CN111647527A - Method for improving saline-alkali soil and preserving fertilizer by utilizing bacillus subtilis - Google Patents
Method for improving saline-alkali soil and preserving fertilizer by utilizing bacillus subtilis Download PDFInfo
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Abstract
The invention provides a method for improving saline-alkali soil and preserving fertilizer by utilizing bacillus subtilis, which is characterized in that the physicochemical property of the saline-alkali soil is improved and the utilization efficiency of phosphate fertilizer by crops is obviously improved in a high-salinity environment of the saline-alkali soil by utilizing a fermentation product extracted from a fermentation bacterium liquid of the bacillus subtilis. The method has the advantages of simple process, environmental protection, harmlessness and obvious effect; the microorganism is fully utilized to improve the saline-alkali soil, the content of available phosphorus in the soil is increased, and the utilization efficiency of crops on the phosphorus in the soil is improved.
Description
Technical Field
The invention relates to the field of saline-alkali soil improvement, in particular to a method for improving saline-alkali soil and improving the utilization efficiency of phosphate fertilizer in saline-alkali soil by utilizing bacillus subtilis.
Background
The salinized soil in China has wide distribution range, large area and various types, the total area is about 1 hundred million hectares, and soluble salts in the salinized soil mainly comprise sulfates, chlorides, carbonates and bicarbonates of sodium, potassium, calcium, magnesium and the like. Because the soil is salinized, the soil is hardened, and the effectiveness of applying fertilizers in the soil is greatly reduced, especially phosphate fertilizers. Most soils are phosphorus deficient soils, and phosphorus is an essential nutrient element for crops, which seriously affects the growth quality and yield of crops. The content circulation of phosphorus in nature is a unidirectional mode, the consumption of the phosphorus is continuously increased, and the content of phosphorus available for recovery is little. In addition, soil salinization is the greatest stress causing the deterioration of arable land, the reduction of the yield of grain crops and the reduction of the quality. 20% of world arable land and 30% of irrigation agriculture are subject to high salt stress.
The salinity of the soil obviously reduces the absorption of the crops to the nutrition, particularly the loss of phosphorus ions, and the phosphorus ions and calcium ions can be precipitated in the saline soil. The mineralization of organic phosphorus in soil increases with increasing pH, unlike the mineralization of organic carbon, organic N in soil. For most crops, the maximum available pH of phosphorus is 5.5-7.0, where soil microbial activity is also strongest. Up to 90% of mined phosphate minerals are used in the production of fertilizers. Phosphorus in the calcareous soil is difficult to be utilized by crops, and the problems of high total phosphorus content and insufficient available phosphorus content in high-yield soil occur. At present, the application efficiency of global phosphorus is not high for improving crop yield, and 74% cultivated land in China is phosphorus-deficient soil, wherein the phosphorus-deficient soil is soil with effective phosphorus content less than 10 mg/kg.
At present, the technology for repairing saline-alkali soil in large area adopted at home and abroad is mainly a physical and chemical repairing technology. The chemical repairing agent is mainly used for repairing soda soil. Bioremediation includes phytoremediation and microbial remediation, where there are two main mechanisms of phytodesalination, one is by lowering the pH. Researches find that the microorganism is a more economic and efficient repair mode and has great application prospect.
The microorganisms such as bacillus subtilis can produce a series of fermentation products through fermentation culture, and the utilization prospect of the microbial polymer in environmental remediation and agriculture can be further clarified through researching the improvement effect of the microbial fermentation products on saline-alkali soil and the effect of the microbial fermentation products on the utilization efficiency of phosphate fertilizers by crops, so that the method has important scientific significance.
Disclosure of Invention
The invention aims to provide a method for improving saline-alkali soil and improving the utilization efficiency of crops on phosphate fertilizers in saline-alkali soil by utilizing bacillus subtilis (No. BNCC190341) purchased from a Beijing Beinano biological strain storage warehouse in China. The invention can reduce the pH value and the total salt content of the soil by using the secretion product of the bacterial strain in the saline-alkali soil environment, and has the function of activating the phosphorus in the soil. Has important significance for improving the soil of saline-alkali areas and growing crops.
The technical scheme adopted by the invention is as follows: the invention provides a method for improving saline-alkali soil and preserving fertilizer by utilizing bacillus subtilis, which comprises the following specific steps:
(1) fermentation culture of strain and extraction of fermentation product
(1.1) inoculating the bacillus subtilis to a seed culture medium by the inoculation amount of 3-5% (V/V), and carrying out shake cultivation for 40-50h at 30-35 ℃ and 140r/min with 120-. The formula of the seed culture medium is as follows: 10-20g/L of soybean peptone, 30-40g/L of glucose, 25-40g/L of sodium glutamate and 10-30g/L of NaCl.
(1.2) inoculating the bacterial liquid cultured in the step (1.1) into a fermentation culture medium in an inoculation amount of 3-5% (V/V), and performing shake cultivation at 32-38 ℃ and 140r/min of 120-. The formula of the fermentation medium is as follows: soybean peptone 20-30g/L, glucose 30-40g/L, sodium glutamate 30-50g/L, NaCl 15-20g/L, MgSO40.5-1g/L，CaCl20.25-0.5g/L，K2HPO42.0-3.0g/L，KH2PO44.0-5.0g/L and 0.1-0.5 g/L biotin. Then adopting organic solvent (ethanol) precipitation method: adjusting the pH value of fermentation broth to 3-4 with hydrochloric acid, centrifuging at 12000r/min for 10min, continuously centrifuging for 2 times, precipitating supernatant with 95% ethanol at-20 ℃ overnight at a volume ratio of 1: 2-4, centrifuging at 12000r/min at 4 ℃ for 10min to remove the supernatant, fully dissolving the precipitate with deionized water, centrifuging at 12000r/min to remove the precipitate, dialyzing the supernatant with a dialysis bag for desalting until the supernatant is clarified to obtain a desalted solution, and freeze-drying to obtain a fermentation product.
(2) Adjustment of physicochemical properties of saline-alkali soil and fertilizer retention of fermentation product of bacillus subtilis
Planting crops in saline-alkali soil of a certain saline-alkali wasteland, applying 100-150 mg/kg of phosphate fertilizer, 100-200 mg/kg of nitrogen fertilizer and 10-50 mg/kg of potassium fertilizer, adding 10-250mg/L of fermentation products with different concentrations, adding primary fermentation product solution for 5-7 days, and watering the crops with the water irrigation amount of 20-30%. Stopping pouring the fermentation product solution in the last week before harvesting, and harvesting after 30 days. The fermentation product can reduce the pH value and the total salt content of the soil and has an activating effect on phosphorus in the soil.
Further, in the step (1), the used bacillus subtilis can grow well under the salinity condition of 10-30 per thousand in mass fraction, can tolerate the salinity of 10-30 per thousand in mass fraction, and the OD660 in the stable period of the fermentation culture of the strain reaches 2.25.
The invention has the following beneficial effects:
(1) the utilized substances are obtained by a microbial fermentation method, the cost is lower, the pollution is less, and the microbes can tolerate 10-30 per mill of salt and can be used in a high-saline-alkali environment;
(2) the invention further verifies the influence of microorganisms on plant growth in the saline-alkali soil, and performs a potting experiment to explain the function of microorganism fermentation products for regulating the saline-alkali soil and the influence on phosphate fertilizer utilization;
(3) the method is environment-friendly and harmless, has low cost, can improve saline-alkali soil by microbial fermentation products, has the functions of water and fertilizer retention, and has positive significance in agricultural production and environmental remediation and treatment.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the invention provides a method for improving saline-alkali soil and having fertilizer retention effect by using microorganisms (bacillus subtilis). The method comprises the steps of extracting a bacillus subtilis fermentation product, adding the extract into saline-alkali soil, planting crops in the saline-alkali soil in a seedling transplanting mode, and measuring the physicochemical properties of the improved saline-alkali soil and the utilization efficiency of the crops on phosphate fertilizer in the crop planting process.
A. The bacillus subtilis is purchased from a Beijing Beina biological strain storage library (the number is BNCC190341) in China.
B. B, domestication of bacillus subtilis: firstly, inoculating 3-5% of the bacillus subtilis to a seed culture medium and carrying out shake cultivation for 40-50h at 30-35 ℃ and 140 r/min. The formula of the seed culture medium is as follows: 10-20g/L of soybean peptone, 30-40g/L of glucose, 25-40g/L of sodium glutamate and 10-30g/L of NaCl.
C. B, fermentation culture of the bacillus subtilis: inoculating the strain in the seed culture solution to a fermentation culture medium by 3-5% of the inoculum size, and performing shake culture at 32-38 ℃ and 140r/min for 40-50h to obtain a zymogen solution. Fermentation medium components: soybean peptone 20-30g/L, glucose 30-40g/L, sodium glutamate 30-50g/L, NaCl 15-20g/L, MgSO40.5-1g/L，CaCl20.25-0.5g/L，K2HPO42.0-3.0g/L，KH2PO44.0-5.0g/L and 0.1-0.5 g/L biotin.
D. The fermentation product has the following effects of regulating the physicochemical properties of saline-alkali soil and keeping fertilizer: planting crops in a flowerpot filled with saline-alkali soil, applying 100-150 mg/kg of phosphate fertilizer, 100-200 mg/kg of nitrogenous fertilizer and 10-50 mg/kg of potash fertilizer, and adding a primary fermentation product (10-250mg/L) solution for 5-7 days, wherein the irrigation amount is 20-30%. During the period, nutrient solution is applied once, the components of the nutrient solution are 5-10g of urea, 3-5g of monopotassium phosphate, 1-5g of calcium sulfate, 0.5-1g of magnesium sulfate, 0.001-0.01g of zinc sulfate, 0.003-0.005g of ferric sulfate, 0.001-0.01g of copper sulfate, 0.003-0.005g of manganese sulfate and 0.002-0.01g of boric acid powder, and water is added to 10L after dissolution. Harvesting about 30 days, and stopping pouring the fermentation product solution in the last week before harvesting. The sampling determination analysis shows that: substances generated by fermentation of the bacillus subtilis can reduce the pH value and the total salt content of soil and activate phosphorus in the soil, so that the bacillus subtilis has important significance for soil improvement and crop growth in saline-alkali areas.
Example 1
(1) Strain source and domestication culture: the selected strain is bacillus subtilis which is purchased from a Beijing Beina biological strain storage library in China, and the storage number is BNCC 190341. In order to improve the fermentation capacity of the bacillus subtilis, the bacillus subtilis is firstly inoculated into a seed culture medium for acclimatization culture, is inoculated into the seed culture medium with the inoculation amount of 3 percent and is subjected to shake cultivation for 40 hours at the temperature of 30 ℃ and at the speed of 120 r/min. The formula of the seed culture medium is as follows: 10g/L of soybean peptone, 30g/L of glucose, 25g/L of sodium glutamate and 10g/L of NaCl;
(2) b, fermentation culture of the bacillus subtilis: inoculating the strain in the seed culture solution into a fermentation culture medium by 3 percent of inoculation amount, and performing shake culture at 32 ℃ and 120r/min for 40h to obtain a zymogen solution. The fermentation medium comprises the following components: soybean peptone 20g/L, glucose 30g/L, sodium glutamate 30g/L, NaCl 15g/L, MgSO40.5g/L，CaCl20.25g/L，K2HPO42.0g/L，KH2PO44.0g/L and 0.1g/L biotin.
(3) Extracting a bacillus subtilis fermentation product: the extraction of the strain product adopts an organic solvent precipitation method. Adjusting pH of the fermentation liquor to 3 with hydrochloric acid, centrifuging at 12000r/min for 10min, continuously centrifuging for 2 times, precipitating the supernatant with 2 times volume of 95% ethanol at-20 deg.C overnight, centrifuging at 12000r/min at 4 deg.C for 10min to remove the supernatant, dissolving the precipitate with deionized water, centrifuging at 12000r/min to remove the precipitate, dialyzing the supernatant with dialysis bag for desalting, and freeze drying the desalted solution to obtain pure product.
(4) The fermentation product has the following effects of regulating the physicochemical properties of saline-alkali soil and keeping fertilizer: firstly, seeds of the crop beet are planted in a small seedling raising pot, then seedling transplantation is carried out, and the seedlings are transplanted (with soil) into a flowerpot filled with saline-alkali soil. 100mg/kg of phosphate fertilizer, 100mg/kg of nitrogenous fertilizer and 10mg/kg of potash fertilizer are added, 10mg/L of solution of fermentation products is added every 5 days, and the irrigation amount is 20%. During the period, nutrient solution is applied once, the components of the nutrient solution are 5g of urea, 3g of monopotassium phosphate, 1g of calcium sulfate, 0.5g of magnesium sulfate, 0.001g of zinc sulfate, 0.003g of ferric sulfate, 0.001g of copper sulfate, 0.003g of manganese sulfate and 0.002g of boric acid powder, and water is added to 10L after dissolution. Harvesting is carried out for about 30 days, and irrigation is stopped in the last week before harvesting. And taking soil samples and plant samples from 7, 14, 27 and 34 days after seedling transplantation.
Effects on sugar beet: compared with a blank group added with 10mg/L fermentation product, the photosynthetic rate can be improved by 38% at the 7 th day, and is improved by 19% at the 14 th day, and the effect is not obvious at the 27 th day and the 34 th day. Compared with the blank group, the plant height of the final plant is improved by 18%, the fresh weight is improved by 86%, and the dry weight is improved by 6%. The total phosphorus in the beets was increased by an average of 17.9% relative to the blank control group when 10mg/L fermentation product was administered. Influence on soil physicochemical properties: compared with a blank group, the pH value is reduced by 0.5%, the EC is reduced by 18%, the total salt content is reduced by 14%, the influence on the total phosphorus in the soil is not obvious, and the effective phosphorus content is improved by 94% by adding 10mg/L of the fermentation product.
Example 2
(1) Strain source and domestication culture: the selected strain is bacillus subtilis which is purchased from a Beijing Beina biological strain storage library in China, and the storage number is BNCC 190341. Firstly, inoculating the bacillus subtilis into a seed culture medium for acclimatization and culture, inoculating the bacillus subtilis into the seed culture medium with the inoculation amount of 5 percent, and performing shake culture at 35 ℃ and 140r/min for 50 h. The formula of the seed culture medium is as follows: soybean peptone 20g/L, glucose 40g/L, sodium glutamate 40g/L, NaCl 30 g/L;
(2) b, fermentation culture of the bacillus subtilis: inoculating the strain in the seed culture solution into a fermentation culture medium at an inoculation amount of 5%, and performing shake culture at 38 ℃ at 140r/min for 50h to obtain a zymogen solution. The fermentation medium comprises the following components: 30g/L of soybean peptone, 40g/L of glucose, 50g/L of sodium glutamate, 20g/L of NaCl and MgSO41g/L，CaCl20.5g/L，K2HPO43.0g/L，KH2PO45.0g/L and 1.0g/L biotin.
(3) Extracting a bacillus subtilis fermentation product: the extraction of the strain product adopts an organic solvent precipitation method. Adjusting pH of the fermentation liquor to 4 with hydrochloric acid, centrifuging at 12000r/min for 10min, continuously centrifuging for 2 times, precipitating the supernatant with 4 times volume of 95% ethanol at-20 deg.C overnight, centrifuging at 12000r/min at 4 deg.C for 10min to remove the supernatant, dissolving the precipitate with deionized water, centrifuging at 12000r/min to remove the precipitate, dialyzing the supernatant with dialysis bag for desalting, and freeze drying the desalted solution to obtain pure substance.
(4) The fermentation product has the following effects of regulating the physicochemical properties of saline-alkali soil and keeping fertilizer: firstly, seeds of the crop beet are planted in a small seedling raising pot, then seedling transplantation is carried out, and the seedlings are transplanted (with soil) into a flowerpot filled with saline-alkali soil. 150mg/kg of phosphate fertilizer, 200mg/kg of nitrogen fertilizer and 50mg/kg of potassium fertilizer are added, 250mg/L of fermentation product solution is added every 6 days, and the water irrigation amount is 30 percent. During the period, nutrient solution is applied once, the nutrient solution comprises 7g of urea, 5g of monopotassium phosphate, 5g of calcium sulfate, 1g of magnesium sulfate, 0.005g of zinc sulfate, 0.005g of ferric sulfate, 0.005g of copper sulfate, 0.005g of manganese sulfate and 0.001g of boric acid powder, and water is added to 10L after the nutrient solution is dissolved. Harvesting is carried out for about 30 days, and irrigation is stopped in the last week before harvesting. And taking soil samples and plant samples from 7, 14, 27 and 34 days after seedling transplantation.
Effects on sugar beet: compared with the blank group added with 250mg/L fermentation product, the photosynthetic rate can be improved by 26% at the 7 th day, and by the 14 th day, the photosynthetic rate is improved by 19% compared with the blank group, and by 64% compared with the blank group at the 34 th day. Compared with the blank group, the plant height of the final plant is improved by 14%, the fresh weight is improved by 36%, and the dry weight is improved by 7%. The total phosphorus of the beet is increased by 61.2 percent compared with the blank control group when 250mg/L of fermentation product is applied. Influence on soil physicochemical properties: compared with a blank group added with 250mg/L fermentation product, the pH value is reduced by 0.3%, the EC is reduced by 20%, the total salt content is reduced by 20%, and the total phosphorus in the soil is improved by 5%.
Example 3
(1) Strain source and domestication culture: the selected strain is bacillus subtilis which is purchased from a Beijing Beina biological strain storage library in China, and the storage number is BNCC 190341. Firstly, inoculating the bacillus subtilis into a seed culture medium for acclimatization and culture, inoculating the bacillus subtilis into the seed culture medium with the inoculation amount of 4.5%, and performing shake culture at 32 ℃ and 130r/min for 45 hours. The formula of the seed culture medium is as follows: 15g/L of soybean peptone, 35g/L of glucose, 35g/L of sodium glutamate and 20g/L of NaCl;
(2) b, fermentation culture of the bacillus subtilis: inoculating the strain in the seed culture solution into a fermentation culture medium at an inoculum size of 4.5%, and performing shake culture at 35 deg.C and 130r/min for 45h to obtain zymogen solution. The fermentation medium comprises: 25g/L of soybean peptone, 35g/L of glucose, 40g/L of sodium glutamate, 18g/L of NaCl and MgSO40.7g/L，CaCl20.35g/L，K2HPO42.5g/L，KH2PO44.5g/L and 0.3g/L biotin.
(3) Extracting a bacillus subtilis fermentation product: the extraction of the strain product adopts an organic solvent precipitation method. Adjusting pH of the fermentation liquor to 3.5 with hydrochloric acid, centrifuging at 12000r/min for 10min, continuously centrifuging for 2 times, precipitating the supernatant with 3 times volume of 95% ethanol at-20 deg.C overnight, centrifuging at 12000r/min at 4 deg.C for 10min to remove the supernatant, dissolving the precipitate with deionized water, centrifuging at 12000r/min to remove the precipitate, dialyzing the supernatant with dialysis bag for desalting, and freeze drying the desalted solution to obtain pure substance.
(4) The fermentation product has the following effects of regulating the physicochemical properties of saline-alkali soil and keeping fertilizer: firstly, seeds of the crop beet are planted in a small seedling raising pot, then seedling transplantation is carried out, and the seedlings are transplanted (with soil) into a flowerpot filled with saline-alkali soil. 120mg/kg of phosphate fertilizer, 150mg/kg of nitrogen fertilizer and 30mg/kg of potassium fertilizer are added, 100mg/L of fermentation product solution is added every 7 days, and the water irrigation amount is 30%. During the period, nutrient solution is applied once, the nutrient solution comprises 7g of urea, 5g of monopotassium phosphate, 5g of calcium sulfate, 1g of magnesium sulfate, 0.005g of zinc sulfate, 0.005g of ferric sulfate, 0.005g of copper sulfate, 0.005g of manganese sulfate and 0.001g of boric acid powder, and water is added to 10L after the nutrient solution is dissolved. Harvesting is carried out for about 30 days, and irrigation is stopped in the last week before harvesting. And taking soil samples and plant samples from 7, 14, 27 and 34 days after seedling transplantation.
Effects on sugar beet: compared with the blank group, the photosynthetic rate can be improved by 34% at the 7 th day and improved by 20% at the 14 th day by adding 100mg/L fermentation product. Compared with the blank group, the plant height of the final plant is increased by 25%, the fresh weight is increased by 175%, and the dry weight is increased by 12%. The total phosphorus of the beet is increased by 24 percent compared with that of a blank control group when 100mg/L fermentation product is applied. Influence on soil physicochemical properties: compared with a blank group added with 100mg/L fermentation product, the pH value is reduced by 0.7%, the EC is reduced by 19%, and the total salt content is reduced by 23%.
In conclusion, the invention mainly utilizes the bacillus subtilis to improve the saline-alkali soil and improve the utilization efficiency of the phosphate fertilizer of crops in the saline-alkali soil, and the technology improves the physicochemical property of the saline-alkali soil in the high-salinity environment of the saline-alkali soil by utilizing the fermentation product after the microbial fermentation culture, improves the absorption of the crops to the phosphorus in the saline-alkali soil, thereby promoting the growth effect of the crops, and has important effects in soil improvement and agriculture.
The above-described embodiments are intended to illustrate rather than to limit the invention, and any modifications and variations of the present invention are within the spirit of the invention and the scope of the appended claims.
Claims (2)
1. A method for improving saline-alkali soil and preserving fertilizer by utilizing bacillus subtilis is characterized by comprising the following specific steps:
(1) fermentation culture of strain and extraction of fermentation product
(1.1) inoculating the bacillus subtilis to a seed culture medium by the inoculation amount of 3-5% (V/V), and carrying out shake cultivation for 40-50h at 30-35 ℃ and 140r/min with 120-. The formula of the seed culture medium is as follows: 10-20g/L of soybean peptone, 30-40g/L of glucose, 25-40g/L of sodium glutamate and 10-30g/L of NaCl.
(1.2) inoculating the bacterial liquid cultured in the step (1.1) into a fermentation culture medium in an inoculation amount of 3-5% (V/V), and performing shake cultivation at 32-38 ℃ and 140r/min of 120-. The formula of the fermentation medium is as follows: soybean peptone 20-30g/L, glucose 30-40g/L, sodium glutamate 30-50g/L, NaCl 15-20g/L, MgSO40.5-1g/L，CaCl20.25-0.5g/L，K2HPO42.0-3.0g/L，KH2PO44.0-5.0g/L and 0.1-0.5 g/L biotin. Then adopting an organic solvent (ethanol) precipitation method: adjusting the pH value of fermentation broth to 3-4 with hydrochloric acid, centrifuging at 12000r/min for 10min, continuously centrifuging for 2 times, precipitating supernatant with 95% ethanol at-20 ℃ overnight at a volume ratio of 1: 2-4, centrifuging at 12000r/min at 4 ℃ for 10min to remove the supernatant, fully dissolving the precipitate with deionized water, centrifuging at 12000r/min to remove the precipitate, dialyzing the supernatant with a dialysis bag for desalting until the supernatant is clarified to obtain a desalted solution, and freeze-drying to obtain a fermentation product.
(2) Adjustment of physicochemical properties of saline-alkali soil and fertilizer retention of fermentation product of bacillus subtilis
Planting crops in saline-alkali soil of a certain saline-alkali wasteland, applying 100-150 mg/kg of phosphate fertilizer, 100-200 mg/kg of nitrogen fertilizer and 10-50 mg/kg of potassium fertilizer, adding 10-250mg/L of fermentation products with different concentrations, adding primary fermentation product solution for 5-7 days, and watering the crops with the water irrigation amount of 20-30%. Stopping pouring the fermentation product solution in the last week before harvesting, and harvesting after 30 days. The fermentation product can reduce the pH value and the total salt content of the soil and has an activating effect on phosphorus in the soil.
2. The method for improving saline-alkali soil and preserving fertilizer by utilizing bacillus subtilis as claimed in claim 1, wherein in the step (1), the used bacillus subtilis can grow well under the salinity condition of 10-30 per thousand of mass fraction, can tolerate the salinity of 10-30 per thousand of mass fraction, and the OD660 of the strain fermentation culture stationary phase reaches 2.25.
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