Preparation method and application of liquid fertilizer containing nano-selenium and organic acid

Abstract

The invention belongs to the technical field of microbial fermentation, and relates to a preparation method and application of a liquid fertilizer containing nano-selenium and organic acid. The invention provides a preparation method of a liquid fertilizer containing nano-selenium and organic acid. The preparation method comprises the following steps: culturing liquid seeds: respectively culturing to obtain bacillus subtilis liquid seeds, saccharomyces cerevisiae liquid seeds and enterococcus faecalis liquid seeds; fermenting in a fermentation tank: adding corn steep liquor powder, soybean meal powder and brown sugar into water to prepare a fermentation culture medium; inoculating the cultured liquid seeds into a fermentation tank for fermentation to obtain a fermentation bacteria solution; preparing a liquid fertilizer: when the pH value of the fermentation liquid is 4, adding sodium selenite until the concentration of selenium in the fermentation liquid is 500mg / L, and continuing fermentation; and when the content of sodium selenite cannot be detected in the zymophyte liquid, ending the fermentation. The liquid fertilizer prepared by the invention can relieve the stress effect of high salinity and high pH value on crops at the same time.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and relates to a preparation method and application of a liquid fertilizer containing nano-selenium and organic acids. Background Art

[0002] Saline-alkali soil, also known as saline soil or saline-alkali land, refers to a type of soil in which the salt content is too high, resulting in inhibited plant growth. Such soils usually contain excessive soluble salts and may also contain too much exchangeable sodium ions. Saline-alkali soil inhibits the water absorption capacity of plant roots, leading to slow plant growth or even death. Excessive sodium ions also damage the soil structure, causing degradation of the soil ecosystem function.

[0003] Currently, the main methods for improving saline-alkali soil include leaching salts, screening salt-tolerant crops, and improving the salt tolerance of crops. Existing research has shown that there are various substances that can help plants alleviate salt stress and improve the salt tolerance of crops. For example, citric acid and malic acid can form complexes with sodium ions, reduce the absorption of sodium ions by roots, and promote the absorption of potassium ions. The trace element selenium can improve photosynthetic efficiency, activate the antioxidant defense system, induce up-regulation of salt-regulated gene expression, and alleviate salt stress by maintaining ion homeostasis through increasing the content of selenium and potassium ions and reducing sodium ion absorption.

[0004] There have been reports on using nano-selenium to alleviate salt stress and promote the growth of crops. However, in actual existence, saline-alkali soil often has the problem of high soil pH in addition to high salt content, usually around 8.0, which is not suitable for the growth of most crops.

[0005] Therefore, it is particularly important to provide a fertilizer containing nano-selenium that can simultaneously alleviate the stress effects of high salt and high pH on crops. Summary of the Invention

[0006] The present invention uses microorganisms to ferment and produce a liquid fertilizer containing nano-selenium and organic acids, which can simultaneously alleviate the stress effects of high salt and high pH on crops.

[0007] To achieve the above object, the present invention provides the following technical solutions: In the first aspect, the present invention provides a preparation method of a liquid fertilizer containing nano-selenium and organic acids, and the preparation method includes the following steps: S1. Liquid seed culture: Inoculate Bacillus subtilis into the first medium and culture it at a temperature of 30 °C and a rotation speed of 200 rpm for 12 h to obtain a Bacillus subtilis liquid seed; Inoculate Saccharomyces cerevisiae into the second medium and culture it at a temperature of 28 °C and a rotation speed of 150 rpm for 12 h to obtain a Saccharomyces cerevisiae liquid seed; Inoculate Enterococcus faecalis into the third medium and culture it at a temperature of 30 °C and a rotation speed of 100 rpm for 24 h to obtain Enterococcus faecalis liquid seeds; S2. Fermentation in a fermenter: Add corn steep liquor powder, soybean meal powder and brown sugar into water to prepare a fermentation medium; Under the condition of controlling the volume of the fermentation medium to be 80% of the volume of the fermenter, inoculate Bacillus subtilis liquid seeds, Saccharomyces cerevisiae liquid seeds and Enterococcus faecalis liquid seeds into the fermenter, keep the stirring speed at 100 rpm, and the ventilation volume at 0.1 m 3 / h, and ferment at a constant temperature of 32 °C for 48 h to obtain a fermented bacterial liquid; S3. Preparation of liquid fertilizer: When the pH value of the fermented bacterial liquid is 4, add sodium selenite, and the final concentration of selenium element in the fermented bacterial liquid is 500 mg / L. Continue fermentation, adjust the stirring speed to 60 rpm, and the ventilation volume to 0.05 m 3 / h, and continue to ferment at a constant temperature of 32 °C for 36 h; When the content of sodium selenite in the fermented bacterial liquid cannot be detected, end the fermentation.

[0008] Preferably, after the fermentation is ended, the fermented bacterial liquid is filtered through an 80-mesh sieve to filter out macromolecular substances such as raw material residues, and the obtained filtrate is the liquid fertilizer.

[0009] After filtration, macromolecular substances such as undigested raw material residues are discarded. The main components of the filtrate are beneficial microorganisms, organic matter, amino acids, organic acids, and various microbial metabolites beneficial to crops. Except for live bacteria, other components are all liquid-soluble small-molecule substances and can be quickly absorbed by crops.

[0010] Preferably, the formula of the first medium is: glucose 10 g / L, peptone 10 g / L, NaCl 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.2 g / L, MnSO4 0.01 g / L, pH 7.0 - 7.2, sterilized at 115 °C for 30 min.

[0011] Preferably, the formula of the second medium is: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, (NH4)2SO4 3 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.3 g / L, biotin 0.2 mg / L, pH 5.5 - 6.0, sterilized at 115 °C for 30 min.

[0012] Preferably, the formula of the third culture medium is: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, beef extract 5 g / L, sodium acetate 3 g / L, ammonium citrate 2 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.2 g / L, Tween 80 1 mL, pH 6.2 - 6.5, sterilized at 115°C for 30 min.

[0013] Preferably, the effective viable count of the Bacillus subtilis liquid seed, the Saccharomyces cerevisiae liquid seed, and the Enterococcus faecalis liquid seed is all 1×10 7 cfu / mL.

[0014] Preferably, the Bacillus subtilis liquid seed, the Saccharomyces cerevisiae liquid seed, and the Enterococcus faecalis liquid seed are inoculated into the fermenter according to an inoculation amount with a volume ratio of 1:1:1.

[0015] Preferably, the effective viable count in the liquid fertilizer is ≥1 billion / mL, the organic matter is ≥100 g / L, the total amino acid content is ≥100 g / L, the total organic acid content is ≥100 g / L, and the nano-selenium content is 500 mg / L.

[0016] In a second aspect, a liquid fertilizer containing nano-selenium and organic acids is provided, and the liquid fertilizer is prepared by the preparation method of the present invention.

[0017] Preferably, the particle size of the nano-selenium is 20 - 300 nm.

[0018] Preferably, the application method of the liquid fertilizer is irrigation.

[0019] In addition, a fertilizer for alleviating the stress of high salt and high pH on crops is provided, and the fertilizer includes the liquid fertilizer of the present invention.

[0020] Then, the application of the liquid fertilizer or fertilizer of the present invention in alleviating the stress of high salt and high pH on crops is provided.

[0021] Furthermore, a livestock and poultry feed containing nano-selenium and organic acids is provided, and the livestock and poultry feed is prepared by the preparation method of the present invention.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: Different fermentation purposes are achieved by controlling fermentation conditions in stages. In the early stage, through the synergistic action of Bacillus subtilis, Saccharomyces cerevisiae, and Enterococcus faecalis, macromolecular substances in corn steep liquor and soybean meal powder are decomposed into small-molecule amino acids and absorbable small-molecule organic matter, and abundant organic acids are also produced. At the same time, a sufficient number of live microorganisms and abundant metabolites are accumulated. In the later stage, sodium selenite is added, and fermentation parameters are adjusted to create a low dissolved oxygen environment, creating favorable conditions for microorganisms to convert sodium selenite into nano-selenium. Because the conversion of selenite ions to zero-valent elemental nano-selenium is a chemical reduction process, the dissolved oxygen content in the fermentation broth cannot be too high.

[0023] If a high concentration of sodium selenite is added at the beginning of fermentation, it will inhibit the growth of microorganisms and affect the decomposition of macromolecular organic matter and proteins. The staged fermentation, first accumulating the number of live bacteria and metabolites, especially the polysaccharide substances produced by metabolism, can also enhance the stability of the nano-selenium particles produced in the later stage of fermentation.

[0024] The liquid fertilizer of the present invention contains high concentrations of beneficial microorganisms, organic matter, amino acids, and organic acids, providing comprehensive nutritional support for crops; especially through the transformation of microorganisms, highly toxic inorganic selenium is converted into low-toxic nano-selenium, which not only improves the safety of product use but also enhances the absorption and utilization rate of selenium by crops through nano-sizing, enhancing the antioxidant activity of crops. These components help to improve the growth rate, yield, and quality of crops, while enhancing their adaptability to adverse conditions such as high salt and high pH. The liquid fertilizer of the present invention contributes to improving the yield and stability of crops by alleviating the stress of high salt and high pH on crops, and makes contributions to the sustainable development of agriculture.

[0025] Specifically, The present invention realizes efficient and stable microbial fermentation by precisely controlling the culture conditions and fermentation parameters (such as temperature, rotation speed, ventilation volume, etc.) of different strains, thereby obtaining a fermentation nutrient solution containing abundant organic matter, amino acids, organic acids, and nano-selenium.

[0026] The synergistic action of Bacillus subtilis, Saccharomyces cerevisiae, and Enterococcus faecalis helps to secrete amylase and protease, decomposing starch and protein in corn steep liquor powder and soybean meal into various amino acids, organic matter, and organic acids that crops can utilize, improving the nutritional value and biological activity of fermentation products. Nano-selenium can enhance the antioxidant capacity of crops and alleviate the stress of high salt environment on crops. Description of the Drawings

[0027] Figure 1 It is a scanning electron microscope result of the fermentation broth after fermentation.

[0028] Figure 2This is another scanning electron microscope result of the fermented bacterial liquid after fermentation.

[0029] Figure 3 This is yet another scanning electron microscope result of the fermented bacterial liquid after fermentation. Detailed implementation mode

[0030] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0031] Bacillus subtilis, Saccharomyces cerevisiae and Enterococcus faecalis were all purchased from the China Center for Industrial Culture Collection.

[0032] Example 1 This example provides a preparation method of a liquid fertilizer containing nano-selenium and organic acids. The preparation method includes the following steps: S1. Liquid seed culture: Inoculate Bacillus subtilis into the first medium and culture it at a temperature of 30 °C and a rotation speed of 200 rpm for 12 h to obtain 100 mL of Bacillus subtilis liquid seeds; the effective viable count of the Bacillus subtilis liquid seeds is 1×10 7 cfu / mL; Inoculate Saccharomyces cerevisiae into the second medium and culture it at a temperature of 28 °C and a rotation speed of 150 rpm for 12 h to obtain 100 mL of Saccharomyces cerevisiae liquid seeds; the effective viable count of the Saccharomyces cerevisiae liquid seeds is 1×10 7 cfu / mL; Inoculate Enterococcus faecalis into the third medium and culture it at a temperature of 30 °C and a rotation speed of 100 rpm for 24 h to obtain 100 mL of Enterococcus faecalis liquid seeds; the effective viable count of the Enterococcus faecalis liquid seeds is 1×10 7 cfu / mL.

[0033] S2. Fermentation tank fermentation: Add the culture medium raw materials into water according to the ratio of 10% (mass / volume ratio) of corn steep liquor powder, 5% (mass / volume ratio) of soybean meal powder and 5% (mass / volume ratio) of brown sugar, adjust the pH value to 7.0, and sterilize at 121 °C for 20 min to obtain the fermentation medium; Under the condition of controlling the volume of the fermentation medium to be 80% of the volume of the fermentation tank, inoculate the Bacillus subtilis liquid seeds, Saccharomyces cerevisiae liquid seeds and Enterococcus faecalis liquid seeds into the fermentation tank, keep the stirring speed at 100 rpm, and the ventilation volume at 0.1 m 3 / h, and ferment at a constant temperature of 32 °C for 48 h to obtain the fermented bacterial liquid.

[0034] It should be noted that this step is a facultative anaerobic fermentation step, aiming to utilize the amylase and protease secreted by three strains of bacteria to decompose the starch and protein in corn flour and soybean meal into amino acids, organic matter and organic acids that various crops can utilize.

[0035] S3. Liquid fertilizer preparation: Ferment for about 48 h, take samples to detect the pH value of the fermented bacterial liquid. When the pH value of the fermented bacterial liquid is about 4, add sodium selenite under aseptic operation, and the final concentration of selenium element in the fermented bacterial liquid is 500 mg / L. Reduce the stirring speed and ventilation volume of the fermenter, keep the stirring speed of the fermenter at 60 rpm, and the ventilation volume at 0.05 m 3 / h, and continue to ferment at a constant temperature of 32 °C for 36 h; This step is to create a low dissolved oxygen environment, creating favorable conditions for the microorganism to convert sodium selenite into nano selenium.

[0036] Use the o-phenylenediamine method of ultraviolet spectrophotometer to detect the content of sodium selenite in the fermented bacterial liquid. When the detected value of sodium selenite in the fermented bacterial liquid is zero, it is judged that the fermentation is over.

[0037] Take the fermented bacterial liquid and observe it with a scanning electron microscope. Nano selenium particles with a particle size of 20 - 300 nm can be seen floating around the bacterial cells, as Figure 1 , Figure 2 , Figure 3 shown. The short rod-shaped ones are bacterial cells, and the tiny particles around the bacterial cells or floating in the liquid medium are nano selenium particles.

[0038] The formula of the first medium is: glucose 10 g / L, peptone 10 g / L, NaCl 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.2 g / L, MnSO4 0.01 g / L, pH 7.0 - 7.2, sterilized at 115 °C for 30 min.

[0039] The formula of the second medium is: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, (NH4)2SO4 3 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.3 g / L, biotin 0.2 mg / L, pH 5.5 - 6.0, sterilized at 115 °C for 30 min.

[0040] The formulation of the third culture medium is as follows: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, beef extract 5 g / L, sodium acetate 3 g / L, ammonium citrate 2 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.2 g / L, Tween 80 1 mL, pH 6.2 - 6.5, sterilized at 115 °C for 30 min.

[0041] The Bacillus subtilis liquid seeds, the Saccharomyces cerevisiae liquid seeds, and the Enterococcus faecalis liquid seeds were inoculated into the fermenter at an inoculation amount with a volume ratio of 1:1:1.

[0042] The effective viable bacteria count in the liquid fertilizer is ≥ 1 billion / mL, the organic matter is ≥ 100 g / L, the total amino acid content is ≥ 100 g / L, and the total organic acid content is ≥ 100 g / L.

[0043] Among them, The effective viable bacteria count was detected by the method in "GB20287 - 2006 Agricultural microbial inoculants"; The detection method of organic matter was carried out by the dichromate volumetric method according to GB / T 17767.1 - 2023 "Organic fertilizers - Part 1: Determination of organic matter".

[0044] The amino acid content was detected by the detection method in NY / T 1977 - 2023 "Water-soluble fertilizers - Determination of free amino acid content", and detected with an amino acid analyzer.

[0045] The organic acid content was detected by the method in NY / T 4063 - 2021 "Determination of 8 organic acids such as citric acid and tartaric acid in fertilizers - Ion chromatography method".

[0046] Comparative example 1 This comparative example was the same as Example 1, except that Bacillus subtilis was replaced with Lactobacillus graminis HX (purchased from the General Microbiological Center of the China Committee for Culture Collection of Microorganisms).

[0047] Comparative example 2 This comparative example was the same as Example 1, except that soybean meal powder was replaced with wheat flour.

[0048] Comparative example 3 This comparative example was the same as Example 1, except that the formulation of the first culture medium was adjusted to: glucose 20 g / L, beef extract 10 g / L, NaCl 3 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, MnSO4 0.02 g / L, pH 7.0 - 7.2.

[0049] Effect verification 1. Experimental object: Liquid fertilizers containing nano-selenium and organic acids prepared according to Example 1 and Comparative Examples 1-3; 2. Experimental method: A field plot experiment on Chinese cabbage cultivation was carried out in the saline-alkali soil of Dawang Village, Li County, Baoding City, Hebei Province. The pH value of the tested soil was 7.88, the EC value was 1017 us / cm, and the salt content was 3.9 g / kg. The area of each plot was 3m * 6m = 18m 2 , and 5 treatments were set, namely the control and the products of Example 1 of the present invention (hereinafter referred to as nano-selenium amino acid fertilizer) and the products of Comparative Examples 1-3, with 3 replicates. The fertilization method was irrigation. The control was irrigation with clear water, and the nano-selenium amino acid fertilizer was diluted 500 times with clear water and then irrigated. The fertilization time was to irrigate once when watering the fixed root water after transplanting Chinese cabbage, and then irrigate once again at the rosette stage according to the above fertilization amount. The usage amount of chemical fertilizers during the planting period was the same for all 5 treatments. Calculated as per mu, 30 kg of 20-20-20 compound fertilizer was used as base fertilizer. Chinese cabbage was transplanted on May 8 and harvested on June 11. After harvesting Chinese cabbage, the physical and chemical properties of the soil were detected respectively, and the yield of Chinese cabbage was measured.

[0050] 3. Sample collection and determination: For soil samples, the soil drilling method was used. The sampling depth was 20 cm. 5 points were taken from each plot and mixed evenly as a soil sample, air-dried, picked out of sundries, and ground for testing.

[0051] The EC value, pH value, and total water-soluble salts of the soil were determined by extraction with a soil-water ratio of 1:5.

[0052] Yield determination: The weighing method was used for determination. After all the Chinese cabbages in each plot were picked, they were weighed.

[0053] 4. Test results: As shown in Table 1.

[0054] Table 1 Test results

[0055] It can be seen from the results in Table 1 that after using the nano-selenium amino acid fertilizer of the present invention, the pH value of the test plot can be significantly reduced, the soil pH value can be restored to neutral, and the soil salt content also drops significantly, with a drop amplitude of 36.6%. The yield of Chinese cabbage has increased significantly. The yield per mu of irrigating with clear water is only 2018 kg, which belongs to low yield, indicating that the saline-alkali land has a great impact on the yield of Chinese cabbage. After using the nano-selenium amino acid fertilizer of the present invention, the yield has increased by 62.3%, and the yield per mu has reached 3275 kg, with a very significant increase.

[0056] Compared with the control, for Comparative Examples 1-3, the EC value, salt content, and pH value of the soil have all decreased, but the decrease amplitude is not as large as that of the treatment with the product of the present invention, and the yield increase is also the most obvious with the product of the present invention.

[0057] Based on the above results, it can be seen that the product of the present invention can well alleviate the stress of high salt and high pH on crops.

[0058] It should be understood that the present invention disclosed is not limited to the specific methods, schemes and substances described, as these can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which is limited only by the appended claims.

Claims

1. A method for preparing a liquid fertilizer containing nano-selenium and organic acid, characterized in that, The preparation method includes the following steps: S1. Liquid seed culture: Inoculate Bacillus subtilis into the first medium and culture it at a temperature of 30°C and a rotation speed of 200 rpm for 12 h to obtain Bacillus subtilis liquid seeds; Inoculate Saccharomyces cerevisiae into the second medium and culture it at a temperature of 28°C and a rotation speed of 150 rpm for 12 h to obtain Saccharomyces cerevisiae liquid seeds; Inoculate Enterococcus faecalis into the third medium and culture it at a temperature of 30°C and a rotation speed of 100 rpm for 24 h to obtain Enterococcus faecalis liquid seeds; S2. Fermentation in a fermenter: Add corn steep powder, soybean meal powder and brown sugar into water to prepare a fermentation medium; Under the condition of controlling the volume of the fermentation medium to 80% of the volume of the fermenter, the liquid seeds of Bacillus subtilis, Saccharomyces cerevisiae and Enterococcus faecalis were inoculated into the fermenter, the stirring speed was maintained at 100 rpm, and the ventilation volume was 0.1 m 3 / h, and fermentation was carried out at a constant temperature of 32 °C for 48 h to obtain the fermented bacterial liquid; S3. Preparation of liquid fertilizer: When the pH value of the fermented bacterial liquid is 4, sodium selenite is added, and the final concentration of selenium element in the fermented bacterial liquid is 500 mg / L. Adjust the stirring speed to 60 rpm and the ventilation volume to 0.05 m 3 / h, and continue fermentation at a constant temperature of 32 °C for 36 h; When the content of sodium selenite cannot be detected in the fermented bacterial liquid, end the fermentation.

2. The preparation method according to claim 1, characterized in that, The formula of the first medium is: glucose 10 g / L, peptone 10 g / L, NaCl 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.2 g / L, MnSO4 0.01 g / L, pH 7.0 - 7.

2.

3. The preparation method according to claim 1, wherein, The formula of the second medium is: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, (NH4)2SO4 3 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.3 g / L, biotin 0.2 mg / L, pH 5.5 - 6.

0.

4. The preparation method according to claim 1, characterized in that, The formula of the third medium is: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, beef extract 5 g / L, sodium acetate 3 g / L, ammonium citrate 2 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.2 g / L, Tween 80 1 mL, pH 6.2 - 6.

5.

5. The preparation method according to claim 1, characterized in that, The effective viable count of the Bacillus subtilis liquid seed, the Saccharomyces cerevisiae liquid seed, and the Enterococcus faecalis liquid seed is all 1×10 7 cfu / mL.

6. The preparation method according to claim 1, characterized in that, Inoculate the Bacillus subtilis liquid seeds, the Saccharomyces cerevisiae liquid seeds and the Enterococcus faecalis liquid seeds into the fermenter according to the inoculation amount with a volume ratio of 1:1:

1.

7. The preparation method according to claim 1, characterized in that, The effective viable bacteria count in the liquid fertilizer ≥ 1 billion / mL, the organic matter ≥ 100 g / L, the total amino acid content ≥ 100 g / L, and the total organic acid content ≥ 100 g / L.

8. Liquid fertilizer containing nano-selenium and organic acid, characterized in that, The liquid fertilizer is prepared by the preparation method described in any one of claims 1 - 7.

9. A fertilizer for alleviating the stress of high salt and high pH on crops, characterized in that, The fertilizer includes the liquid fertilizer described in claim 8.

10. Application of the liquid fertilizer described in claim 8 or the fertilizer described in claim 9 in alleviating the stress of high salt and high pH on crops.

Images