h / d. Two days after transplanting, the control group was irrigated with 10 mL of sterile water near the root zone; the test group was irrigated with 10 mL of 150 mg / L Cr(VI) solution near the root zone, with irrigation performed every 2 days. Five parallel groups were set up for each rice crop, with 6 rice plants per pot. After 7 days of growth, a second root irrigation was performed as described above. The rice was harvested after 20 days of Cr(VI) stress. Six rice plants were randomly selected from each group, and the aboveground fresh weight, aboveground dry weight, plant height, and root length were measured. The roots were washed in tap water to remove soil, then dried with filter paper, and the length of the stems and roots were measured, along with their fresh and dry weights. The washed and dried rice roots were blanched in a hot air oven at 105℃ for 30 minutes, and then dried at 80℃ for 72 hours. The dry weight was then measured, and the results are shown in Table 1.

[0047] Table 1. Determination of plant growth-promoting ability of strains

[0048]

[0049] As shown in Table 1, in the comparative experiment on rice seed germination rate and seedling growth and development, a control group (CK group) and an experimental group treated with strain LXY-3 (LXY-3 group) were set up. The experimental results showed that the germination rate of rice seeds in the CK group was 70%, while the germination rate of rice seeds in the experimental group treated with strain LXY-3 increased to 80%, which was significantly higher than that of the CK group by 14.3% (calculated as: (80%-70%) / 70%×100%≈14.3%). This data clearly reflects that strain LXY-3 has a positive promoting effect on rice seed germination and can effectively improve the germination ability of seeds.

[0050] In investigating the effects of strain LXY-3 on the growth and development of rice seedlings, the experimental data also showed significant differences. Compared with the control group (CK), the LXY-3 group showed substantial improvements in key growth indicators such as aboveground fresh weight, aboveground dry weight, plant height, and root length. Specifically, aboveground fresh weight increased by 150%, aboveground dry weight by 75%, plant height by 20.3%, and root length by 54.8%. These data fully demonstrate that strain LXY-3 not only promotes the growth of the aboveground parts of rice seedlings, increasing their fresh and dry weight, but also significantly promotes root development, resulting in a significant increase in root length. In summary, strain LXY-3 exhibits significant effects in improving plant germination rate, promoting the accumulation of plant fresh and dry weight, increasing plant height, and promoting root growth, demonstrating a very good growth-promoting effect on plants.

[0051] As can be seen from the above embodiments, the Bacillus subtilis LXY-3 provided by this invention possesses significant phytoremediation characteristics. This unique characteristic enables Bacillus subtilis LXY-3 to maintain effective growth activity and continue to exert its excellent phytoremediation function even when faced with an environment contaminated with Cr(VI) heavy metal at a concentration of 150 mg / L. This discovery is of great significance, as it not only expands the application scope of Bacillus subtilis LXY-3, demonstrating its enormous application potential in the field of phytoremediation and phytoremediation of heavy metal contaminated soil, but also provides new ideas and methods for addressing the negative impacts of heavy metal pollution on agricultural production.

[0052] Through in-depth research and rational regulation of the growth conditions of Bacillus subtilis LXY-3, including optimizing the culture medium formula and controlling key factors such as culture temperature, pH value, and aeration conditions, large-scale controlled production of this bacterium can be achieved. This controllable large-scale production capability lays a solid foundation for the industrial application of Bacillus subtilis LXY-3. Its application in the preparation of microbial fertilizers can not only improve the quality and efficacy of microbial fertilizers but also provide agricultural production with a green, environmentally friendly, and efficient bio-fertilizer option. This helps promote sustainable agricultural development, reduce the use of chemical fertilizers, and lower environmental pollution, resulting in significant economic, social, and ecological benefits.

[0053] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A Bacillus-like bacterium, characterized in that, The classification name of this type of Bacillus is: Paenibacillus sp. LXY-3. The depositary institution is: China General Microbiological Culture Collection Center (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; deposit date: October 18, 2024; deposit number: CGMCC No. 1.64949.

2. A microbial fertilizer, characterized in that, It includes the Bacillus subtilis as described in claim 1.

3. The application of the Bacillus subtilis as described in claim 1 in Cr(VI) contaminated areas.

4. The application of the Bacillus subtilis as described in claim 1 in promoting crop growth and improving crop yield and quality in Cr(VI) contaminated areas.

5. The application as described in claim 3 or 4, characterized in that, The Cr(VI) concentration in the Cr(VI) polluted areas is 0-200 mg / L.

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