A kind of MOF-based halophilic microbial inoculant and its preparation method and application

Abstract

This invention provides a MOF-based halophilic microbial inoculant, its preparation method, and its application, relating to the field of microbial technology. The preparation method of the MOF-based halophilic microbial inoculant of this invention includes the following steps: mixing a halophilic bacterial suspension with a metal-organic framework (MOF) for reaction; then coating the surface of the microbial cell with the MOF to form a MOF shell with a thickness of 0.01–6.0 μm, thereby obtaining the MOF-based halophilic microbial inoculant. The MOF-based halophilic microbial inoculant of this invention, through the organic combination between the halophilic bacterial strain and the metal-organic framework, can reduce the impact of external stimuli on the activity of the halophilic strain, maintain the activity of the halophilic strain under high salt stress conditions, and thus alleviate the damage caused by salt stress to plants.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a MOF-based halophilic microbial agent, its preparation method, and its application. Background Technology

[0002] Currently, secondary soil salinization caused by improper irrigation and irrational land reclamation is expanding, and salt stress is becoming increasingly prominent. Excessive salt in the soil inhibits crop growth, reduces crop yield, and leads to crop losses. Salt stress has become a significant problem facing global agricultural development. Plant roots not only participate in the absorption of water and nutrients but are also the main site of interaction between plants and soil microorganisms. Studies have shown that salt stress hinders the absorption of water and inorganic salts by plants, significantly reducing root length, root mass, and root surface area. This stunted root growth and development leads to slow plant development and even death. In recent years, the impact of salt stress on the composition of plant root microbial communities and changes in their interactions with plant roots have received considerable attention.

[0003] Current methods for addressing salt stress include establishing comprehensive farmland irrigation facilities, applying chemical amendments to reduce salt accumulation, and planting salt-tolerant plants to improve saline soil utilization. Soil microorganisms play a crucial role in the soil ecological environment, especially those in the rhizosphere, which are essential for the remediation of saline-alkali land. Under salt stress, rhizosphere growth-promoting bacteria reduce stress damage to plants by inducing systemic tolerance. Improving salt stress through ecological and biological measures not only promotes lower soil salinity and increases crop yields but is also low-cost and environmentally friendly, making it a preferred technical measure for farmers. Microbial organic fertilizers offer advantages such as low input, high output, high efficiency, high quality, abundant raw materials, simple production processes, and ease of promotion. In agricultural production, they increase fertilizer utilization, improve crop quality, improve soil structure, enhance crop resistance and disease resistance, and increase crop yields. This is an inevitable trend in agricultural production and fertilizer development, and the development of microbial fertilizers perfectly aligns with the requirements of modern ecological agriculture and sustainable agricultural development.

[0004] Although there are salt-tolerant microbial strains that can be used to alleviate salt-alkali stress, these strains have low activity in severely saline-alkali soils and cannot effectively reduce the damage caused by salt stress to plants. Summary of the Invention

[0005] The purpose of this invention is to provide a MOF-based halophilic microbial inoculant, its preparation method, and its application, which solves the problem that halophilic microbial strains have low activity in severely saline-alkali soils and cannot effectively reduce the damage to plants caused by salt stress.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing MOF-based halophilic microbial inoculants, comprising the following steps:

[0008] S1. The halophilic strains were inoculated separately into the culture medium and cultured, then mixed to obtain a mixed bacterial solution;

[0009] S2. Mix ZrCl4·8H2O with DMF, then add H4-TCPP-H2 and water, and react at 115-125℃ for 10-15 h to obtain Zr6O4(OH)4(TCPP-H2)3.

[0010] S3. Mix Zr6O4(OH)4(TCPP-H2)3 with the bacterial solution and react at 23-27℃ for 110-130 min to obtain MOF-based halophilic microbial inoculum.

[0011] Preferably, the halophilic strains include Bacillus subtilis, Bacillus lateralis, and Bacillus polymyxa.

[0012] Preferably, the volume ratio of Bacillus subtilis culture, Bacillus retroflexus culture, and Bacillus polymyxa culture is 0.8–1.2:0.8–1.2:1.

[0013] Preferably, the ratio of ZrCl4·8H2O to DMF is 1.5 mmol / L: 12–17 mL.

[0014] Preferably, the molar ratio of ZrCl4·8H2O to H4-TCPP-H2 is 0.8–1.2:1; and the ratio of H4-TCPP-H2 to water is 1.5 mmol: 2.5–3.5 mL.

[0015] Preferably, the spore concentrations in the Bacillus subtilis culture, Bacillus lateralis culture, and Bacillus polymyxa culture are 1–9 × 10⁻⁶. 8 CFU / mL.

[0016] Preferably, the mass-to-volume ratio of Zr6O4(OH)4(TCPP-H2)3 to the mixed bacterial solution is 0.01–0.1 mg: 1 mL.

[0017] The present invention also provides an MOF-based halophilic microbial agent prepared by the aforementioned preparation method.

[0018] The present invention also provides a microbial fertilizer fermented from the MOF-based halophilic microbial agent.

[0019] The present invention also provides the application of the MOF-based halophilic microbial inoculant and / or the microbial fertilizer in all or part of the following (1) to (3):

[0020] (1) Application in improving saline-alkali land;

[0021] (2) Application in alleviating salt and alkali stress in plants;

[0022] (3) Application in the preparation of growth promoters for plants under salt stress.

[0023] By adopting the above technical solution, the present invention has the following beneficial effects:

[0024] (1) The halophilic bacteria provided by the present invention include Bacillus subtilis, Bacillus lateralis, and Bacillus mucilaginosus, which can improve the tolerance of plants to oxidative stress in high-salt and alkaline pH environments, effectively reduce the damage caused to plants by salt and alkali stress, enhance the soil's permeability, water retention and aeration, promote the rapid growth of roots, and increase the yield of crops in saline-alkali land.

[0025] (2) The preparation method of the MOF-based halophilic microbial agent of the present invention includes the following steps: mixing bacterial solution with metal-organic framework (MOF) material for reaction, coating the cell surface with MOF material to form a MOF shell with a thickness of 0.01-6.0 μm, and obtaining the MOF-based halophilic microbial agent. The MOF-based halophilic microbial agent of the present invention, through the organic combination between halophilic strains and metal-organic framework material, can reduce the influence of external stimuli on the activity of halophilic strains, maintain the activity of halophilic strains under high salt stress conditions, and thus alleviate the damage caused by salt stress to plants. Detailed Implementation

[0026] This invention provides a method for preparing MOF-based halophilic microbial inoculants, comprising the following steps:

[0027] S1. The halophilic strains were inoculated separately into the culture medium and cultured, then mixed to obtain a mixed bacterial solution;

[0028] S2. Mix ZrCl4·8H2O with DMF, then add H4-TCPP-H2 and water, and react at 115-125℃ for 10-15 h to obtain Zr6O4(OH)4(TCPP-H2)3.

[0029] S3. Mix Zr6O4(OH)4(TCPP-H2)3 with the mixed bacterial solution and react at 23-27℃ for 110-130 min to obtain MOF-based halophilic microbial inoculum.

[0030] This invention first inoculates halophilic bacterial strains separately into a culture medium for cultivation, then mixes them to obtain a mixed bacterial solution. The halophilic strains include *Bacillus subtilis*, *Bacillus laterosporus*, and *Bacillus polymyxa*. The culture medium is preferably LB medium. The cultivation temperature is 35–39°C, more preferably 36–38°C, and even more preferably 37°C. The cultivation time is 22–26 h, more preferably 23–25 h, and even more preferably 24 h. The cultivation is preferably a shaking culture, with a shaking speed of 120–180 rpm / min, more preferably 140–160 rpm / min, and even more preferably 150 rpm / min. After cultivation, the spore concentrations in the *Bacillus subtilis*, *Bacillus laterosporus*, and *Bacillus polymyxa* bacterial solutions are 1–9 × 10⁻⁶. 8 CFU / mL, more preferably 3–8 × 10⁻⁶ 8 CFU / mL, more preferably 5×10⁻⁶ 8 CFU / mL. Then, the Bacillus subtilis, Bacillus brevis, and Bacillus polymyxa bacterial suspensions are mixed, with the volume ratio of the Bacillus subtilis, Bacillus brevis, and Bacillus polymyxa bacterial suspensions being 0.8–1.2:0.8–1.2:1, preferably 0.9–1.1:0.9–1.1:1, and more preferably 1:1:1.

[0031] In this invention, ZrCl4·8H2O is dissolved in DMF, with a ZrCl4·8H2O to DMF ratio of 1.5 mmol:12-17 mL, more preferably 1.5 mmol:14-16 mL, and even more preferably 1.5 mmol:mL; then H4-TCPP-H2 and water are added, with a ZrCl4·8H2O to H4-TCPP-H2 molar ratio of 0.8-1.2:1, more preferably 0.9-1.1:1, and even more preferably 1:1; the ratio of H4-TCPP-H2 to water is 1.5 mmol. The reaction mixture consists of 2.5–3.5 mL, more preferably 1.5 mmol / L: 2.7–3.2 mL, and more preferably 1.5 mmol / L: 2.5 mL; the reaction is then carried out at a temperature of 115–125 °C, more preferably 117–122 °C, and more preferably 120 °C; the reaction time is 10–15 h, more preferably 11–14 h, and more preferably 12 h, with stirring during the reaction process at a speed of 300–700 rpm, more preferably 400–600 rpm, and more preferably 500 rpm. After the reaction is complete, the mixture is cooled to room temperature, filtered to obtain the precipitate, and washed 2-3 times with DMF and water (washed once with DMF and then twice with distilled water). The precipitate is then vacuum dried at a temperature of 58-63°C, more preferably 59-62°C, and even more preferably 60°C. The vacuum drying time is 11-13 hours, more preferably 12 hours, to obtain the metal-organic framework material Zr6O4(OH)4(TCPP-H2)3.

[0032] This invention involves reacting a metal-organic framework (MOF) material Zr6O4(OH)4(TCPP-H2)3 with a mixed bacterial solution. The mass-to-volume ratio of Zr6O4(OH)4(TCPP-H2)3 to the bacterial solution is 0.01–0.1 mg:1 mL, more preferably 0.03–0.07 mg:1 mL, and even more preferably 0.05 mg:1 mL. The reaction temperature is 23–27°C, more preferably 24–26°C, and even more preferably 25°C. The reaction time is 110–130 min, more preferably 115–125 min, and even more preferably 120 min. After the reaction is complete, the MOF shell coats the surfaces of Bacillus subtilis, Bacillus laterosporus, and Bacillus polymyxa, thus forming a MOF-based halophilic microbial agent. The thickness of the MOF shell is 0.01–6.0 μm, more preferably 1.0–5.0 μm, and even more preferably 3.0 μm.

[0033] The present invention also provides an MOF-based halophilic microbial agent prepared by the aforementioned preparation method.

[0034] This invention also provides a microbial fertilizer, which is fermented from the MOF-based halophilic microbial inoculant. During fermentation, the inoculant amount is 6–10 mL / kg substrate, more preferably 7–9 mL / kg substrate, and even more preferably 8 mL / kg substrate; the number of spores of Bacillus subtilis, Bacillus laterosporus, or Bacillus polymyxa in the MOF-based halophilic microbial inoculant is independently 1–9 × 10⁻⁶. 8 CFU / g, further preferably 3 to 7 × 10⁻⁶ 8 CFU / g, more preferably 5×10 8 CFU / g; the substrate is a raw material commonly used in the preparation of organic fertilizers, such as agricultural waste, animal manure, etc. This invention does not impose any special limitations on the fermentation process; fertilizer fermentation can be carried out according to conventional methods in the art.

[0035] The present invention also provides the application of the MOF-based halophilic microbial inoculant and / or the microbial fertilizer in all or part of the following (1) to (3):

[0036] (1) Application in improving saline-alkali land;

[0037] (2) Application in alleviating salt and alkali stress in plants;

[0038] (3) Application in the preparation of growth promoters for plants under salt stress.

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] The salt-tolerant strains of Bacillus subtilis, Bacillus lateralis, and Bacillus polymyxa of the present invention were purchased from the China General Microbiological Culture Collection Center, with accession numbers CGMCC NO.17213, CGMCC No.21217, and CGMCC NO.14481, respectively. Other reagents can be purchased through conventional commercial channels unless otherwise specified.

[0041] Example 1

[0042] (1) Bacterial culture: Bacillus subtilis, Bacillus laterosporus, and Bacillus polymyxa were added to pre-sterilized LB medium and cultured at 35°C with shaking at 120 rpm for 23 h. The cell concentrations of Bacillus subtilis, Bacillus laterosporus, and Bacillus polymyxa at the end of the culture were 1×10⁻⁶. 8 The concentration of CFU / mL was then increased, and the bacterial solutions of Bacillus subtilis, Bacillus lateralis, and Bacillus polymyxa were mixed at a volume ratio of 0.8:0.8:1 to obtain a mixed bacterial solution.

[0043] (2) Synthesis of Zr6O4(OH)4(TCPP-H2)3: 1.5 mmol of ZrCl4·8H2O was dissolved in 15 mL of DMF, and then 1.5 mmol of H4-TCPP-H2 and 3 mL of water were added. The mixture was stirred and heated to 115 °C at 300 rpm and kept for 15 h. After the reaction was completely cooled to room temperature, the mixture was filtered and washed with DMF and water, and then dried under vacuum at 58 °C for 13 h.

[0044] (3) MOF hybridization: Zr6O4(OH)4(TCPP-H2)3 and mixed bacterial solution were mixed at a ratio of 0.01mg:1mL and reacted at 23℃ for 130min to obtain MOF-based halophilic microbial inoculum.

[0045] (4) Preparation of microbial fertilizer: MOF-based halophilic microbial agent is mixed with fermentation substrate at a ratio of 6 mL / kg substrate. The fermentation substrate is a mixture of cow manure, pig manure and straw. After mixing evenly, it is composted and fermented. The first fermentation lasts for 7 days and the second fermentation lasts for 10 days. After the fermentation is completed, microbial fertilizer is obtained.

[0046] Example 2

[0047] (1) Bacterial culture: Bacillus subtilis, Bacillus laterosporus, and Bacillus polymyxa were added to pre-sterilized LB medium and cultured at 37°C with shaking at 150 rpm for 24 h. The cell concentrations of Bacillus subtilis, Bacillus laterosporus, and Bacillus polymyxa at the end of the culture were 5 × 10⁻⁶. 8 CFU / mL, then mix Bacillus subtilis, Bacillus lateralis, and Bacillus polymyxa in a volume ratio of 1:1:1 to obtain a mixed bacterial solution.

[0048] (2) Synthesis of Zr6O4(OH)4(TCPP-H2)3: 1.5 mmol of ZrCl4·8H2O was dissolved in 15 mL of DMF, and then 1.5 mmol of H4-TCPP-H2 and 3 mL of water were added. The mixture was stirred and heated to 120 °C at 500 rpm and kept for 12 h. After the reaction was completely cooled to room temperature, the mixture was filtered and washed with DMF and water, and then dried under vacuum at 60 °C for 12 h.

[0049] (3) MOF hybridization: Zr6O4(OH)4(TCPP-H2)3 and mixed bacterial solution were mixed at a ratio of 0.05mg:1mL and reacted at 25℃ for 120min to obtain MOF-based halophilic microbial inoculum.

[0050] (4) Preparation of microbial fertilizer: MOF-based halophilic microbial agent is mixed with fermentation substrate at a ratio of 8 mL / kg substrate. The fermentation substrate is a mixture of cow manure, pig manure and straw. After mixing evenly, it is composted and fermented. The first fermentation lasts for 7 days and the second fermentation lasts for 10 days. After the fermentation is completed, microbial fertilizer is obtained.

[0051] Example 3

[0052] (1) Bacterial culture: Bacillus subtilis, Bacillus laterosporus, and Bacillus polymyxa were added to pre-sterilized LB medium and cultured at 39°C with shaking at 180 rpm for 25 h. The cell concentrations of Bacillus subtilis, Bacillus laterosporus, and Bacillus polymyxa at the end of the culture were 9 × 10⁻⁶. 8 The concentration of CFU / mL was then increased, and the bacterial solutions of Bacillus subtilis, Bacillus lateralis, and Bacillus polymyxa were mixed at a volume ratio of 1.2:1.2:1 to obtain a mixed bacterial solution.

[0053] (2) Synthesis of Zr6O4(OH)4(TCPP-H2)3: 1.5 mmol of ZrCl4·8H2O was dissolved in 15 mL of DMF, and then 1.5 mmol of H4-TCPP-H2 and 3 mL of water were added. The mixture was stirred and heated to 125 °C at 700 rpm and kept for 10 h. After the reaction was completely cooled to room temperature, the mixture was filtered and washed with DMF and water, and then dried under vacuum at 63 °C for 11 h.

[0054] (3) MOF hybridization: Zr6O4(OH)4(TCPP-H2)3 and mixed bacterial solution were mixed at a ratio of 0.1mg:1mL and reacted at 27℃ for 110min to obtain MOF-based halophilic microbial inoculum.

[0055] (4) Preparation of microbial fertilizer: MOF-based halophilic microbial agent is mixed with fermentation substrate at a ratio of 10 mL / kg substrate. The fermentation substrate is a mixture of cow manure, pig manure and straw. After mixing evenly, it is composted and fermented. The first fermentation lasts for 7 days and the second fermentation lasts for 10 days. After the fermentation is completed, microbial fertilizer is obtained.

[0056] Comparative Example 1

[0057] Unlike Example 1, the microbial fertilizer in Comparative Example 1 was prepared by composting the mixed bacterial solution from step (1) with the fermentation substrate.

[0058] Comparative Example 2

[0059] Unlike Example 2, the microbial fertilizer in Comparative Example 2 was prepared by composting the mixed bacterial solution from step (1) with the fermentation substrate.

[0060] Comparative Example 3

[0061] Unlike Example 3, the microbial fertilizer in Comparative Example 3 was prepared by composting the mixed bacterial solution from step (1) with the fermentation substrate.

[0062] Experimental Example

[0063] Application in maize cultivation on saline-alkali land:

[0064] The experiment was conducted in the experimental farmland of Guangxi University, covering an area of ​​2 mu (approximately 0.33 hectares). The maize variety used was "Xinyu 62". The soil salinity of the farmland was 1.6%, the pH was 8.5, the soil salinity type was sulfate-chloride, the soil organic matter was 11.6 g / kg, available nitrogen was 74 mg / kg, available phosphorus was 13 mg / kg, available potassium was 178 mg / kg, and the soil fertility was moderate.

[0065] The experiment was divided into 6 groups. Before corn sowing, the microbial fertilizer of experimental groups 1-3 and control groups 1-3 was applied at a depth of 30-40cm. Experimental groups 1-3 were applied with the fertilizer of Examples 1-3, and control groups 1-3 were applied with the fertilizer of Comparative Examples 1-3. The fertilizer application rate was 35kg / mu. The experiment was repeated 3 times. The experimental results are shown in Table 1.

[0066] Table 1. Effects of different fertilization treatments on various indicators of maize.

[0067]

[0068]

[0069] The experimental results show that during the use of the microbial fertilizer provided above, the saline-alkali soil no longer experienced surface salt return during the growing season, and the overall germination rate and survival rate of the experimental group were higher than those of the control group. This indicates that the MOF-based halophilic microbial agent of the present invention can maintain the activity of the strain under high salt stress conditions, reduce the impact of external stimuli on the activity of the strain, and thus alleviate the damage caused by salt stress to plants.

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a MOF-based halophilic microbial inoculant, characterized in that, Includes the following steps: S1. The halophilic strains were separately inoculated into a culture medium and cultured, then mixed to obtain a mixed bacterial solution. The halophilic strains included Bacillus subtilis, Bacillus laterosporus, and Bacillus polymyxa. The volume ratio of the Bacillus subtilis, Bacillus laterosporus, and Bacillus polymyxa bacterial solutions was 0.8–1.2:0.8–1.2:

1. The spore concentrations in the Bacillus subtilis, Bacillus laterosporus, and Bacillus polymyxa bacterial solutions were 1–9 × 10⁻⁶. 8 CFU / mL; S2. Mix ZrCl4·8H2O with DMF, then add H4-TCPP-H2 and water, and react at 115-125℃ for 10-15 h to obtain Zr6O4(OH)4(TCPP-H2)3. The ratio of ZrCl4·8H2O to DMF is 1.5 mmol: 12-17 mL, the molar ratio of ZrCl4·8H2O to H4-TCPP-H2 is 0.8-1.2:1, and the ratio of H4-TCPP-H2 to water is 1.5 mmol: 2.5-3.5 mL. S3. Mix Zr6O4(OH)4(TCPP-H2)3 with the mixed bacterial solution, wherein the mass-to-volume ratio of Zr6O4(OH)4(TCPP-H2)3 to the mixed bacterial solution is 0.01-0.1 mg: 1 mL, and react at 23-27°C for 110-130 min to obtain MOF-based halophilic microbial inoculant.

2. The MOF-based halophilic microbial inoculant prepared by the method of claim 1.

3. A microbial fertilizer, characterized in that, It is produced by fermentation of the MOF-based halophilic microbial agent as described in claim 2.

4. The use of the MOF-based halophilic microbial agent of claim 2 and / or the microbial fertilizer of claim 3 in all or part of the following (1) to (3): (1) Application in improving saline-alkali land; (2) Application in alleviating salt and alkali stress in plants; (3) Application in the preparation of growth promoters for plants under salt stress.

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