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

Abstract

The present invention discloses the application of sodium alginate-carboxymethyl chitosan composite hydrogel in the preparation of biological fertilizer. The present invention uses sodium alginate-carboxymethyl chitosan composite hydrogel for the preparation of biological fertilizer, and further uses it for the preparation of biological fertilizer whose effective ingredient is plant rhizosphere growth-promoting bacteria. It can be stored for a long time and stably plays a growth-promoting role. The present invention effectively encapsulates and efficiently delivers plant rhizosphere growth-promoting bacteria through nano means to enhance the growth-promoting effect on crops, effectively improves the adaptability of plant rhizosphere growth-promoting bacteria to acidic environments, and expands the advantages of plant rhizosphere growth-promoting bacteria 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 enhancement, and improving crop stress resistance, thereby achieving the purpose of "weight loss and efficiency enhancement".

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 fertilizer application can lead to soil acidification, soil structure damage, the accumulation of heavy metals in the soil, and the leaching of nutrients such as nitrogen and phosphorus into water bodies through rainwater, causing eutrophication. Therefore, reducing fertilizer input and improving nutrient utilization efficiency are key issues that need to be urgently addressed in agriculture.

[0003] Microorganisms, as interactors with plant roots, play a vital role in plant growth. Microbial fertilizers made from effective microorganisms not only promote plant growth but also reduce the use of chemical fertilizers, improve soil heavy metal contamination, help plants obtain nutrients, and, to a certain extent, enhance crop resistance to pathogens. The use of microbial fertilizers combined with changes in farming practices can reduce carbon footprints and greenhouse gas emissions, which aligns with the country's dual carbon goals. Therefore, the development of microbial fertilizers is an important way to reduce chemical fertilizer inputs and improve nutrient utilization efficiency, and holds great promise in modern agricultural production.

[0004] However, one of the major bottlenecks hindering the large-scale use of biofertilizers and a major pain point in the industry is how to ensure that plant growth-promoting rhizobacteria (PGPR) can be stored in encapsulated materials for long periods of time and stably exert their growth-promoting effects. Therefore, there is an urgent need to develop new encapsulated materials with strong environmental adaptability, facilitate microbial colonization, and improve growth-promoting efficiency to mitigate the toxic effects of acidic soil on 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 glutinosa 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 Agrobacterium or 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 a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of 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 sodium alginate-carboxymethyl chitosan composite hydrogel.

[0012] Preferably, the plant growth-promoting rhizobacteria are of the genus Sagittium or 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 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) The bacterial cells were then 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: the frozen plant rhizosphere growth-promoting bacteria are streaked on a solid culture medium (purchased from Amida Biotechnology Co., Ltd.) and cultured at 25-30°C for 12 hours, and then transferred to a triangular flask containing LB liquid culture medium (purchased from Amida Biotechnology Co., Ltd.) and cultured at 200 r / min for 12 hours to obtain a bacterial solution, and the OD is adjusted. 600 =0.7, centrifuge and take the precipitate to obtain the bacterial cells.

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

[0021] Preferably, the dosage ratio of the bacterial cells, sodium alginate solution, carboxymethyl chitosan solution, and calcium chloride solution is 11 mL: 5 mL: 5 mL: 1 mL; the sodium alginate solution is obtained by dissolving sodium alginate in 0.2 mol / L sodium chloride solution, with a concentration of 7 g / L; the carboxymethyl chitosan solution is obtained by dissolving carboxymethyl chitosan in water, with a concentration of 3 g / L; the calcium chloride solution is obtained by dissolving calcium chloride in water, with a concentration of 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 using 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 uses it for the preparation of biological fertilizer whose effective ingredient is plant rhizosphere growth-promoting bacteria. It can be stored for a long time and stably exerts a growth-promoting effect. Specifically, the plant rhizosphere growth-promoting bacteria are first cultured to obtain the bacteria; then the bacteria are mixed evenly 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 product. The biological fertilizer has the characteristics of being multifunctional, environmentally friendly, efficient, and green. 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 enhancement, and crop stress resistance improvement, thereby achieving the purpose of "weight loss and efficiency enhancement".

[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) The Ca in SA gel is too high. 2+ It is easy to exchange ions with monovalent ions in the environment, making the strength of the scaffold unstable.

[0026] CMCS is the only naturally occurring, positively charged alkaline polysaccharide. It boasts safety, non-toxicity, biodegradability, excellent biocompatibility, film-forming properties, plant growth promotion, and moderate hydrophobicity. It is primarily used for fruit preservation, growth regulation, drug delivery, and soil improvement, making it an ideal raw material for plant protection. However, single CMCS hydrogels generally have low strength and rapid degradation, significantly limiting their application in agriculture.

[0027] The present invention combines SA with CMCS, making ion exchange and gel dispersion more difficult through electrostatic attraction hydrogen bonds and intermolecular entanglement between SA and CMCS. In addition, the addition of CMCS can reduce the dissolution of SA hydrogels by external factors and improve the interaction between SA and cells. By adjusting the ratio and uniformity between SA and CMCS and the positive charge density of CMCS molecules, the present invention makes the CMCS-SA composite hydrogel plastic, which can be better applied to the production of biofertilizer in the future.

[0028] CMCS and SA are both 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 to obtain a polymeric hydrogel (PMH), further improving the mechanical strength.

[0029] The present invention further combines PGPR with this polymeric hydrogel (PMH), which not only maintains the physiological activity of PGPR at a high level but also offers many unexpected benefits. For example, it possesses multiple biological activities, such as inducing crop defense responses and film-forming properties, helping to enhance crop immunity and defense against plant pathogens. Furthermore, chitosan can serve as a carrier for trace mineral elements, significantly increasing their longevity and reducing their adverse environmental impact, creating a remarkable 1+1>2 effect.

[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. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is 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 active microcapsules to encapsulate agricultural microorganisms with the characteristics of nitrogen fixation, phosphorus and potassium solubilization, 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 with soil microorganisms, PGPR colonizes inside plants, 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 coating agent is simple, highly operational, and easy to use, and all agricultural practitioners can easily master it.

[0037] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may 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 with reference to the accompanying drawings, in which:

[0039] Figure 1 This is a representative image of Ensifer biofertilizer acting on rapeseed under greenhouse potting conditions.

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

[0041] Preservation Information

[0042] Classification name: Arrow fungus

[0043] Latin name: Ensifer adhaerens.

[0044] Name of depository institution: 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 with reference to specific embodiments. The accompanying drawings are for illustrative purposes only and are schematic, not actual, representations. They should not be construed as limiting this patent. To better illustrate the embodiments of the present invention, some components in the drawings may be omitted, enlarged, or reduced, and do not represent the actual dimensions of the product. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted from the drawings.

[0048] The PGPR used in the present invention includes Bacillus and Ensifer. Specific examples include: 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 three times with sterile water; then disinfected with 10% sodium hypochlorite solution by mass for 10 minutes and washed five times with sterile water.

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

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

[0052] 1. Streak the growth-promoting strains of Bacillus and Ensifer from a frozen glycerol tube onto solid culture medium and culture at 25-30°C for 12 hours. Then inoculate the bacteria into a 250 mL Erlenmeyer flask containing LB liquid medium and culture at 200 rpm for 12 hours before use.

[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-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 11 mL of bacterial broth solution at 5500 r / min for 6 minutes, discard the supernatant and take the bacteria, mix with 5 mL of SA solution, then add 5 mL of CMCS solution and mix, and finally add 1 mL 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 confirmed that BnC5 belongs to the genus Ensifer adhaerens. The partial gene sequence is shown in SEQID NO.1.

[0057] Example 1

[0058] Mix soil, vermiculite, and perlite in a volume ratio of 6:3:1, sterilize twice with high-pressure steam, and distribute into seedling pots. Sow seeds in the prepared soil and incubate in a light incubator for one month. During this period, add biofertilizer and regularly water with sterile water and a nutrient solution (or low-concentration solution) containing phosphorus-deficient MS powder from Amida Biotech. After one month, measure fresh weight, dry weight, stem diameter, and other relevant physiological and biochemical indicators.

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

[0060] Experimental results

[0061] like Figure 1 As shown in the results, after rapeseed was inoculated with the growth-promoting bacteria Ensifer wrapped in the composite hydrogel, 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, compared with the treatment with the growth-promoting bacteria Ensifer. This shows that the use of composite hydrogel to wrap Ensifer can significantly improve its growth-promoting effect.

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

[0063]

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

[0065]

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

[0067]

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

[0069]

[0070]

[0071] Table 5 Effects of Ensifer biofertilizer on rapeseed stem diameter under greenhouse pot conditions

[0072]

[0073] like Figure 2 As shown in the results, after rapeseed was inoculated with the growth-promoting bacteria Bacillus wrapped in the composite hydrogel, 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, compared with the treatment with the growth-promoting bacteria Bacillus. This indicates 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 potting conditions

[0075]

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

[0077]

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

[0079]

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

[0081]

[0082] Example 2

[0083] Sterilized rapeseed seeds were sown on a medium containing 1 / 2 MS and 1% agar (purchased from Amida Biotech). The pH of the plate was adjusted to 5.5, and four treatments were set: CK, PMH, Ensifer, and Ensifer-PMH. The plates were placed vertically in a 22°C light incubator (16 hours light / 8 hours dark). The light intensity was 20,000 Lux. Physiological parameters were collected at 14 days of seedling growth.

[0084] Experimental results

[0085] Under acidic conditions, after rapeseed was inoculated with the growth-promoting bacteria Ensifer wrapped in the composite hydrogel, the aboveground fresh weight (Table 10), underground fresh weight (Table 11), lateral root length (Table 12), number of lateral roots (Table 13), and lateral root density (Table 14) of the plant 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 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 Effects of Ensifer biofertilizer on the fresh weight of rapeseed underground parts under acidic conditions

[0089]

[0090] Table 12 Effects 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 solutions of the present invention and are not limiting. 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 solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a biological fertilizer, 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; The plant rhizosphere growth-promoting bacteria are the Ensifer adhaerens strain BnC5, which was deposited in the China General Microbial Culture Collection Center on July 13, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.27880.

2. The preparation method according to claim 1, 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.

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

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