A microbial remediation agent for desertified soil and its preparation method

Abstract

This invention relates to a microbial remediation agent for sandy soil and its preparation method, belonging to the technical field of microbial remediation agents. The invention modifies lignin by preparing a crosslinking agent containing quaternary ammonium salt and phosphate ester structures. The modified lignin has a more complex three-dimensional network structure, enabling it to store more water, achieve water retention and slow release, and provide the necessary moisture environment for microbial reproduction activities in a long-term stable manner. Furthermore, the modified lignin of this invention also has good microbial enrichment capacity, effectively adsorbing and anchoring microorganisms, reducing the diffusion and loss of microorganisms in the microbial remediation agent in sandy soil.

Description

Technical Field

[0001] This invention belongs to the field of microbial remediation agent technology, specifically, it relates to a microbial remediation agent for sandy soil and its preparation method. Background Technology

[0002] Soil desertification leads to grassland degradation and soil erosion, reduces vegetation cover, causes a decrease in arable land and significant degradation of pastures, and poses serious environmental problems, threatening human living space. There are three main methods for remediating desertified soil: physical remediation, chemical remediation, and bioremediation. Physical remediation requires significant human and material resources, while chemical remediation is not only costly but may also have environmental impacts. Bioremediation, through the synergistic effect of plants and microorganisms, is less costly and environmentally friendly. Therefore, this invention provides a microbial remediation agent for desertified soil and its preparation method. Summary of the Invention

[0003] The purpose of this invention is to provide a microbial remediation agent for sandy soil and its preparation method, in order to solve the problems mentioned in the background art.

[0004] The objective of this invention can be achieved through the following technical solutions:

[0005] A microbial remediation agent for sandy soil comprises the following raw materials in parts by weight: 1-2.5 parts Bacillus, 1.5-3 parts Trichoderma, 2-4 parts humic acid, 10-15 parts modified lignin, and 3-5 parts organic fertilizer.

[0006] Furthermore, the Bacillus is at least one of Bacillus subtilis, Bacillus mucilaginosus, Bacillus licheniformis, and Bacillus megaterium.

[0007] Furthermore, the Trichoderma is at least one of Trichoderma harzianum, Trichoderma viride, and Trichoderma simonii.

[0008] Furthermore, the organic fertilizer is animal manure organic fertilizer.

[0009] Furthermore, the modified lignin is prepared by the following steps:

[0010] Step 1: 2,3-Dihydroxypyrazine is subjected to phosphoric acid esterification reaction with phosphorus oxychloride, followed by hydrolysis to obtain the crosslinking agent precursor;

[0011]

[0012]

[0013] The second step involves reacting the crosslinking agent precursor with a halo-fatty aldehyde via a quaternary ammonium salt reaction to obtain the crosslinking agent.

[0014]

[0015] The third step involves reacting the crosslinking agent with lignin to produce acetal, thereby obtaining modified lignin.

[0016] Furthermore, the halogenated fatty aldehyde is at least one of 4-chlorobutyraldehyde, 5-chloropentanaldehyde, and 6-chlorohexanaldehyde.

[0017] Furthermore, the mass ratio of 2,3-dihydroxypyrazine to phosphorus oxychloride used is 1.7–2.6:8–12.

[0018] Furthermore, the mass ratio of the crosslinking agent precursor to the haloalfatty aldehyde used is 2–3.2:3–5.

[0019] Furthermore, the mass ratio of the crosslinking agent to lignin used is 3 to 6:15.

[0020] A method for preparing a microbial remediation agent for sandy soil includes the following steps:

[0021] Bacillus and Trichoderma were formulated to a viable bacterial concentration ≥5×10⁻⁶. 8 The mixed bacterial solution of CFU / mL was prepared, and then the modified lignin, which had been dried and sterilized, was immersed in the mixed bacterial solution. The solution was shaken at room temperature and a speed of 50-150 rpm for 6-20 hours. After the shaking adsorption was completed, the solution was allowed to stand for 12-24 hours for colonization. The modified lignin was then separated by filtration, and after vacuum freeze-drying, it was mixed evenly with humic acid and organic fertilizer to obtain the microbial remediation agent.

[0022] The beneficial effects of this invention are:

[0023] This invention uses lignin as a microbial carrier. Lignin is a natural organic cementing substance that can bind sand particles, promote soil aggregation, and improve soil stability. In addition, lignin itself is a natural organic polymer and can also serve as a carbon source to provide nutrients for microorganisms.

[0024] This invention modifies lignin by cross-linking and introducing a quaternary ammonium salt structure into it. The modified lignin has a more complex three-dimensional network structure, which can store more water. The water retention performance of the modified lignin can be effectively improved by utilizing the hydrogen bond adsorption capacity of the quaternary ammonium salt structure for water molecules, thus achieving water retention and slow release. Using the modified lignin of this invention as a carrier for microorganisms can provide the necessary water environment for microbial reproduction activities in a long-term and stable manner.

[0025] This invention modifies lignin by introducing positively charged quaternary ammonium salt and phosphate ester structures. The positively charged quaternary ammonium salt structure can attract the negatively charged cell membranes of microorganisms, improving the adsorption and colonization efficiency of the modified lignin on microorganisms in the microbial remediation agent during preparation. In addition, the phosphate ester structure in the modified lignin has good affinity with the phospholipid bilayer of the cell membrane, which can regulate the interfacial properties of the modified lignin, reduce the stimulating effect of the quaternary ammonium salt structure on microorganisms, and effectively anchor microorganisms, reducing the diffusion and loss of microorganisms in the microbial remediation agent in sandy soil. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0028] It should be understood that the use of “including,” “having,” or “containing,” including its grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0029] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0030] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0031] Example 1

[0032] A microbial remediation agent for sandy soil comprises the following raw materials in parts by weight: 1 part Bacillus, 1.5 parts Trichoderma, 6 parts humic acid, 30 parts modified lignin, and 12 parts organic fertilizer.

[0033] In this embodiment, the Bacillus used is a mixture of 0.4 parts Bacillus subtilis, 0.4 parts Bacillus mucilaginosus, and 0.2 parts Bacillus megaterium. The Trichoderma used is Trichoderma harzianum. The organic fertilizer used is chicken manure organic fertilizer. The modified lignin used is prepared by the following steps:

[0034] Step 1: Mix 1.7 parts of 2,3-dihydroxypyrazine, 0.36 parts of aluminum chloride, and 8 parts of phosphorus oxychloride in a reaction vessel according to the mass fraction. React at 75°C for 4 hours. After the reaction is completed, remove excess phosphorus oxychloride by rotary evaporation. Wash the remaining solid with 1M dilute hydrochloric acid and deionized water in sequence and then dry to obtain the crosslinking agent precursor.

[0035] Step 2: According to the mass fraction, 2 parts of crosslinking agent precursor, 3 parts of 4-chlorobutyraldehyde and 20 parts of acetonitrile are mixed in a reaction vessel and reacted at 40℃ for 10 hours. After the reaction is completed, the solvent is removed by rotary evaporation. The remaining solid is washed with diethyl ether and deionized water in sequence and then dried to obtain the crosslinking agent.

[0036] Step 3: According to the mass fraction, mix 3 parts of crosslinking agent, 15 parts of lignin, 2 parts of N,N-diisopropylethylamine, and 80 parts of dimethyl sulfoxide in a reaction vessel and react at 70℃ for 6 hours. After the reaction is completed, pour the reaction solution into deionized water to precipitate. After filtering to separate the solid, wash with methanol and deionized water in sequence and then dry to obtain modified lignin.

[0037] A method for preparing a microbial remediation agent for sandy soil includes the following steps:

[0038] S1: By mass fraction, 1 part of Bacillus and 1.5 parts of Trichoderma were mixed in 47.5 parts of an aqueous solution containing 5% (w / v) trehalose and 1% (w / v) glycerol to obtain a mixed bacterial solution. The viable cell concentration of the mixed bacterial solution was measured to be 5.4 × 10⁻⁶. 8 CFU / mL;

[0039] S2: The modified lignin, after being dried and sterilized, is immersed in a mixed bacterial solution and shaken at room temperature and 50 rpm for 20 hours. After the shaking adsorption is completed, it is allowed to stand for 12 hours for colonization. Then, the modified lignin is separated by filtration, and after vacuum freeze-drying, it is mixed evenly with humic acid and organic fertilizer to obtain the microbial remediation agent.

[0040] Example 2

[0041] A microbial remediation agent for sandy soil comprises the following raw materials in parts by weight: 1.75 parts Bacillus, 2.25 parts Trichoderma, 3 parts humic acid, 12.5 parts modified lignin, and 4 parts organic fertilizer.

[0042] In this embodiment, the Bacillus used is a mixture of 0.55 parts Bacillus subtilis, 0.5 parts Bacillus mucilaginosus, 0.4 parts Bacillus licheniformis, and 0.3 parts Bacillus megaterium; the Trichoderma used is a mixture of 1.5 parts Trichoderma harzianum and 0.75 parts Trichoderma viride; the organic fertilizer used is sheep manure organic fertilizer; and the modified lignin used is prepared by the following steps:

[0043] Step 1: According to the mass fraction, 2.1 parts of 2,3-dihydroxypyrazine, 0.45 parts of aluminum chloride, and 10 parts of phosphorus oxychloride were mixed in a reaction vessel and reacted at 85°C for 3.5 hours. After the reaction was completed, excess phosphorus oxychloride was removed by rotary evaporation. The remaining solid was washed successively with 1M dilute hydrochloric acid and deionized water and then dried to obtain the crosslinking agent precursor.

[0044] Step 2: According to the mass fraction, 2.5 parts of crosslinking agent precursor, 3.6 parts of 5-chloropentanal and 24 parts of acetonitrile are mixed in a reaction vessel and reacted at 50°C for 8 hours. After the reaction is completed, the solvent is removed by rotary evaporation. The remaining solid is washed with diethyl ether and deionized water in sequence and then dried to obtain the crosslinking agent.

[0045] Step 3: According to the mass fraction, 4.5 parts of crosslinking agent, 15 parts of lignin, 2.5 parts of N,N-diisopropylethylamine, and 90 parts of dimethyl sulfoxide are mixed in a reaction vessel and reacted at 80℃ for 5 hours. After the reaction is completed, the reaction solution is poured into deionized water to precipitate. After filtering to separate the solid, it is washed with methanol and deionized water in sequence and then dried to obtain modified lignin.

[0046] A method for preparing a microbial remediation agent for sandy soil includes the following steps:

[0047] S1: By mass fraction, 1.75 parts of Bacillus and 2.25 parts of Trichoderma were dissolved in 58.5 parts of a preservative solution to prepare a mixed bacterial solution. The preservative solution contained 1% glycerol and 5% trehalose by mass. The viable cell concentration of the mixed bacterial solution was measured to be 6.8 × 10⁻⁶. 8 CFU / mL;

[0048] S2: The modified lignin, after being dried and sterilized, is immersed in a mixed bacterial solution and shaken at room temperature and 100 rpm for 13 hours. After the shaking adsorption is completed, it is allowed to stand for 18 hours for colonization. Then, the modified lignin is separated by filtration, and after vacuum freeze-drying, it is mixed evenly with humic acid and organic fertilizer to obtain the microbial remediation agent.

[0049] Example 3

[0050] A microbial remediation agent for sandy soil comprises the following raw materials in parts by weight: 2.5 parts Bacillus, 3 parts Trichoderma, 4 parts humic acid, 15 parts modified lignin, and 5 parts organic fertilizer.

[0051] In this embodiment, the Bacillus used is a mixture of 1 part Bacillus subtilis, 1 part Bacillus mucilaginosus, and 0.5 parts Bacillus megaterium; the Trichoderma used is a mixture of 2 parts Trichoderma harzianum and 1 part Trichoderma simonii; the organic fertilizer used is pig manure organic fertilizer; and the modified lignin used is prepared by the following steps:

[0052] Step 1: According to the mass fraction, 2.6 parts of 2,3-dihydroxypyrazine, 0.56 parts of aluminum chloride, and 12 parts of phosphorus oxychloride were mixed in a reaction vessel and reacted at 95°C for 3 hours. After the reaction was completed, excess phosphorus oxychloride was removed by rotary evaporation. The remaining solid was washed successively with 1M dilute hydrochloric acid and deionized water and then dried to obtain the crosslinking agent precursor.

[0053] Step 2: According to the mass fraction, 3.2 parts of crosslinking agent precursor, 5 parts of haloallofatty aldehyde and 32 parts of acetonitrile are mixed in a reaction vessel and reacted at 60°C for 6 hours. After the reaction is completed, the solvent is removed by rotary evaporation. The remaining solid is washed with diethyl ether and deionized water in sequence and then dried to obtain the crosslinking agent.

[0054] Step 3: According to the mass fraction, 6 parts of crosslinking agent, 15 parts of lignin, 3 parts of N,N-diisopropylethylamine, and 100 parts of dimethyl sulfoxide are mixed in a reaction vessel and reacted at 90℃ for 4 hours. After the reaction is completed, the reaction solution is poured into deionized water to precipitate. After filtering to separate the solid, it is washed with methanol and deionized water in sequence and then dried to obtain modified lignin.

[0055] A method for preparing a microbial remediation agent for sandy soil includes the following steps:

[0056] S1: By mass fraction, 2.5 parts of Bacillus and 3 parts of Trichoderma were dissolved in 69.5 parts of a preservative solution to prepare a mixed bacterial solution. The preservative solution contained 1% glycerol and 5% trehalose by mass. The viable cell concentration of the mixed bacterial solution was measured to be 7.8 × 10⁻⁶. 8 CFU / mL.

[0057] S2: The modified lignin, after being dried and sterilized, is immersed in a mixed bacterial solution and shaken at room temperature and 150 rpm for 6 hours. After the shaking adsorption is completed, it is allowed to stand for 24 hours for colonization. Then, the modified lignin is separated by filtration, and after vacuum freeze-drying, it is mixed evenly with humic acid and organic fertilizer to obtain the microbial remediation agent.

[0058] Comparative Example 1

[0059] The difference between this comparative example and Example 2 is that the lignin is not modified; instead, an equal mass of unmodified lignin is used to replace the modified lignin.

[0060] A microbial remediation agent for sandy soil comprises the following raw materials in parts by weight: 1.75 parts Bacillus, 2.25 parts Trichoderma, 3 parts humic acid, 12.5 parts lignin, and 4 parts organic fertilizer.

[0061] In this embodiment, the Bacillus used is a mixture of 0.55 parts Bacillus subtilis, 0.5 parts Bacillus mucilaginosus, 0.4 parts Bacillus licheniformis, and 0.3 parts Bacillus megaterium. The Trichoderma used is a mixture of 1.5 parts Trichoderma harzianum and 0.75 parts Trichoderma citrinum. The organic fertilizer used is sheep manure organic fertilizer.

[0062] A method for preparing a microbial remediation agent for sandy soil includes the following steps:

[0063] S1: By mass fraction, 1.75 parts of Bacillus and 2.25 parts of Trichoderma were dissolved in 58.5 parts of a preservative solution to prepare a mixed bacterial solution. The preservative solution contained 1% glycerol and 5% trehalose by mass. The viable cell concentration of the mixed bacterial solution was measured to be 6.9 × 10⁻⁶. 8 CFU / mL;

[0064] S2: The modified lignin, after being dried and sterilized, is immersed in a mixed bacterial solution and shaken at room temperature and 100 rpm for 13 hours. After the shaking adsorption is completed, it is allowed to stand for 18 hours for colonization. Then, the lignin is separated by filtration, and after vacuum freeze-drying, it is mixed evenly with humic acid and organic fertilizer to obtain the microbial remediation agent.

[0065] Experimental Example 1

[0066] The waste bacterial solutions from Examples 1-3 and Comparative Example 1 after lignin adsorption were sampled, and the concentration of remaining free viable bacteria was tested. The adsorption rate was calculated by (original viable bacteria concentration - viable bacteria concentration after adsorption) / original viable bacteria concentration. The test results are shown in Table 1.

[0067] Table 1

[0068]

[0069] As can be seen from Table 1, the modified lignin obtained in Examples 1 to 3 has a much higher loading efficiency for microbial culture than the unmodified lignin in Comparative Example 1, indicating that the modification of lignin in this invention effectively improves its loading efficiency for microorganisms.

[0070] Experiment Example 2

[0071] The microbial remediation agents obtained in Examples 1-3 and Comparative Example 1 were applied to the remediation of desertified soil. Four groups of test land with similar morphologies were established, with desertified areas ≥80% and soil moisture content ≤1%. Each group of test land was treated with the microbial remediation agents obtained in Examples 1-3 and Comparative Example 1 at a rate of 5 kg per mu (approximately 0.067 hectares), and supplemented every six months at a rate of 3 kg per mu. After one year of microbial remediation (i.e., after two supplementary applications), quinoa was sown in each group of test land. The germination rate, soil moisture content, and relative compaction of the quinoa in each group of test land were recorded. The test results are shown in Table 2.

[0072] Table 2

[0073]

[0074] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention, including the best mode, and also to enable any person skilled in the art to practice the present invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A microbial remediation agent for a desertified soil, characterized by, The preparation method of the microbial repair agent for the desertification soil comprises the following steps: The preparation method of the microbial repair agent for the desertification soil comprises the following steps: The preparation method of the microbial repair agent for the desertification soil comprises the following steps: The preparation method of the microbial repair agent for the desertification soil comprises the following steps: The preparation method of the microbial repair agent for the desertification soil comprises the following steps: The preparation method of the microbial repair agent for the desertification soil comprises the following steps: The preparation method of the microbial repair agent for the desertification soil comprises the following steps: The preparation method of the microbial repair agent for the desertification soil comprises the following steps:

2. A microbial remediation agent for a desertifying soil according to claim 1, characterized in that, The preparation method of the microbial repair agent for the desertification soil comprises the following steps:

3. A microbial remediation agent for a desertifying soil according to claim 1, characterized in that, The preparation method of the microbial repair agent for the desertification soil comprises the following steps:

4. The microbial remediation agent for a desertifying soil according to claim 1, characterized by, The preparation method of the microbial repair agent for the desertification soil comprises the following steps:

5. The microbial remediation agent for a desertifying soil according to claim 1, characterized by, The preparation method of the microbial repair agent for the desertification soil comprises the following steps:

6. 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Then, the modified lignin after drying and sterilization is immersed in the mixed bacteria solution, and oscillated at a room temperature and a rotation speed of 50-150 rpm for 6-20 h. After oscillation and adsorption, the modified lignin is left to stand for 12-24 h for colonization, and then filtered and separated. After vacuum freeze-drying, the modified lignin is mixed with humic acid and organic fertilizer to obtain a microbial remediation agent.

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