MOF-based halophilic microbial agent as well as preparation method and application thereof

Abstract

The invention provides an MOF-based halophilic microbial agent as well as a preparation method and application thereof, and relates to the technical field of microorganisms. The preparation method of the MOF-based halophilic microbial agent comprises the following steps: mixing a bacteria solution of a halophilic strain with a metal organic framework (MOF) for reaction, and then coating the metal organic framework (MOF) on the surface of a microbial cell to form an MOF shell with the thickness of 0.01-6.0 mu m, so as to obtain the MOF-based halophilic microbial agent. According to the MOF-based halophilic flora microbial agent disclosed by the invention, through organic combination of the halophilic strains and the metal organic framework material, the influence of external stimulation on the activity of the halophilic strains can be reduced, and the activity of the halophilic strains is maintained under the condition of high salt stress, so that the damage of salt stress to plants is reduced.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a MOF-based halophilic microbial agent and a preparation method and application thereof. Background Art

[0002] At present, the area of ​​secondary soil salinization caused by improper irrigation and unreasonable reclamation is constantly expanding, and the problem of salt stress is becoming more and more prominent. Excessive salt in the soil will inhibit the growth of crops, reduce crop yields, and cause crop yield reduction. Salt stress has become an important issue facing global agricultural development. The plant root system is not only involved in the absorption of water and nutrients by plants, but also the main place for plants to interact with soil microorganisms. Studies have shown that salt stress hinders the absorption of water and inorganic salts by plants, resulting in a significant reduction in plant root length, root mass and root surface area. The growth and development of plant roots is hindered, resulting in slow plant development or even death. In recent years, the effects of salt stress on the composition of plant root microbial communities and changes in their interactions with plant roots have attracted much attention.

[0003] In response to salt stress, the main methods currently include establishing complete farmland irrigation and water conservancy facilities, applying chemical amendments to reduce salt accumulation, and planting salt-tolerant plants to improve the utilization rate of saline soil. Soil microorganisms play an important role in the soil ecological environment, especially microorganisms in the rhizosphere of plants are crucial to the restoration of saline-alkali land. Under salt stress, rhizosphere growth-promoting bacteria can reduce the stress damage to plants by inducing systemic tolerance. Improving salt stress through ecological and biological measures can not only promote the reduction of soil salt content and increase crop yields, but also be low-cost and green and environmentally friendly, and is the preferred technical measure for farmers. Microbial organic fertilizers have the advantages of low input, high output, high efficiency, high quality, sufficient raw materials, simple production process, and easy promotion. In agricultural production, increasing the utilization rate of chemical fertilizers, improving crop quality, improving soil structure, increasing crop resistance and disease resistance, and increasing crop yields are the inevitable trends in agricultural production and fertilizer development. The development of microbial fertilizers is very consistent with the requirements of modern ecological agriculture and sustainable agricultural development.

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

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

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0008] S1. inoculating the halophilic strains into culture medium respectively for cultivation, and then mixing them 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°C for 10-15h to obtain Zr6O4(OH)4(TCPP-H2)3.

[0010] S3. Mix Zr6O4(OH)4(TCPP-H2)3 with the bacterial solution, react at 23-27°C for 110-130 minutes, and obtain a MOF-based halophilic bacterial microbial agent.

[0011] Preferably, the halophilic strains include Bacillus subtilis, Brevibacillus laterosporus and Paenibacillus polymyxa.

[0012] Preferably, the volume ratio of the Bacillus subtilis solution, the Brevibacillus laterosporus solution and the Paenibacillus polymyxa solution is 0.8-1.2:0.8-1.2:1.

[0013] Preferably, the ratio of ZrCl4·8H2O to DMF is 1.5mmoL:12-17mL.

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

[0015] Preferably, the spore concentrations in the Bacillus subtilis solution, the Bacillus laterosporus solution and the Bacillus polymyxa solution are 1 to 9×10 8 CFU / mL.

[0016] Preferably, the mass 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 a MOF-based halophilic bacteria microbial agent prepared by the preparation method.

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

[0019] The present invention also provides the use of the MOF-based halophilic bacteria microbial agent 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 saline-alkali stress in plants;

[0022] (3) Application in the preparation of promoters for promoting plant growth 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 laterosporus, and Bacillus mucilaginosus, which can improve the tolerance of plants to oxidative stress in high-salt environments and alkaline pH environments, effectively reduce the damage caused by salt-alkali stress to plants, enhance the water permeability, water retention and air permeability of the soil, promote the rapid growth of the root system, and increase the yield of crops in saline-alkali land.

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

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

[0027] S1. inoculating the halophilic strains into culture medium respectively for cultivation, and then mixing them 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°C for 10-15h to obtain Zr6O4(OH)4(TCPP-H2)3.

[0029] S3. Mix Zr6O4(OH)4(TCPP-H2)3 with the mixed bacterial solution, react at 23-27°C for 110-130 minutes, and obtain a MOF-based halophilic bacterial microbial agent.

[0030] The present invention first inoculates the halophilic strains into a culture medium for culture, and then mixes them to obtain a mixed bacterial solution; the halophilic strains include Bacillus subtilis, Brevibacillus laterosporus and Paenibacillus polymyxa, and the culture medium is preferably LB culture medium; the culture temperature is 35-39°C, more preferably 36-38°C, and more preferably 37°C; the culture time is 22-26h, more preferably 23-25h, and more preferably 24h; the culture is preferably an oscillating culture, and the oscillating speed is 120-180rpm / min, more preferably 140-160rpm / min, and more preferably 150rpm / min. After the culture is completed, the spore concentrations in the Bacillus subtilis culture, the Brevibacillus laterosporus culture, and the Paenibacillus polymyxa solution are 1-9×10 8 CFU / mL, more preferably 3 to 8×10 8 CFU / mL, more preferably 5×10 8 Then, the Bacillus subtilis solution, the Brevibacillus laterosporus solution and the Paenibacillus polymyxa solution are mixed, wherein the volume ratio of the Bacillus subtilis solution, the Brevibacillus laterosporus solution and the Paenibacillus polymyxa solution is 0.8-1.2:0.8-1.2:1, and the asynchronous ratio is preferably 0.9-1.1:0.9-1.1:1, and more preferably 1:1:1.

[0031] The present invention dissolves ZrCl4·8H2O in DMF, the ratio of ZrCl4·8H2O to DMF is 1.5mmoL:12-17mL, more preferably 1.5mmoL:14-16mL, more preferably 1.5mmoL:mL; then H4-TCPP-H2 and water are added, the molar ratio of ZrCl4·8H2O to H4-TCPP-H2 is 0.8-1.2:1, more preferably 0.9-1.1:1, more preferably 1:1; the ratio of H4-TCPP-H2 to water is 1.5mmoL : 2.5~3.5mL, more preferably 1.5mmoL:2.7~3.2mL, more preferably 1.5mmoL:2.5mL; then react, the reaction temperature is 115~125℃, more preferably 117~122℃, more preferably 120℃; the reaction time is 10~15h, more preferably 11~14h, more preferably 12h, the reaction process is accompanied by stirring, the stirring speed is 300~700rpm, more preferably 400~600rpm, more preferably 500rpm. After the reaction is completed, cool to room temperature, filter and collect the precipitate, wash the precipitate with DMF and water 2 to 3 times (first wash once with DMF, then wash twice with distilled water), and then vacuum dry. The vacuum drying temperature is 58 to 63°C, more preferably 59 to 62°C, and more preferably 60°C; the vacuum drying time is 11 to 13h, more preferably 12h, to obtain the metal organic framework material Zr6O4(OH)4(TCPP-H2)3.

[0032] The present invention mixes the metal organic framework material Zr6O4(OH)4(TCPP-H2)3 with the mixed bacterial solution for reaction, wherein the mass volume ratio of the metal organic framework material 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 more preferably 0.05 mg:1 mL. The reaction temperature is 23-27°C, more preferably 24-26°C, and more preferably 25°C; the reaction time is 110-130 min, more preferably 115-125 min, and more preferably 120 min. After the reaction is completed, the MOF shell is coated on the surface of Bacillus subtilis, Bacillus laterosporus, and Bacillus polymyxa, which is a MOF-based halophilic bacterial microbial agent. The thickness of the MOF shell is 0.01-6.0 μm, more preferably 1.0-5.0 μm, and more preferably 3.0 μm.

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

[0034] The present invention also provides a microbial fertilizer, which is fermented by the MOF-based halophilic bacteria microbial agent. During fermentation, the inoculation amount of the MOF-based halophilic bacteria microbial agent is 6 to 10 mL / kg substrate, more preferably 7 to 9 mL / kg substrate, and more preferably 8 mL / kg substrate; the number of spores of Bacillus subtilis, Brevibacillus laterosporus or Paenibacillus polymyxa in the MOF-based halophilic bacteria microbial agent is independently 1 to 9×10 8 CFU / g, more preferably 3 to 7×10 8 CFU / g, more preferably 5×10 8 CFU / g; the substrate is a conventional raw material for preparing organic fertilizer, such as agricultural waste, animal feces, etc. The present invention has no special limitation on the fermentation, and the fertilizer fermentation can be carried out according to conventional methods in the art.

[0035] The present invention also provides the use of the MOF-based halophilic bacteria microbial agent 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 saline-alkali stress in plants;

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

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

[0040] The salt-tolerant strains of Bacillus subtilis, Brevibacillus laterosporus and Paenibacillus polymyxa are purchased from the China General Microbiological Culture Collection, and the collection numbers are 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 liquid culture: Bacillus subtilis, Brevibacillus laterosporus, and Paenibacillus polymyxa were added to the pre-sterilized LB medium, respectively, and cultured at 35°C and 120 rpm for 23 h. The bacterial cell concentrations of Bacillus subtilis, Brevibacillus laterosporus, and Paenibacillus polymyxa at the end of the culture were 1×10 8 CFU / mL, and then the Bacillus subtilis bacterial solution, the Brevibacillus laterosporus bacterial solution, and the Paenibacillus polymyxa bacterial solution were mixed in 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 heated to 115°C with stirring at 300 rpm and maintained 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 in vacuum at 58°C for 13 h.

[0044] (3) MOF hybridization: Zr6O4(OH)4(TCPP-H2)3 was mixed with the mixed bacterial solution at a ratio of 0.01 mg:1 mL, and reacted at 23 °C for 130 min to obtain a MOF-based halophilic bacterial microbial agent.

[0045] (4) Preparation of microbial fertilizer: MOF-based halophilic bacteria microbial agent and fermentation matrix are mixed at a ratio of 6 mL / kg matrix, wherein the fermentation matrix is ​​a mixture of cow dung, pig dung and straw. After mixing evenly, composting and fermentation are carried out. 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 liquid culture: Bacillus subtilis, Brevibacillus laterosporus, and Paenibacillus polymyxa were added to a pre-sterilized LB medium and cultured at 37°C with shaking at 150 rpm for 24 h. The bacterial cell concentrations of Bacillus subtilis, Brevibacillus laterosporus, and Paenibacillus polymyxa at the end of the culture were 5×10 8 CFU / mL, and then the Bacillus subtilis bacterial solution, the Brevibacillus laterosporus bacterial solution, and the Paenibacillus polymyxa bacterial solution were mixed 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 heated to 120°C with stirring at 500 rpm and maintained for 12 h. The reaction mixture was completely cooled to room temperature, filtered, washed with DMF and water, and dried in vacuum at 60°C for 12 h.

[0049] (3) MOF hybridization: Zr6O4(OH)4(TCPP-H2)3 was mixed with the mixed bacterial solution at a ratio of 0.05 mg:1 mL, and reacted at 25 °C for 120 min to obtain a MOF-based halophilic bacterial microbial agent.

[0050] (4) Preparation of microbial fertilizer: MOF-based halophilic bacteria microbial agent and fermentation matrix are mixed at a ratio of 8 mL / kg matrix, wherein the fermentation matrix is ​​a mixture of cow dung, pig dung and straw. After mixing evenly, composting and fermentation are carried out. 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 liquid culture: Bacillus subtilis, Brevibacillus laterosporus, and Paenibacillus polymyxa were added to a pre-sterilized LB medium and cultured at 39°C with shaking at 180 rpm for 25 h. The bacterial cell concentrations of Bacillus subtilis, Brevibacillus laterosporus, and Paenibacillus polymyxa at the end of the culture were 9×10 8 CFU / mL, and then the Bacillus subtilis bacterial solution, the Brevibacillus laterosporus bacterial solution, and the Paenibacillus polymyxa bacterial solution were mixed in 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 heated to 125°C with stirring at 700 rpm and maintained 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 in vacuum at 63°C for 11 h.

[0054] (3) MOF hybridization: Zr6O4(OH)4(TCPP-H2)3 was mixed with the mixed bacterial solution at a ratio of 0.1 mg:1 mL, and reacted at 27 °C for 110 min to obtain a MOF-based halophilic bacterial microbial agent.

[0055] (4) Preparation of microbial fertilizer: MOF-based halophilic bacteria microbial agent and fermentation matrix are mixed at a ratio of 10 mL / kg matrix, wherein the fermentation matrix is ​​a mixture of cow dung, pig dung and straw. After mixing evenly, composting and fermentation are carried out. 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] Different from Example 1, the microbial fertilizer of Comparative Example 1 is prepared by mixing the mixed bacterial solution of step (1) with the fermentation matrix for composting.

[0058] Comparative Example 2

[0059] Different from Example 2, the microbial fertilizer of Comparative Example 2 is prepared by mixing the mixed bacterial solution of step (1) with the fermentation matrix for composting.

[0060] Comparative Example 3

[0061] Different from Example 3, the microbial fertilizer of Comparative Example 3 is prepared by mixing the mixed bacterial solution of step (1) with the fermentation matrix for composting.

[0062] Experimental example

[0063] Application in corn planting in saline-alkali land:

[0064] The experiment was arranged in the experimental farmland of Guangxi University, with an area of ​​2 mu. The corn variety selected was "Xinyu No. 62". The soil salt content of the farmland was 1.6%, the pH was 8.5, the soil salt type was sulfate-chloride type, the soil organic matter was 11.6g / kg, the alkaline nitrogen was 74mg / kg, the available phosphorus was 13mg / kg, the available potassium was 178mg / kg, and the soil fertility was medium.

[0065] The experiment was divided into 6 groups. Before corn sowing, the microbial fertilizers of experimental groups 1 to 3 and control groups 1 to 3 were applied 30-40 cm deep, the fertilizers of embodiments 1 to 3 were applied to experimental groups 1 to 3, and the fertilizers of comparative examples 1 to 3 were applied to control groups 1 to 3. The fertilizer application rate was 35 kg / mu. The experiment was repeated 3 times. The test results are shown in Table 1.

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

[0067]

[0068]

[0069] The test results show that during the use of the experimental group microbial fertilizer provided above, the saline-alkali soil no longer has the problem of surface salt return during the growth period, and the overall germination rate and survival rate of the experimental group are higher than those of the control group. This shows that the MOF-based halophilic bacteria microbial agent of the present invention can maintain the activity of the strain under high salt stress conditions, reduce the influence of external stimuli on the activity of the strain, and thus reduce the damage caused by salt stress to plants.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a MOF-based halophilic microbial agent, characterized in that: The following steps are involved: S1. inoculating the halophilic strains into culture medium respectively for cultivation, and then mixing them to obtain a mixed bacterial solution; S2. Mix ZrCl4·8H2O with DMF, then add H4-TCPP-H2 and water, and react at 115-125°C for 10-15h to obtain Zr6O4(OH)4(TCPP-H2)3. S3. Mix Zr6O4(OH)4(TCPP-H2)3 with the mixed bacterial solution, react at 23-27°C for 110-130 minutes, and obtain a MOF-based halophilic bacterial microbial agent.

2. The preparation method according to claim 1, characterized in that: The halophilic strains include Bacillus subtilis, Brevibacillus laterosporus and Paenibacillus polymyxa.

3. The preparation method according to claim 2, characterized in that: The volume ratio of the Bacillus subtilis solution, the Brevibacillus laterosporus solution and the Paenibacillus polymyxa solution is 0.8-1.2:0.8-1.2:

1.

4. The preparation method according to claim 1, characterized in that: The ratio of ZrCl4·8H2O to DMF is 1.5mmoL:12~17mL.

5. The preparation method according to claim 4, characterized in that: The molar ratio of ZrCl4·8H2O to H4-TCPP-H2 is 0.8-1.2:1; the ratio of H4-TCPP-H2 to water is 1.5mmoL:2.5-3.5mL.

6. The preparation method according to claim 5, characterized in that: The spore concentrations in the Bacillus subtilis solution, the Brevibacillus laterosporus solution and the Paenibacillus polymyxa solution are 1 to 9×10 8 CFU / mL.

7. The preparation method according to claim 6, characterized in that: The mass volume ratio of Zr6O4(OH)4(TCPP-H2)3 to the mixed bacterial solution is 0.01-0.1 mg:1 mL.

8. A MOF-based halophilic bacteria microbial agent prepared by any one of the preparation methods of claims 1 to 7.

9. A microbial fertilizer, characterized in that: It is fermented by the MOF-based halophilic bacteria microbial agent.

10. Use of the MOF-based halophilic bacteria microbial agent according to claim 8 and / or the microbial fertilizer according to claim 9 in all or part of the following (1) to (3): (1) Application in improving saline-alkali land; (2) Application in alleviating saline-alkali stress in plants; (3) Application in the preparation of promoters for promoting plant growth under salt stress.

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