Salt-tolerant wormcast-based dual-network microbial sustained-release gel microspheres as well as preparation method and application thereof

Abstract

The invention relates to the technical field of soil conditioning materials, in particular to salt-tolerant wormcast-based double-network microbial sustained-release gel microspheres as well as a preparation method and application thereof. The wormcast is selected as a filler, sodium alginate is selected as an embedding agent, and desulfurized gypsum is selected as a cross-linking agent, so that the salt-tolerant wormcast-based double-network microbial slow-release gel microspheres with high mechanical strength and high microbial encapsulation efficiency are constructed, and the gel microspheres can improve the soil quality by increasing the soil nutrient and organic matter content. The microbial survival rate is increased, the pH and salinity of the soil are reduced, the saline-alkali soil is effectively improved, and the method has the advantages of safety, high efficiency, low cost, simplicity in operation and the like, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of soil conditioning materials, and in particular to a salt-tolerant earthworm castings-based double-network microbial slow-release gel microsphere and a preparation method and application thereof. Background Art

[0002] Saline-alkali soil is an important type of salt-induced soil degradation. In recent years, soil salinization caused by climate change has become a key issue in global soil degradation and hindering agricultural development. Globally, salt affects about 935 million hectares of land in arid and semi-arid regions, accounting for more than 20% of the world's total irrigated area.

[0003] At present, many soil conditioners have been used to mitigate the negative impact of saline-alkali soil. This includes the use of materials or fertilizers such as gypsum and biochar, but improper use of the above conditioners can lead to soil acidification, imbalance of nitrogen and phosphorus nutrients, and reduced quality of agricultural products. In recent years, the use of salt-alkali tolerant microorganisms has shown good prospects in buffering and improving soil conditions. Brown ball nitrogen-fixing bacteria are a type of salt-tolerant autotrophic nitrogen-fixing bacteria that can use nitrogenase to reduce nitrogen molecules in the air into ammonium nitrogen that can be absorbed and utilized by plants, enriching soil nitrogen nutrients. However, the life activities of brown ball nitrogen-fixing bacteria are still limited by the soil environment. The lack of soil nutrients and high pH values ​​will lead to a significant reduction in the number and function of microbial populations, affecting the improvement effect. In order for the brown ball nitrogen-fixing bacteria introduced into the soil to survive, a carrier is needed to assist it in resisting adverse external environmental factors. Sodium alginate is a polymer polysaccharide extracted from brown algae. It has good biodegradability and is a perfect substrate for encapsulating microorganisms. Its rich carboxyl groups can cross-link with calcium ions in the solution to form water-absorbent gel microspheres with a 3D mesh structure, which can effectively alleviate soil water stress, solve the problems of soil water shortage caused by water stress and soil salt accumulation caused by capillary action, and effectively Ca 2+ However, the mechanical strength of sodium alginate microspheres is low and they are easily damaged during application, resulting in the rapid release and loss of microorganisms.

[0004] Therefore, how to provide a gel microsphere with high mechanical strength, high encapsulation rate for microorganisms and the ability to effectively improve saline-alkali soil has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres, the raw materials of which include earthworm castings, sodium alginate, desulfurized gypsum, microbial agents and solvents.

[0006] Worm castings are the product of earthworms engulfing and digesting organic matter. It is a natural organic fertilizer rich in nutrients such as nitrogen, phosphorus, potassium, protein, humus, etc. It has a large specific surface area, good aeration and drainage, and high water holding capacity. It is also rich in a large number of beneficial microorganisms.

[0007] The present invention constructs a more stable double network hydrogel by selecting earthworm castings as a filler, sodium alginate as an embedding agent, and desulfurized gypsum as a cross-linking agent, thereby improving the mechanical strength of the gel microspheres and the encapsulation rate of microorganisms. In addition, earthworm castings, as a nutrient source for microorganisms, can effectively improve the survival rate of microorganisms. When applied to the soil, the gel microspheres can also swell by absorbing water and salt, releasing some nutrients in the earthworm castings into the soil, improving the soil nutrient conditions, providing a more favorable environment for microorganisms, and improving their survival rate and biological effectiveness.

[0008] Among them, using industrial waste desulfurization gypsum instead of commonly used calcium chloride as a cross-linking agent can realize the resource utilization of waste and reduce the preparation cost.

[0009] In some embodiments, the microbial inoculant includes microorganisms and Mg2SO4 solution.

[0010] In some embodiments, the microorganism is Azotobacter chlorosphaeroides.

[0011] In some embodiments, the microbial agent is prepared by culturing a microorganism (preferably azotobacter sphaeroides) in a nutrient medium (preferably LB medium) and then resuspending the microorganism in a Mg2SO4 solution.

[0012] In some embodiments, the concentration of microorganisms in the microbial inoculant is 3.8×10 8 cfu / ml and above.

[0013] In some embodiments, the co-solvent is gluconolactone.

[0014] In some embodiments, the weight ratio of earthworm castings, sodium alginate and microbial agent is (0.5~2):1:50.

[0015] In some embodiments, the weight ratio of desulfurized gypsum to co-solvent is 1.5:3.

[0016] Furthermore, the present invention provides a method for preparing the salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres, comprising: mixing sterile water, earthworm castings, sodium alginate and microbial agents to obtain a suspension; mixing sterile water, desulfurized gypsum and a solvent to obtain a cross-linking agent solution; and dropping the suspension into the cross-linking agent solution to obtain salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres by cross-linking reaction.

[0017] In some embodiments, the weight ratio of sterile water, earthworm castings, sodium alginate and microbial agent is 50:(0.5-2):1:50.

[0018] In some embodiments, the weight ratio of sterile water, desulfurized gypsum and co-solvent is 300:1.5:3.

[0019] In some embodiments, the preparation method further comprises: washing the salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres with water after the cross-linking reaction.

[0020] Furthermore, the present invention provides the use of the salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres or the preparation method in at least one of the following aspects: (1) Preparation of soil conditioners; (2) Improve saline-alkali soil.

[0021] In some embodiments, the purpose of improving saline-alkali soil is achieved by increasing soil nutrient and organic matter content, improving microbial survival rate, and reducing soil pH and salinity.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention selects earthworm castings as fillers, sodium alginate as embedding agents, and desulfurized gypsum as cross-linking agents to construct salt-tolerant earthworm casting-based double-network microbial slow-release gel microspheres with high mechanical strength and high microbial encapsulation rate. The gel microspheres can effectively improve saline-alkali soil by increasing soil nutrient and organic matter content, improving microbial survival rate, and reducing soil pH and salinity. The microspheres have the advantages of safety, high efficiency, low cost, simple operation, etc., and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the swelling performance of different gel microspheres in acid / base environments.

[0024] Figure 2 This is the release curve of salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres IV within 28 days.

[0025] Figure 3 This is a test result chart of the live bacterial count and mortality rate of brown spherical nitrogen-fixing bacteria.

[0026] Figure 4 This is a graph showing the effects of different gel microspheres on soil pH.

[0027] Figure 5 This is a graph showing the effects of different gel microspheres on the total salt content of the soil.

[0028] Figure 6 This is a graph showing the effects of different gel microspheres on soil total nitrogen content.

[0029] Figure 7 This is a graph showing the effects of different gel microspheres on soil organic matter content. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The "parts" in the following embodiments represent parts by weight.

[0031] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0032] Example 1 This embodiment provides a salt-tolerant earthworm castings-based double-network microbial slow-release gel microsphere, the preparation method of which is as follows: (1) The brown spherical nitrogen-fixing bacteria were cultured in a nutrient medium (LB medium, containing 10 g / L sodium hydroxide, 10 g / L tryptone, and 5 g / L yeast extract) for 16 h, and then the OD of the bacterial solution was increased to 0.03% using a 0.03% Mg2SO4 solution. 600 The value was adjusted to 1.532, and the brown ball nitrogen-fixing bacteria were resuspended to obtain the microbial agent. The concentration of microorganisms in the microbial agent was 3.8×10 8 cfu / ml; (2) Air-dry the earthworm castings (filler) and pass through a 100-mesh sieve. Take 0.5 parts of filler and 1 part of sodium alginate (embedding agent) and mix with 50 parts of microbial agent (50 mL) and 50 parts of sterile water and stir for 1 hour to form a uniform microbial agent-embedding agent-filler suspension; (3) Mix 1.5 parts of a cross-linking agent (desulfurized gypsum), 3 parts of a co-solvent (gluconolactone) and 300 parts of sterile water and stir for 1 hour, collect the liquid by suction filtration, and prepare a cross-linking agent solution; (4) Add the above-mentioned microbial agent-embedding agent-filler suspension into a 50 ml syringe and slowly drip it into the cross-linking agent solution to cross-link into gel microspheres; (5) After cross-linking and curing at room temperature for 1 h, the synthesized gel microspheres were washed three times with deionized water and dried overnight at room temperature. The obtained salt-tolerant earthworm castings-based double-network microbial sustained-release gel microspheres were named gel microspheres II and stored in a centrifuge tube for later use.

[0033] Example 2 This embodiment provides a salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres, and the preparation method is different from that of Example 1 only in that the amount of earthworm castings is adjusted to 1 part, and the obtained salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres are named gel microspheres III.

[0034] Example 3 This embodiment provides a salt-tolerant earthworm casting-based double-network microbial slow-release gel microspheres, and the preparation method is different from that of Example 1 only in that the amount of earthworm castings is adjusted to 2 parts, and the obtained salt-tolerant earthworm casting-based double-network microbial slow-release gel microspheres are named gel microspheres IV.

[0035] Control group This control group provides a microbial slow-release gel microsphere, the preparation method of which is different from that of Example 1 only in that the amount of earthworm manure is adjusted to 0 parts, and the obtained microbial slow-release gel microsphere is named gel microsphere I.

[0036] Comparative Example The comparative examples are from published literature, as shown in Table 1.

[0037] Test Example 1 This test example tests the swelling properties and mechanical properties of the gel microspheres prepared in the above different embodiments, and the steps are as follows: 1. The swelling performance test method is as follows: Swelling analysis was performed by weight swelling method. The dried gel microspheres (weight W0) were immersed in 0.9% sterile sodium chloride solution for 24 h and removed, while the excess water was wiped off with a paper towel, weighed (weight W1), and the swelling ratio was calculated as: swelling ratio (ER, %) = (W1− W0) / W0× 100%. Each treatment was performed in triplicate.

[0038] The swelling performance test results are as follows: The test results of swelling properties are as follows: Figure 1 As shown, A1 to A4 represent the gel microspheres of the control group, Example 1, Example 2, and Example 3, respectively. The swelling ability of the gel microspheres under alkaline conditions is significantly higher than that under acidic conditions, but the swelling rate decreases from 6992.72% to 4580.43% with the increase of the amount of earthworm manure added. According to the experimental results, the salt and alkaline environments have a synergistic effect on the swelling of the gel microspheres. Under acidic conditions, the hydrophilic groups -COO- that have not been completely reacted inside the gel spheres can bind to water molecules, causing the material to swell. Under alkaline conditions, OH -The presence of will accelerate the conversion between -COOH and -COO-, promote the expansion of the water adsorbent polymer network chain, and the release of microorganisms depends on the swelling or relaxation of the gel polymer system. However, as the filler content increases, the filler and the embedding agent are more closely combined, resulting in a decrease in hydrophilic groups, thereby reducing the swelling properties of the material.

[0039] 2. The test method of mechanical strength is as follows: The dried gel microspheres were immersed in 0.9% sterile sodium chloride solution for 24 h and removed, while the excess water was wiped off with a paper towel, and the mechanical strength was judged by the apparent phenomenon.

[0040] The test results of mechanical strength are as follows: When the gel microspheres were taken out from a 0.9% sterile sodium chloride solution, the gel microspheres without fillers were soft, fragile, and had poor elasticity, while the gel microspheres with 0.5-2 parts of fillers had a certain elasticity and were not easy to break. Moreover, the mechanical strength was the highest when the amount of earthworm manure added was two parts (Example 3).

[0041] Test Example 2 The microbial encapsulation efficiency, sustained-release effect of brown-globose nitrogen-fixing bacteria, number of viable bacteria and mortality rate of the gel microspheres prepared by the above different methods were measured.

[0042] 1. The test method for microbial encapsulation rate is as follows: The encapsulation efficiency is the actual number of microorganisms in the gel microspheres. The number of live bacteria in the sodium alginate mixture (N0) and the live bacteria not encapsulated in the crosslinker (N1) were calculated using plate counts. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency (EE, %) = (N0− N1) / N0 × 100%.

[0043] The type of filler has a significant effect on the microbial encapsulation rate. The test results of the microbial encapsulation rate of gel microspheres and the comparison with existing studies are shown in Table 1. In previous studies, adding trehalose or starch as a filler to gel microspheres can significantly improve the encapsulation efficiency of microorganisms, and the highest encapsulation efficiency can reach 84.91% and 70.83%, respectively. In the present invention, the gel microspheres prepared with earthworm castings as fillers have an encapsulation efficiency of up to 90% for microorganisms. Compared with gel microspheres without earthworm castings, the addition of earthworm castings significantly increased the encapsulation rate of microorganisms from 63.61% to more than 90%. When the amount of earthworm castings added was 2 parts, the microbial encapsulation rate was as high as 100%. This shows that earthworm castings as a filler can significantly improve the microbial encapsulation rate, and the optimal addition amount is 2 parts (Example 3). Therefore, Example 3 is used as a representative to determine the microbial sustained release effect and the number of viable bacteria in the subsequent.

[0044] Table 1 Microbial encapsulation efficiency test results

[0045] 2. The test method for the slow-release effect of brown ball nitrogen-fixing bacteria is as follows: 0.5 g of gel microspheres were immersed in 10 mL of sterile sodium chloride solution (0.9%) and stored at room temperature for 28 days. Samples (0.1 mL) were taken out at 1, 3, 5, 7, 14, and 28 days, and the number of viable bacteria in the solution was determined by the dilution plate method. All experiments were performed in triplicate.

[0046] Taking the gel microspheres of Example 3 as an example, the results of testing the slow-release effect of brown spherical nitrogen-fixing bacteria are as follows: Figure 2 It can be seen that the gel microspheres have a sustained release effect, and the number of live bacteria in the first 5 days increased significantly, reaching 10 7 cfu -1 The number of viable bacteria reached a peak on the 14th day, about 10 8 cfu -1 .

[0047] 3. The test method for the viable count and mortality rate of brown ball nitrogen-fixing bacteria is as follows: Viable count: Dried gel microspheres (1.0 g) were immersed in 10 mL of sterile phosphate buffer (pH 7.0) for 1 hour. The microspheres were then ground into the solution, resulting in the release of bacteria entrapped or covered by the microspheres. After serial dilution, all viable bacteria were counted for colony counts.

[0048] The microbial mortality rate is calculated as: mortality rate = (total bacterial count - viable bacterial count) ÷ total bacterial count × 100%‌‌.

[0049] Taking the gel microspheres of Example 3 as an example, the results of the determination of the viable bacterial count and mortality rate of brown spherical nitrogen-fixing bacteria are as follows: Figure 3 As shown. It can be seen that on the first day, the number of live bacteria of brown ball nitrogen-fixing bacteria in the salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres was more than twice that of the original bacterial solution. After 28 days, the number of live bacteria in the original bacterial solution decreased significantly, the mortality rate of brown ball nitrogen-fixing bacteria was 98.8%, while the number of live bacteria in the salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres only decreased by 36.7%. The above results show that the salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres can provide a carbon source for brown ball nitrogen-fixing bacteria, increase the activity of microorganisms, and effectively prevent the microorganisms from being interfered by external environmental factors.

[0050] Test Example 3 This test example uses the gel microspheres prepared in the above example and the control group to treat moderate coastal saline-alkali soil, including the following steps: The experiment adopted a randomized experimental design, including: control group (CK, blank soil), treatment group 1 (gel microsphere I, A1), treatment group 2 (gel microsphere II, A2), treatment group 3 (gel microsphere III, A3), treatment group 4 (gel microsphere IV, A4), each group contained 4 replicates. 300g of air-dried soil passed through a 2mm sieve was weighed for each replicate, mixed evenly with gel microspheres and placed in a flower pot. The soil moisture content was maintained at 60% of the field water holding capacity during the entire experiment.

[0051] 1. Impact on soil pH After 30 days of treatment, the soil pH values ​​of different treatment groups were tested. Figure 4 As shown. The pH value of the soil treated with different gel microspheres was between 8.10 and 7.12. Compared with the control group, the pH value of the soil added with 0 (A1), 0.5 (A2), 1 (A3), and 2 (A4) parts of earthworm manure showed an overall downward trend, and the pH value decreased by 3.7%, 2.0%, 5.34%, and 4.6%, respectively. Except for the soil in the A4 and A3 treatment groups, there were significant differences between the other treatments. The buffering effect of gel microspheres on soil pH may be related to the acidic functional groups they carry. In a high pH environment, the acidic functional groups will undergo deprotonation behavior (-COOH when pH>4; -OH when pH>8), triggering a neutralization reaction, thereby reducing the soil pH. In addition, in a high saline-alkali environment, the gel microspheres swell, and the organic acids contained in the earthworm manure are released into the soil through the pores, reacting with alkaline substances such as carbonates and bicarbonates in the soil, and reducing the soil pH.

[0052] 2. Impact on total salt content of soil After 30 days of treatment, the soil total salt content of different treatment groups was tested. The changes in soil total salt content under different treatments are as follows: Figure 5 As shown, the total salt content of each treatment ranged from 3.60 to 4.33 g kg -1 The total salt content of the soil in the A1, A2, A3, and A4 treatments was significantly lower than that in the control group, with the total salt content reduced by 14.0%, 10.8%, 11.6%, and 17%, respectively. Gel microspheres can provide a calcium source for the soil and reduce the content of exchangeable sodium in saline-alkali soil. The salt in the soil can also penetrate into the microspheres under the action of osmotic pressure until the osmotic pressure balance is reached. The possible reason why the soil salt removal efficiency in the A4 treatment was the highest was that the earthworm manure content in the A4 treatment was the highest, which could provide sufficient carbon source for microorganisms and maintain the activity of brown ball nitrogen-fixing bacteria to the maximum extent, thereby exerting the synergistic effect of microorganisms and gel microspheres.

[0053] 3. Impact on soil total nitrogen content After 30 days of treatment, the soil total nitrogen content of different treatment groups was tested. The changes in soil total nitrogen content of different treatment groups were as follows: Figure 6 As shown in the figure, compared with the control group, the gel microspheres with different amounts of earthworm manure added had an effect on improving the total nitrogen content in the soil. With the increase of earthworm manure content, the total nitrogen content in the soil increased. When the amount of earthworm manure added was 2 parts, the total nitrogen content in the soil was significantly different from that in the control group, and the total nitrogen content increased by 14.0%. This may be because the increase in earthworm manure content increased its cross-linking density with sodium alginate, achieving a higher encapsulation efficiency of brown ball nitrogen-fixing bacteria, and earthworm manure can provide a carbon source for brown ball nitrogen-fixing bacteria and increase its activity. Under the action of nitrogenase, brown ball nitrogen-fixing bacteria can fix nitrogen in the air into the soil and increase the nutrient content of the soil.

[0054] 4. Impact on soil organic matter content After 30 days of treatment, the soil organic matter content of different treatment groups was tested. The changes in soil organic matter content of different treatments were as follows: Figure 7 As shown in the figure, gel microspheres with different amounts of earthworm manure added effectively improved the content of soil organic matter, which was increased by 6.2% to 8.2% compared with the blank. The treatment with 2 portions of earthworm manure had the best effect on improving the content of soil organic matter. Under saline-alkali conditions, the gel microspheres swelled to relax the molecular chains, promoting the release of brown spherical nitrogen-fixing bacteria, which participated in important processes such as soil organic matter decomposition and nutrient conversion, and increased the content of soil organic matter to a certain extent.

[0055] In summary, the best ratio is when the earthworm manure is added in two parts, which can maintain the microbial activity to the maximum extent. At this time, the salt-tolerant earthworm manure-based double-network microbial slow-release gel microspheres have the best saline-alkali soil improvement effect.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A salt-tolerant earthworm castings-based double-network microbial slow-release gel microsphere, characterized in that: Its raw materials include: earthworm castings, sodium alginate, desulfurized gypsum, microbial agents and solvents.

2. The salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres according to claim 1, characterized in that: The microbial inoculant includes microorganisms and Mg2SO4 solution.

3. The salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres according to claim 2, characterized in that: The microorganism is brown spherical nitrogen-fixing bacteria.

4. The salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres according to claim 1, characterized in that: The concentration of microorganisms in the microbial agent is 3.8×10 8 cfu / ml and above.

5. The salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres according to claim 1, characterized in that: The cosolvent is gluconolactone.

6. The salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres according to claim 1, characterized in that: The weight ratio of earthworm castings, sodium alginate and microbial agent is (0.5-2):1:50; and / or the weight ratio of desulfurized gypsum and solvent is 1.5:

3.

7. The method for preparing the salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres according to any one of claims 1 to 6, characterized in that: include: Mixing sterile water, earthworm castings, sodium alginate and microbial agents to obtain a suspension; Mixing sterile water, desulfurized gypsum and a co-solvent to obtain a cross-linking agent solution; The suspension is added dropwise to the cross-linking agent solution to undergo a cross-linking reaction to obtain salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres.

8. The preparation method according to claim 7, characterized in that: Also includes: The salt-tolerant vermicompost-based double-network microbial slow-release gel microspheres were washed with water after the cross-linking reaction.

9. Use of the salt-tolerant earthworm castings-based double-network microbial slow-release gel microspheres according to any one of claims 1 to 6 or the preparation method according to claim 7 or 8 in at least one of the following aspects: (1) Preparation of soil conditioners; (2) Improve saline-alkali soil.

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