Application of sodium alginate-carboxymethyl chitosan composite hydrogel in preparation of biological bacterial fertilizer

Abstract

The invention discloses application of sodium alginate-carboxymethyl chitosan composite hydrogel in preparation of a biological bacterial fertilizer, the sodium alginate-carboxymethyl chitosan composite hydrogel is used for preparation of the biological bacterial fertilizer, and is further used for preparation of the biological bacterial fertilizer with plant growth-promoting rhizobacteria as an effective component, and the sodium alginate-carboxymethyl chitosan composite hydrogel can be stored for a long time and has a good application prospect. The growth promoting effect is stably exerted. The plant growth-promoting bacteria are effectively wrapped and efficiently delivered through a nanometer means to enhance the growth-promoting effect on crops, the adaptability of the plant growth-promoting bacteria to the acid environment is effectively improved, and the advantage of the plant growth-promoting bacteria for promoting plant growth in acid soil is expanded. The biological bacterial fertilizer is suitable for neutral and acidic environments, and the effects of the biological bacterial fertilizer in the aspects of soil remediation and improvement, crop quality and efficiency improvement, crop stress resistance improvement and the like are fully exerted, so that the purposes of losing weight and improving efficiency are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fertilizers and provides application of sodium alginate-carboxymethyl chitosan composite hydrogel in the preparation of biological fertilizers. Background Art

[0002] my country is a major producer and consumer of fertilizers. With the development of agricultural production, the demand for fertilizers is also increasing. However, excessive use of fertilizers can lead to soil acidification, soil structure damage, soil heavy metal accumulation, and nitrogen and phosphorus nutrients entering the water body through rainwater leaching, thus causing eutrophication of the water body. Therefore, reducing fertilizer input and improving nutrient utilization are key issues that need to be urgently addressed in agriculture.

[0003] As interactors of plant roots, microorganisms play an important role in plant growth. Microbial fertilizers made from effective microorganisms can not only promote plant growth, but also reduce the use of chemical fertilizers, help improve soil heavy metal pollution, help plants obtain nutrients, and to a certain extent enhance the resistance of crops to pathogens. The use of microbial fertilizers combined with changes in farming methods can reduce carbon footprints and greenhouse gas emissions. It can be seen that the development of microbial fertilizers is one of the important ways to reduce fertilizer inputs and improve nutrient utilization efficiency, and has great prospects in modern agricultural production.

[0004] However, how to store plant growth-promoting rhizobacteria (PGPR) in encapsulants for a long time and stably play a growth-promoting role is one of the main bottlenecks hindering the large-scale use of biofertilizers, which is also the pain point of the current industry. Therefore, there is an urgent need to develop new encapsulants with strong environmental adaptability, assist microbial colonization and improve growth-promoting efficiency, so as to reduce the toxicity of acidic soil to plants and microorganisms. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a biological fertilizer comprising a Strigosa spp. strain or a Bacillus strain and a preparation method thereof, which can be stored for a long time and stably exert a growth-promoting effect.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] 1. Application of sodium alginate-carboxymethyl chitosan composite hydrogel in the preparation of biological fertilizer.

[0008] Preferably, the active ingredient of the biological fertilizer is plant rhizosphere growth-promoting bacteria.

[0009] Further preferably, the active ingredient of the biological fertilizer is from the genus Sagittaria or the genus Bacillus.

[0010] Further preferably, the effective ingredient of the biological fertilizer is Ensiferadhaerens BnC5 or Bacillus sp. RPA1, wherein Ensiferadhaerens BnC5 was deposited in the China General Microbiological Culture Collection Center on July 13, 2023, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.27880; Bacillus sp. RPA1 comes from patent application CN116814507A.

[0011] 2. A biological fertilizer is obtained by encapsulating plant rhizosphere growth-promoting bacteria using a sodium alginate-carboxymethyl chitosan composite hydrogel.

[0012] Preferably, the plant rhizosphere growth-promoting bacteria are of the genus Sagittaria or the genus Bacillus.

[0013] Further preferably, the plant rhizosphere growth-promoting bacteria are Ensifer adhaerens BnC5 or Bacillus sp. RPA1, wherein Ensifer adhaerens BnC5 was deposited in the China General Microbiological Culture Collection Center on July 13, 2023, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.27880; Bacillus sp. RPA1 comes from patent application CN116814507A.

[0014] 3. The preparation method of the aforementioned biological fertilizer comprises the following specific steps:

[0015] (1) First, culture the plant rhizosphere growth-promoting bacteria to obtain the bacteria;

[0016] (2) Then the bacteria are mixed evenly with sodium alginate (SA) solution;

[0017] (3) Add carboxymethyl chitosan (CMCS) solution and mix well;

[0018] (4) Finally, add calcium chloride (CaCl2) solution and mix well.

[0019] Preferably, the specific method of step (1) is as follows: streak the frozen plant rhizosphere growth-promoting bacteria on a solid culture medium (purchased from Amida Biotech), culture at 25-30°C for 12 hours, then transfer to a triangular flask containing LB liquid culture medium (purchased from Amida Biotech), culture at 200r / min for 12 hours to obtain a bacterial solution, and adjust the OD 600 =0.7, centrifuge and take the precipitate to obtain the bacterial cells.

[0020] Further preferably, the centrifugal process conditions are: 5500r / min centrifugation for 6 minutes.

[0021] Preferably, the dosage ratio of the bacterial cells, the sodium alginate solution, the carboxymethyl chitosan solution, and the calcium chloride solution is 11 mL: 5 mL: 5 mL: 1 mL; the sodium alginate solution is obtained by dissolving sodium alginate in a 0.2 mol / L sodium chloride solution, and the concentration is 7 g / L; the carboxymethyl chitosan solution is obtained by dissolving carboxymethyl chitosan in water, and the concentration is 3 g / L; the calcium chloride solution is obtained by dissolving calcium chloride in water, and the concentration is 0.07 mol / L.

[0022] Preferably, the sodium alginate solution and the calcium chloride solution are sterilized by high pressure steam at 121° C., and the carboxymethyl chitosan solution is sterilized by filtration through a 0.45 μm filter membrane.

[0023] The beneficial effects of the present invention are:

[0024] The present invention uses sodium alginate-carboxymethyl chitosan composite hydrogel for the preparation of biological fertilizer, and further for the preparation of biological fertilizer whose effective ingredient is plant rhizosphere growth-promoting bacteria, which can be stored for a long time and stably play a growth-promoting role. Specifically, the plant rhizosphere growth-promoting bacteria are first cultured to obtain the bacterial body; then the bacterial body is evenly mixed with the sodium alginate solution; then the carboxymethyl chitosan solution is added and mixed evenly; finally, the calcium chloride solution is added and mixed evenly to obtain. The biological fertilizer has the characteristics of multifunctionality, environmental friendliness, high efficiency, and greenness. The present invention effectively encapsulates and efficiently delivers plant rhizosphere growth-promoting bacteria (PGPR) through nano means to enhance the growth-promoting effect on crops, effectively improve the adaptability of plant rhizosphere growth-promoting bacteria (PGPR) to acidic environments, and expand the advantages of plant rhizosphere growth-promoting bacteria (PGPR) in promoting plant growth in acidic soils. The biological fertilizer is suitable for neutral and acidic environments, and gives full play to the role of biological fertilizer in soil remediation and improvement, crop quality improvement and efficiency improvement, and crop stress resistance improvement, thereby achieving the purpose of "weight loss and efficiency improvement".

[0025] SA is a negatively charged natural polysaccharide with good biocompatibility and can be absorbed by divalent ions such as Ca 2+SA hydrogel can be used as a signal molecule, store carbohydrates and stress protectants (e.g., drought, cold and salt stress), and has a certain gel strength. It is a raw material for encapsulation in the field of microbial agent research. However, it has the following disadvantages: (1) The excessive hydrophilicity makes SA unable to interact with cells. For most cells, cell-cell and cell-matrix interactions are important factors in regulating cell proliferation, differentiation and specific expression; (2) Ca in SA gel is not high. 2+ The ion exchange with the monovalent ions in the environment easily makes the strength of the scaffold unstable.

[0026] CMCS is the only alkaline polysaccharide with positive charge that exists in large quantities in nature. It has the advantages of safety, non-toxicity, biodegradability, good biocompatibility, film-forming ability, plant growth promotion, and certain hydrophobicity. It is mainly used for fruit preservation, growth regulation, drug carrier, soil improvement, etc. It is an ideal raw material for application in the field of plant protection. However, the strength of a single CMCS hydrogel is generally low and it degrades quickly, which greatly limits its application in the agricultural field.

[0027] The present invention combines SA with CMCS, and through the electrostatic attraction hydrogen bond and intermolecular entanglement between SA and CMCS, ion exchange and gel discretization are difficult to occur. In addition, the addition of CMCS can reduce the dissolution effect of external factors on SA hydrogel, and improve the interaction between SA and cells. The present invention makes the CMCS-SA composite hydrogel plastic by adjusting the ratio, uniformity and positive charge density of CMCS molecules between SA and CMCS, and can be better applied to the production of biological fertilizer in the future.

[0028] Both CMCS and SA are natural polysaccharides, non-toxic, biocompatible, and biodegradable natural polymer materials. Since there are a large number of primary amino groups (positively charged) on the CMCS molecular chain and a large number of carboxyl groups (negatively charged) on the SA molecular chain, CMCS and SA can complement each other and form a stable polyelectrolyte membrane through the attraction of positive and negative charges. The formed hydrogel has good air permeability, which is conducive to the exchange of O2 and nutrients with the outside world. At the same time, the added Ca 2+ Cross-linking with SA forms a chelate with a three-dimensional spatial structure, so that the composite hydrogel forms a solid and obtains nanoparticles (NP), further improving the mechanical strength.

[0029] The present invention further combines PGPR with the nanoparticle (NP), which can not only maintain the physiological activity of PGPR at a high level, but also bring many unexpected benefits, such as multiple biological activities such as inducing crop defense response and film-forming properties, which help to enhance crop immunity and defense against plant pathogens. In addition, chitosan can also be used as a carrier to load trace mineral elements, significantly increase the duration of mineral elements, and reduce adverse effects on the environment), with a miraculous effect of 1+1>2.

[0030] The present invention further prepares a biological fertilizer whose active ingredient is the Ensifer adhaerens strain, and the Ensifer adhaerens strain is the Ensifer adhaerens strain (Ensifer adhaerens) BnC5, which was deposited in the China General Microbiological Culture Collection Center on July 13, 2023, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCCNO.27880.

[0031] The material of the present invention has a wide range of agricultural applications. The material of the present invention can be used to prepare nano-scale active microcapsules to encapsulate agricultural microorganisms with the characteristics of nitrogen fixation, phosphorus and potassium dissolution, disease resistance and growth promotion to form new bacterial fertilizers and other agricultural fields. It can embody unique properties such as maintaining bacterial vitality, directional binding to the root surface and precise and efficient fertilization. It is suitable for modern agricultural production methods such as mechanized sowing, integrated water and fertilizer drip irrigation and soilless cultivation, and can replace the high-cost and low-efficiency traditional agricultural microbial inoculation method.

[0032] The present invention can significantly improve the biological activity and survival ability of the strain, and at the same time has unique properties such as maintaining bacterial vitality, directional binding to the root surface, and precise and efficient fertilization. It is suitable for modern agricultural production methods such as mechanized sowing, integrated water and fertilizer drip irrigation, and soilless cultivation, and has a wide range of applications.

[0033] The present invention has the following advantages:

[0034] (1) Plant growth-promoting rhizobacteria (PGPR) are the basic materials for developing biofertilizers. Compared with biofertilizers commonly developed from soil microorganisms, PGPR colonizes inside the plant, has a stable ecological niche, and can play a sustainable and stable role in promoting crop growth.

[0035] (2) It was confirmed that the composite hydrogel can effectively protect crop growth-promoting bacteria and resist other adverse factors in the soil (such as acidic environment), thereby amplifying the growth-promoting effect of the growth-promoting bacteria.

[0036] (3) The preparation method of the encapsulation agent is simple, highly operational, and easy to use, and all agricultural practitioners can easily master it.

[0037] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0039] Figure 1 This is a representative image of the effect of Ensifer biofertilizer on rapeseed under greenhouse potted conditions.

[0040] Figure 2 This is a representative image of the effect of Bacillus biofertilizer on rapeseed under greenhouse potted conditions.

[0041] Collection Information

[0042] Classification name: Glue arrow fungus

[0043] Latin name: Ensifer adhaerens.

[0044] Name of depository: China General Microbiological Culture Collection Center (CGMCC)

[0045] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0046] Deposit date: July 13, 2023 DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with specific implementation methods. The accompanying drawings are only used for exemplary descriptions and are only schematic diagrams, not actual drawings, and cannot be understood as limiting this patent; in order to better illustrate the embodiments of the present invention, some parts of the accompanying drawings may be omitted, enlarged or reduced, and do not represent the size of the actual product; for those skilled in the art, it is understandable that some well-known structures and their descriptions in the accompanying drawings may be omitted.

[0048] The PGPR used in the present invention includes Bacillus and Ensifer. The specific examples are: Bacillus sp. RPA1 and Ensifer adhaerens BnC5, which are abbreviated as Bacillus and Ensifer, respectively.

[0049] Test seeds: ZS11 seeds (purchased from Wuhan Zhongyou Seed Technology Co., Ltd.) were disinfected with 70% alcohol by volume for 1 minute and washed with sterile water 3 times; then disinfected with 10% sodium hypochlorite solution by mass for 10 minutes and washed with sterile water 5 times.

[0050] Test soil: The red soil was collected from Impression Tobacco Village, Taichuang Garden, Shilin Yi Autonomous County, Kunming, Yunnan. The basic physical and chemical properties of the soil are pH 6.51, total potassium 7.62g / kg, total phosphorus content 1.07g / kg, and alkaline hydrolyzable nitrogen 106.3mg / kg.

[0051] The preparation method of biological fertilizer comprises the following steps:

[0052] 1. Streak the growth-promoting strains of Bacillus and Ensifer from the frozen glycerol tube on a solid culture medium, culture at 25-30°C for 12 hours, then inoculate the bacteria into a 250 mL Erlenmeyer flask containing LB liquid culture medium, culture at 200 r / min for 12 hours, and set aside;

[0053] 2. Sterilize the prepared SA solution (7 g / L, obtained by dissolving sodium alginate in 0.2 mol / L sodium chloride solution) and CaCl2 solution (0.07 mol / L, obtained by dissolving calcium chloride in water) by high pressure steam at 121°C for 15 minutes, and filter and sterilize the CMCS solution (3 g / L, obtained by dissolving carboxymethyl chitosan in water);

[0054] 3. First, the cultured bacterial solution OD 600 Adjust to 0.7, then centrifuge 11mL of bacterial broth solution at 5500r / min for 6 minutes, discard the supernatant and take the bacteria, mix with 5mL of SA solution, then add 5mL of CMCS solution and mix, and finally add 1mL of CaCl2 solution and mix.

[0055] Reference Examples

[0056] A new low-phosphorus growth-promoting bacterium screened from the rhizosphere of rapeseed was obtained by the applicant from rapeseed roots. 16SrDNA sequence similarity analysis determined that BnC5 belonged to the genus Ensifer adhaerens., and a partial gene sequence was shown in SEQID NO.1.

[0057] Example 1

[0058] Soil: vermiculite: perlite = 6:3:1 (volume ratio) was mixed evenly, sterilized twice with high pressure steam, and dispensed into seedling pots. Seeds were sown in the prepared soil and placed in a light incubator for one month, during which biological fertilizer was added once, and sterile water and nutrient solution (or low concentration) (prepared with phosphorus-deficient MS powder from Amida Biotech) were regularly irrigated. After one month, fresh weight, dry weight, stem diameter and other related physiological and biochemical indicators were counted.

[0059] Four treatment schemes were set up: Treatment 1, control group CK; Treatment 2, application of composite hydrogel group NP; Treatment 3, application of growth-promoting bacteria group Bacillus or Ensifer; Treatment 4, application of biological fertilizer group Bacillus-NP or Ensifer-NP.

[0060] Experimental Results

[0061] like Figure 1 As shown, after rapeseed was inoculated with the growth-promoting bacteria Ensifer wrapped in the composite hydrogel, compared with the treatment with the growth-promoting bacteria Ensifer, the aboveground fresh weight (Table 1), aboveground dry weight (Table 2), underground fresh weight (Table 3), underground dry weight (Table 4), and stem diameter (Table 5) of the plant increased by 20.65%, 24.91%, 45.17%, 46.26%, and 21.97%, respectively, indicating that the use of composite hydrogel to wrap Ensifer can significantly improve its growth-promoting effect.

[0062] Table 1 Effect of Ensifer biofertilizer on the fresh weight of rapeseed aboveground parts under greenhouse potted conditions

[0063]

[0064] Table 2 Effect of Ensifer biofertilizer on the aboveground dry weight of rapeseed under greenhouse potted conditions

[0065]

[0066] Table 3 Effect of Ensifer biofertilizer on the fresh weight of rapeseed underground under greenhouse potted conditions

[0067]

[0068] Table 4 Effect of Ensifer biofertilizer on underground dry weight of rapeseed under greenhouse potted conditions

[0069]

[0070]

[0071] Table 5 Effect of Ensifer biofertilizer on rapeseed stem diameter under greenhouse potted conditions

[0072]

[0073] like Figure 2 As shown in the figure, after rapeseed was inoculated with the growth-promoting bacteria Bacillus wrapped in the composite hydrogel, compared with the treatment with the growth-promoting bacteria Bacillus, the aboveground fresh weight (Table 6), aboveground dry weight (Table 7), underground fresh weight (Table 8), and underground dry weight (Table 9) of the plant increased by 27.66%, 25.53%, 47.53%, and 52.27%, respectively, indicating that the use of composite hydrogel to wrap Bacillus can significantly improve its growth-promoting effect.

[0074] Table 6 Effects of Bacillus biofertilizer on the fresh weight of rapeseed aboveground parts under greenhouse potted conditions

[0075]

[0076] Table 7 Effects of Bacillus biofertilizer on the aboveground dry weight of rapeseed under greenhouse potted conditions

[0077]

[0078] Table 8 Effect of Bacillus biofertilizer on the fresh weight of rapeseed underground under greenhouse potted conditions

[0079]

[0080] Table 9 Effects of Bacillus biofertilizer on the underground dry weight of rapeseed under greenhouse potted conditions

[0081]

[0082] Example 2

[0083] The sterilized rapeseed seeds were sown in a medium containing 1 / 2MS and 1% agar (purchased from Amida Biotech). The pH of the plate was adjusted to 5.5, and four treatments were set: CK, NP, Ensifer, and Ensifer-NP. The plate was placed vertically in a light incubator at 22°C (16 hours of light / 8 hours of darkness). The light intensity was 20,000 Lux. Physiological indicators were statistically analyzed when the seedlings grew for 14 days.

[0084] Experimental Results

[0085] Under acidic conditions, after rapeseed was inoculated with the growth-promoting bacteria Ensifer wrapped in the composite hydrogel, the fresh weight of the aboveground part of the plant (Table 10), the fresh weight of the underground part (Table 11), the lateral root length (Table 12), the number of lateral roots (Table 13), and the lateral root density (Table 14) increased by 40.48%, 51.42%, 34.52%, 32.20%, and 33.51%, respectively, compared with the treatment with the growth-promoting bacteria Ensifer. However, there was no significant difference in the number of lateral roots and the lateral root density between the Ensifer group and the CK group, indicating that the composite hydrogel can help Ensifer resist the acidic environment and thus stabilize its growth-promoting effect.

[0086] Table 10 Effect of Ensifer biofertilizer on the fresh weight of rapeseed aboveground parts under acidic conditions

[0087]

[0088] Table 11 Effect of Ensifer biofertilizer on the fresh weight of rapeseed underground under acidic conditions

[0089]

[0090] Table 12 Effect of Ensifer biofertilizer on lateral root length of rapeseed under acidic conditions

[0091]

[0092] Table 13 Effect of Ensifer biofertilizer on the number of lateral roots of rapeseed under acidic conditions

[0093]

[0094] Table 14 Effect of Ensifer biofertilizer on lateral root density of rapeseed under acidic conditions

[0095]

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. Application of sodium alginate-carboxymethyl chitosan composite hydrogel in the preparation of biological fertilizer.

2. The use according to claim 1, characterized in that: The effective component of the biological fertilizer is plant rhizosphere growth-promoting bacteria.

3. The use according to claim 2, characterized in that: The effective component of the biological fertilizer is Sagittaria or Bacillus.

4. The use according to claim 3, characterized in that: The effective ingredient of the biological fertilizer is Ensifer adhaerens BnC5 or Bacillus sp. RPA1, wherein Ensifer adhaerens BnC5 was deposited in the China General Microbiological Culture Collection Center on July 13, 2023, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.27880; Bacillus sp. RPA1 comes from patent application CN116814507A.

5. A biological fertilizer, characterized in that: The plant rhizosphere growth-promoting bacteria are encapsulated by sodium alginate-carboxymethyl chitosan composite hydrogel.

6. A biological fertilizer according to claim 5, characterized in that: The plant rhizosphere growth-promoting bacteria are of the genus Sagittaria or the genus Bacillus.

7. A biological fertilizer according to claim 6, characterized in that: The plant rhizosphere growth-promoting bacteria are Ensifer adhaerens BnC5 or Bacillus sp. RPA1, wherein Ensifer adhaerens BnC5 was deposited in the China General Microbiological Culture Collection Center on July 13, 2023, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.27880; Bacillus sp. RPA1 comes from patent application CN116814507A.

8. The method for preparing a biological fertilizer according to any one of claims 5 to 7, characterized in that: The specific steps are as follows: (1) First, culture the plant rhizosphere growth-promoting bacteria to obtain the bacteria; (2) then mixing the bacterial cells and the sodium alginate solution evenly; (3) adding carboxymethyl chitosan solution and mixing evenly; (4) Finally, add calcium chloride solution and mix well to obtain the product.

9. The preparation method according to claim 8, characterized in that: The specific method of step (1) is as follows: streak the frozen plant rhizosphere growth-promoting bacteria on a solid culture medium, culture at 25-30°C for 12 hours, then transfer to a triangular flask containing LB liquid culture medium, culture at 200 r / min for 12 hours, obtain a bacterial solution, and adjust the OD 600 =0.7, centrifuge and take the precipitate to obtain the bacterial cells.

10. The preparation method according to claim 8, characterized in that: The dosage ratio of the bacterial cells, the sodium alginate solution, the carboxymethyl chitosan solution and the calcium chloride solution is 11 mL: 5 mL: 5 mL: 1 mL; the sodium alginate solution is obtained by dissolving sodium alginate in a 0.2 mol / L sodium chloride solution, and the concentration is 7 g / L; the carboxymethyl chitosan solution is obtained by dissolving carboxymethyl chitosan in water, and the concentration is 3 g / L; the calcium chloride solution is obtained by dissolving calcium chloride in water, and the concentration is 0.07 mol / L.

Images