A method for preparing acidified soil conditioner based on microbial fermentation

Abstract

The invention relates to the technical field of microbial fermentation, and discloses a method for preparing an acidified soil conditioner based on microbial fermentation, comprising the following steps: inoculating an acidified soil conditioner with a phosphorus-solubilizing effect into a fermentation culture medium, performing a first fermentation, and obtaining a primary fermentation liquid; adding mussel shell powder to the primary fermentation liquid, performing a second fermentation, and obtaining a secondary fermentation liquid; and separating products from the secondary fermentation liquid to obtain an acidified soil conditioner. The acidified soil conditioner prepared by the method of the invention can quickly increase the pH of the acidified soil, and at the same time can also increase the soluble phosphorus content in the soil, and has a good acidified soil repair effect.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a method for preparing an acidified soil conditioner based on microbial fermentation. Background Art

[0002] Soil acidification refers to the phenomenon that soil pH decreases and acidity increases. Soil acidification can be caused by both natural processes and human influences, such as the secretion of organic acids by plant roots, the production of acidic substances during the decomposition of plant residues or litter, and the formation of strong acids after the residual anions after the application of physiologically acidic fertilizers combine with hydrogen ions in the soil. Soil acidification is one of the main manifestations of soil degradation, which will cause the effectiveness of soil nutrients and soil fertility to decrease, and the activity of heavy metals to increase. In addition, it will affect the soil microbial structure and enzyme activity, thereby hindering the growth and development of plants.

[0003] Acidification soil conditioners can be used to repair acidified soils and increase soil pH. Commonly used acidification soil conditioners include chemical conditioners and microbial conditioners. Among them: chemical conditioners (such as mineral lime) have a slow improvement rate on the acidity of the subsoil, and their excessive use will cause the bioavailability of trace nutrients in the soil to decrease; in contrast, microbial conditioners use the metabolic activities of microorganisms to repair acidified soils, which has less adverse effects on the environment, and therefore has received greater attention in recent years. At present, microbial conditioners are limited by factors such as the reproduction rate of microorganisms in the soil, and the rate of increasing soil pH is slow, and it is difficult to quickly increase the available phosphorus content in acidified soils. Summary of the invention

[0004] In order to solve the technical problem that microbial conditioners are difficult to quickly increase the pH and effective phosphorus content of soil, the present invention provides a method for preparing an acidified soil conditioner based on microbial fermentation. The acidified soil conditioner prepared by the method of the present invention can quickly increase the pH of acidified soil and at the same time increase the soluble phosphorus content in the soil, and has a good acidified soil repair effect.

[0005] The specific technical scheme of the present invention is:

[0006] In a first aspect, the present invention provides a method for preparing an acidified soil conditioner based on microbial fermentation, comprising the following steps:

[0007] (1) inoculating an acidified soil improving bacterium having a phosphate-solubilizing function into a fermentation culture solution, performing a first fermentation, and obtaining a primary fermentation solution;

[0008] (2) adding mussel shell powder to the primary fermentation liquid to perform a second fermentation to obtain a secondary fermentation liquid;

[0009] (3) Separating the products from the secondary fermentation liquid to obtain an acidified soil conditioner.

[0010] In the present invention, the acidified soil improver with phosphorus solubilizing effect is used for the first fermentation, which can make the strain rapidly expand and secrete substances that can increase the pH value of the soil. Then, mussel shell powder is added for the second fermentation, and the phosphorus solubilizing effect of the acidified soil improver can be used to convert the insoluble phosphorus in the mussel shell powder into soluble phosphorus, so that the acidified soil improver of the present invention can increase the soluble phosphorus content in the acidified soil to a greater extent; in addition, the mussel shell powder contains calcium carbonate and calcium oxide, which can neutralize the acidity of the soil, thereby improving the acidified soil improvement effect. When the acidified soil improver prepared by the present invention is applied to the acidified soil, the acidified soil improver and the substances that can increase the pH value of the soil produced during the first fermentation, as well as the mussel shell powder, can be used to quickly increase the pH value of the soil. At the same time, the acidified soil improver can also be used to convert the insoluble phosphorus in the soil into soluble phosphorus, and the soluble phosphorus released from the mussel shell powder during the second fermentation process can be used to quickly increase the effective phosphorus content in the soil.

[0011] Preferably, in step (1), the strain name of the acidified soil improving bacteria is XHZG06-95A3, classified as Candida palmioleophila, and deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, the deposit address of the depository is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is December 4, 2023, and the deposit number is CGMCC No. 29215

[0012] Candida palmioleophila is a species in the genus Candida, and there is no unified Chinese translation at present. There has been no report of a strain that can improve acidified soil in this species. The strain XHZG06-95A3 used in the present invention has high acid resistance, and after being applied to acidified soil, it can effectively improve soil pH, and its mechanism may be to reduce soil acidity by using acidic substances in the soil, and to produce amine substances to neutralize hydrogen ions in the soil. In addition, in acidic soil, phosphorus is easily fixed, has low biological effectiveness, and is difficult to be absorbed and utilized by plants. Phosphorus deficiency can cause greater inhibition on plant growth, and the strain XHZG06-95A3 of the present invention has a phosphorus-solubilizing effect, which can convert the fixed insoluble phosphorus into soluble phosphorus (effective phosphorus), so that it can be absorbed and utilized by plants. In addition, at pH 3-8 and 15-35°C, the strain XHZG06-95A3 can grow well and reduce environmental acidity, so it has good adaptability to the environment.

[0013] Preferably, in step (1), the fermentation broth comprises components in the following concentrations: yeast extract 5-10 g / L, glucose 10-20 g / L, peptone 10-20 g / L, K2HPO4 0.5-1 g / L, (NH4)2HPO4 3-6 g / L, Mg2SO4·7H2O0.5-1 g / L, KCl 0-0.3 g / L, the solvent is water, and the pH is 6-7.

[0014] Preferably, in step (1), the OD of the primary fermentation broth is 600 It is 0.7~1.2.

[0015] Preferably, in step (2), the mass volume ratio between the mussel shell powder and the first fermentation liquid is 2-8 g:100 mL.

[0016] Preferably, in step (2), before the second fermentation, a carbon source is added to the first fermentation broth; the carbon source is one or more of glucose, galactose, sucrose and starch.

[0017] By adding a carbon source, the strain XHZG06-95A3 can be promoted to play a role in solubilizing phosphorus during the second fermentation, and the insoluble phosphorus in the mussel shell powder can be converted into soluble phosphorus that can be absorbed and utilized by plants. In addition, for the strain XHZG06-95A3 used in the present invention, when one or more of glucose, galactose, sucrose and starch is used as a carbon source, the effect of promoting its solubilizing phosphorus is better.

[0018] Furthermore, the mass volume ratio between the carbon source and the primary fermentation liquid is 0.5-2.0 g:100 mL.

[0019] Preferably, in step (2), before adding the mussel shell powder to the primary fermentation liquid, the mussel shell powder is first calcined at 550-650° C. for 3-4 hours.

[0020] By calcining mussel shell powder, the organic matter in it is decomposed and the generated gas escapes, which can increase the porosity of the mussel shell powder and expand the pore volume, thereby facilitating its adsorption and reaction with acidic substances in the soil and improving the effect of acidified soil improvement.

[0021] Preferably, in step (1), the temperature of the first fermentation is 20-30°C.

[0022] Preferably, in step (2), the temperature of the second fermentation is 20-30° C. and the time is 1-5 days.

[0023] Preferably, in step (1), the specific process of inoculating the acidified soil improving bacteria into the fermentation culture medium includes the following steps: inoculating the acidified soil improving bacteria into the activation culture medium, activating it, and then picking a single colony from the activation culture medium and inoculating it into the fermentation culture medium.

[0024] Furthermore, the activation medium is LB solid medium.

[0025] Preferably, in step (2), the particle size of the mussel shell powder is less than or equal to 10 μm.

[0026] In a second aspect, the present invention provides an acidified soil conditioner prepared by the method.

[0027] In a third aspect, the present invention provides use of the acidified soil conditioner in improving acidified soil.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] (1) The present invention adopts acidified soil improving bacteria with phosphate solubilizing effect, and cooperates with the first fermentation and the second fermentation process after adding mussel shell powder. The obtained acidified soil conditioner can quickly increase the pH of the acidified soil and increase the soluble phosphorus content in the soil.

[0030] (2) The acidified soil improving bacteria (strain XHZG06-95A3) used in the present invention can increase the pH of acidified soil and can also play a role in solubilizing phosphate, converting the insoluble phosphorus in soil and mussel shell powder into soluble phosphorus. At present, no strains of the same species (Candida palmioleophila) that can be used for acidified soil improvement have been reported. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a colony photo of strain XHZG06-95A3.

[0032] Figure 2 This is the effect of initial pH on the growth of strain XHZG06-95A3.

[0033] Figure 3 This is the effect of the initial pH on the pH-raising effect of strain XHZG06-95A3.

[0034] Figure 4 This is the effect of temperature on the growth of strain XHZG06-95A3.

[0035] Figure 5 This is the effect of temperature on the pH-raising effect of strain XHZG06-95A3.

[0036] Figure 6This is the change of soluble phosphorus concentration in the fermentation liquid during the second fermentation. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the embodiments.

[0038] Overall embodiment

[0039] A method for preparing an acidified soil conditioner based on microbial fermentation comprises the following steps:

[0040] (1) inoculating an acidified soil improving bacterium having a phosphate-solubilizing function into a fermentation culture solution, performing a first fermentation, and obtaining a primary fermentation solution;

[0041] (2) adding mussel shell powder to the primary fermentation liquid to perform a second fermentation to obtain a secondary fermentation liquid;

[0042] (3) Separating the products from the secondary fermentation liquid to obtain an acidified soil conditioner.

[0043] As a specific implementation method, in step (1), the strain name of the acidifying soil improving bacteria is XHZG06-95A3, classified and named Candida palmioleophila, and preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, the address of the depository is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is December 4, 2023, and the deposit number is CGMCC No. 29215.

[0044] As a specific embodiment, in step (1), the fermentation culture medium includes the following components in concentrations: yeast extract 5-10 g / L, glucose 10-20 g / L, peptone 10-20 g / L, K2HPO4 0.5-1 g / L, (NH4)2HPO4 3-6 g / L, Mg2SO4·7H2O 0.5-1 g / L, KCl 0-0.3 g / L, the solvent is water, and the pH is 6-7.

[0045] As a specific implementation, in step (1), the temperature of the first fermentation is 20-30°C.

[0046] As a specific embodiment, in step (1), the OD of the primary fermentation broth is 600 It is 0.7~1.2.

[0047] As a specific implementation, in step (2), the mass volume ratio between the mussel shell powder and the first fermentation liquid is 2-8g:100mL.

[0048] As a specific embodiment, in step (2), before the second fermentation, a carbon source is added to the primary fermentation broth; the carbon source is one or more of glucose, galactose, sucrose and starch; the mass volume ratio between the carbon source and the primary fermentation broth is 0.5-2.0g:100mL.

[0049] As a specific implementation, in step (2), before adding the mussel shell powder to the primary fermentation liquid, the mussel shell powder is first calcined at 550-650° C. for 3-4 hours.

[0050] As a specific implementation, in step (2), the temperature of the second fermentation is 20 to 30° C., and the time is 1 to 5 days.

[0051] As a specific embodiment, in step (1), the specific process of inoculating the acidified soil improving bacteria into the fermentation culture medium includes the following steps: inoculating the acidified soil improving bacteria into the activation medium, activating it, and then picking a single colony from the activation medium and inoculating it into the fermentation culture medium. The activation medium can be selected from LB solid medium.

[0052] As a specific implementation, in step (2), the particle size of the mussel shell powder is less than or equal to 10 μm.

[0053] An acidified soil conditioner prepared by the method.

[0054] Application of the above acidified soil conditioner in improving acidified soil. Specific embodiments

[0056] The present invention is described below by specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0057] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. The raw materials and equipment used in the present invention are conventional raw materials and equipment in the art unless otherwise specified, and can be obtained from conventional commercial channels. The methods used are conventional methods in the art unless otherwise specified.

[0058] The “strain XHZG06-95A3” described in the following examples refers to the strain deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on December 4, 2023, with a deposit number of CGMCC No. 29215, and its microbial classification name is Candida palmioleophila.

[0059] Example 1: Isolation and identification of strain XHZG06-95A3

[0060] Strain XHZG06-95A3 was isolated from seaside soil. Its morphological characteristics and 16S rDNA sequence are as follows:

[0061] (1) Morphological characteristics:

[0062] The colony morphology of strain XHZG06-95A3 is as follows Figure 1 shown.

[0063] (2) 16S rDNA sequence:

[0064] The 16S rDNA sequence of strain XHZG06-95A3 is shown in SEQ ID NO: 1, and is as follows:

[0065]

[0066] After identification, strain XHZG06-95A3 belongs to Candida palmioleophila (there is currently no unified Chinese translation for this species).

[0067] Example 2: Stress resistance and ability to reduce environmental acidity of strain XHZG06-95A3

[0068] 2.1 Preparation of culture medium

[0069] Prepare LB solid medium and fermentation broth according to the following formula:

[0070] (1) LB solid medium:

[0071] Dissolve 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 950 mL of distilled water, then add distilled water to make up to 1 L. Adjust the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize with high-pressure steam at 121°C for 20 min.

[0072] (2) Fermentation broth:

[0073] Yeast extract 10g / L, glucose 15g / L, peptone 20g / L, K2HPO4 0.5g / L, (NH4)2HPO4 5g / L, Mg2SO4·7H2O0.5g / L, KCl 0.1g / L, dissolve in 950mL of distilled water, add distilled water to make up to 1L, adjust the pH to the required value with dilute sulfuric acid and sodium hydroxide solution, and sterilize with high-pressure steam at 121℃ for 20min.

[0074] 2.2 Suitable initial pH range of strain XHZG06-95A3

[0075] The strain XHZG06-95A3 was inoculated on LB solid medium, cultured at 25°C until a single colony grew, picked a single colony, and inoculated into fermentation broth with pH values ​​of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively, cultured in a shaking incubator at 25°C and 180 r / min, and samples were taken at regular intervals to detect the absorbance OD value of the fermentation broth at 600 nm. 600 and pH of the fermentation broth. Figure 2 and Figure 3 The OD of the fermentation broth measured at different initial pH values ​​is shown in 600 Changes of pH value over time.

[0076] Test results analysis and conclusion: Figure 2 and Figure 3 It can be seen that strain XHZG06-95A3 has good acid resistance and can survive at a pH of 1 to 8. It can grow well in an environment of pH 3 to 8 and can effectively increase the pH of the fermentation culture medium.

[0077] 2.3 Suitable temperature range of strain XHZG06-95A3

[0078] The strain XHZG06-95A3 was inoculated on LB solid medium, cultured at 25°C until a single colony grew, picked a single colony, and inoculated into a fermentation medium with a pH of 5.0. The temperature was set to 15°C, 20°C, 25°C, 30°C, and 35°C, respectively, and cultured in a shaking incubator at 180 r / min. Samples were taken at regular intervals to detect the absorbance OD value of the fermentation medium at 600 nm. 600 and pH of the fermentation broth. Figure 4 and Figure 5 The OD of the fermentation broth was measured at different temperatures. 600 Changes of pH value over time.

[0079] Test results analysis and conclusion: Figure 4 and Figure 5It can be seen that when the temperature is 15-35℃, the strain XHZG06-95A3 can grow well and increase the pH of the fermentation culture medium. Therefore, this strain can function in most ambient temperatures.

[0080] Example 3: Phosphorus-dissolving effect of mussel shell powder in the second fermentation

[0081] 3.1 Preparation of culture medium

[0082] Prepare LB solid medium and fermentation broth according to the following formula:

[0083] (1) LB solid medium:

[0084] Dissolve 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 950 mL of distilled water, then add distilled water to make up to 1 L. Adjust the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize with high-pressure steam at 121°C for 20 min.

[0085] (2) Fermentation broth:

[0086] Yeast extract 10g / L, glucose 15g / L, peptone 20g / L, K2HPO4 0.5g / L, (NH4)2HPO4 5g / L, Mg2SO4·7H2O0.5g / L, KCl 0.1g / L, dissolved in 950mL of distilled water, then made up to 1L with distilled water, adjusted the pH to 6.5 with dilute sulfuric acid and sodium hydroxide solution, and sterilized by high-pressure steam at 121℃ for 20min.

[0087] 3.2 Pretreatment of mussel shell powder

[0088] The mussel shell powder with a particle size of 5 to 10 μm is placed in a muffle furnace and calcined at 600° C. for 3 hours to obtain calcined mussel shell powder.

[0089] 3.3 First fermentation

[0090] The strain XHZG06-95A3 was inoculated on LB solid medium and cultured at 25°C until a single colony grew out. A single colony was picked and inoculated into the fermentation medium. The culture was shaken at 25°C and 180 r / min until the OD 600 is 0.8, and a fermentation liquid is obtained.

[0091] 3.4 Second fermentation Add calcined mussel shell powder to the first fermentation liquid at an amount of 50 g / L, then add glucose, galactose, sucrose, and starch as carbon sources at an amount of 5 g / L, and use no carbon source as a control. Stir at 25°C at a speed of 40 r / min, and use ammonium molybdate colorimetry to detect the effective phosphorus concentration in the fermentation liquid at regular intervals. The results are shown in Table 1. Figure 6 .

[0092] A blank control without inoculation of strain XHZG06-95A3 was set up: calcined mussel shell powder was added to the fermentation culture medium at an amount of 50 g / L. Stirring was carried out at 25°C at a speed of 100 r / min, sampling was performed at regular intervals, and after centrifugation, the soluble phosphorus concentration in the supernatant was detected by ammonium molybdate colorimetry. The results are shown in Table 1. Figure 6 .

[0093] Test results analysis and conclusion: Figure 6 It can be seen that compared with the blank control without using strain XHZG06-95A3, the soluble phosphorus concentration in the secondary fermentation liquid can be significantly increased by adding calcined mussel shell powder to the primary fermentation liquid prepared by fermentation of strain XHZG06-95A3 for a second fermentation, indicating that strain XHZG06-95A3 can convert the insoluble phosphorus in mussel shell powder into soluble phosphorus; and, by adding glucose, galactose, sucrose or starch as a carbon source before the second fermentation, the rate at which strain XHZG06-95A3 converts the insoluble phosphorus in mussel shell powder into soluble phosphorus can be accelerated, and further preferred carbon sources are glucose and sucrose.

[0094] Example 4: Preparation of Acidified Soil Conditioner (Sample #1)

[0095] 4.1 Preparation of culture medium

[0096] Prepare LB solid medium and fermentation broth according to the following formula:

[0097] (1) LB solid medium:

[0098] Dissolve 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 950 mL of distilled water, then add distilled water to make up to 1 L. Adjust the pH to 6.5 with dilute sulfuric acid and sodium hydroxide solution, and sterilize with high-pressure steam at 121°C for 20 min.

[0099] (2) Fermentation broth:

[0100] Yeast extract 10g / L, glucose 15g / L, peptone 20g / L, K2HPO4 0.5g / L, (NH4)2HPO4 5g / L, Mg2SO4·7H2O0.5g / L, KCl 0.1g / L, dissolved in 950mL of distilled water, then made up to 1L with distilled water, adjusted the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilized by high-pressure steam at 121℃ for 20min.

[0101] 4.2 Pretreatment of mussel shell powder

[0102] The mussel shell powder with a particle size of 5 to 10 μm is placed in a muffle furnace and calcined at 600° C. for 3 hours to obtain calcined mussel shell powder.

[0103] 4.3 First fermentation

[0104] The strain XHZG06-95A3 was inoculated on LB solid medium and cultured at 25°C until a single colony grew out. A single colony was picked and inoculated into the fermentation medium. The culture was shaken at 25°C and 180 r / min until the OD 600 is 0.8, and a fermentation liquid is obtained.

[0105] 4.4 Second fermentation

[0106] Calcined mussel shell powder was added to the first fermentation liquid in an amount of 50 g / L, and glucose was added in an amount of 5 g / L, and stirred at 40 r / min for 3 days at 25°C to obtain a second fermentation liquid. The second fermentation liquid was concentrated under reduced pressure and freeze-dried to obtain an acidified soil conditioner.

[0107] Example 5: Preparation of Acidified Soil Conditioner (Sample #2)

[0108] 5.1 Preparation of culture medium

[0109] Prepare LB solid medium and fermentation broth according to the following formula:

[0110] (1) LB solid medium:

[0111] Dissolve 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 950 mL of distilled water, then add distilled water to make up to 1 L. Adjust the pH to 6.5 with dilute sulfuric acid and sodium hydroxide solution, and sterilize with high-pressure steam at 121°C for 20 min.

[0112] (2) Fermentation broth:

[0113] Yeast extract 10g / L, glucose 15g / L, peptone 20g / L, K2HPO4 0.5g / L, (NH4)2HPO4 5g / L, Mg2SO4·7H2O0.5g / L, KCl 0.1g / L, dissolve in 950mL of distilled water, add distilled water to make up to 1L, adjust the pH to the required value with dilute sulfuric acid and sodium hydroxide solution, and sterilize with high-pressure steam at 121℃ for 20min.

[0114] 5.2 First fermentation

[0115] The strain XHZG06-95A3 was inoculated on LB solid medium and cultured at 25°C until a single colony grew out. A single colony was picked and inoculated into the fermentation medium. The culture was shaken at 25°C and 180 r / min until the OD 600is 0.8, and a fermentation liquid is obtained.

[0116] 5.3 Second fermentation

[0117] Add mussel shell powder with a particle size of 5 to 10 μm to the first fermentation liquid in an amount of 50 g / L, and then add glucose in an amount of 5 g / L, and stir at 40 r / min for 3 days at 25°C to obtain a second fermentation liquid. The second fermentation liquid is concentrated under reduced pressure and freeze-dried to obtain an acidified soil conditioner.

[0118] Example 6: Preparation of Acidified Soil Conditioner (Sample #3)

[0119] 6.1 Preparation of culture medium

[0120] Prepare LB solid medium and fermentation broth according to the following formula:

[0121] (1) LB solid medium:

[0122] Dissolve 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 950 mL of distilled water, then add distilled water to make up to 1 L. Adjust the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize with high-pressure steam at 121°C for 20 min.

[0123] (2) Fermentation broth:

[0124] Yeast extract 5 g / L, glucose 20 g / L, peptone 20 g / L, K2HPO4 1 g / L, (NH4)2HPO4 3 g / L, Mg2SO4·7H2O0.5 g / L, KCl 0.3 g / L, dissolved in 950 mL of distilled water, then made up to 1 L with distilled water, adjusted the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilized by high-pressure steam at 121 °C for 20 min.

[0125] 6.2 Pretreatment of mussel shell powder

[0126] The mussel shell powder with a particle size of 5 to 10 μm is placed in a muffle furnace and calcined at 550° C. for 4 hours to obtain calcined mussel shell powder.

[0127] 6.3 First fermentation

[0128] The strain XHZG06-95A3 was inoculated on LB solid medium and cultured at 25°C until a single colony grew out. A single colony was picked and inoculated into the fermentation medium. The culture was shaken at 25°C and 180 r / min until the OD 600 is 1.0, and a fermentation liquid is obtained.

[0129] 6.4 Second fermentation

[0130] Calcined mussel shell powder was added to the first fermentation liquid in an amount of 20 g / L, and glucose was added in an amount of 20 g / L, and stirred at 40 r / min for 1 day at 25°C to obtain a second fermentation liquid. The second fermentation liquid was concentrated under reduced pressure and freeze-dried to obtain an acidified soil conditioner.

[0131] Example 7: Preparation of Acidified Soil Conditioner (Sample #4)

[0132] 7.1 Preparation of culture medium

[0133] Prepare LB solid medium and fermentation broth according to the following formula:

[0134] (1) LB solid medium:

[0135] Dissolve 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 950 mL of distilled water, then add distilled water to make up to 1 L. Adjust the pH to 7.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize with high-pressure steam at 121°C for 20 min.

[0136] (2) Fermentation broth:

[0137] Yeast extract 10g / L, glucose 20g / L, peptone 10g / L, K2HPO4 0.5g / L, (NH4)2HPO4 6g / L, Mg2SO4·7H2O1g / L, dissolve in 950mL of distilled water, add distilled water to make up to 1L, adjust the pH to the required value with dilute sulfuric acid and sodium hydroxide solution, and sterilize with high-pressure steam at 121℃ for 20min.

[0138] 7.2 Pretreatment of mussel shell powder

[0139] The mussel shell powder with a particle size of 5 to 10 μm is placed in a muffle furnace and calcined at 650° C. for 3 hours to obtain calcined mussel shell powder.

[0140] 7.3 First fermentation

[0141] The strain XHZG06-95A3 was inoculated on LB solid medium and cultured at 25°C until a single colony grew out. A single colony was picked and inoculated into the fermentation medium. The culture was shaken at 25°C and 180 r / min until the OD 600 is 0.7, and a fermentation liquid is obtained.

[0142] 7.4 Second fermentation

[0143] Calcined mussel shell powder was added to the first fermentation liquid in an amount of 80 g / L, and stirred at 40 r / min for 5 days at 25°C to obtain a second fermentation liquid. The second fermentation liquid was concentrated under reduced pressure and freeze-dried to obtain an acidified soil conditioner.

[0144] Example 8: Effect of acidified soil conditioner on the restoration of acidified soil

[0145] 8.1 Soil sources

[0146] The soil samples were taken from paddy soil after multiple rounds of planting. The pH was 4.9 (soil pH was detected using the water-immersed glass electrode method) and the available phosphorus content was 22.56 mg / kg (soil available phosphorus content was detected using the Olsen method).

[0147] 8.2 Acidified soil conditioner treatment

[0148] In a flower pot with an inner bottom diameter of 21 cm, an inner mouth diameter of 30 cm, and a height of 25 cm, a soil sample was placed in an amount of 8 kg / pot. The acidified soil conditioner (samples #1 to #4) prepared in Examples 4 to 7 was taken, and mussel shell powder and a primary fermentation liquid of strain XHZG06-95A3 (prepared according to the method in Example 4) were used as controls, respectively, and added to the flower pot containing the soil sample. The amount of the acidified soil conditioner and mussel shell powder added was 0.3 kg / pot, and the amount of the primary fermentation liquid added was 10 mL / pot, and the mixture was stirred evenly. After 15 days and 30 days, samples were taken to detect the pH and available phosphorus content of the soil. The results are shown in Table 1.

[0149] Table 1 Acidification soil improvement effect

[0150]

[0151] Test results analysis and conclusion:

[0152] (1) As can be seen from Table 1, the pH and available phosphorus content of acidified soil can be increased by applying the primary fermentation liquid of strain XHZG06-95A3, indicating that the strain has the ability to degrade or neutralize acidic substances in the soil and has a phosphorus-solubilizing effect, which can convert the insoluble phosphorus in the soil into soluble phosphorus that can be absorbed and utilized by plants.

[0153] (2) As can be seen from Table 1, compared with using mussel shell powder or strain XHZG06-95A3 alone, making them into the acidified soil improving bacteria of the present invention can better improve the pH and available phosphorus content of the acidified soil.

[0154] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing an acidified soil conditioner based on microbial fermentation, characterized in that: The following steps are involved: (1) Inoculating an acidified soil-improving bacterium with a phosphate-dissolving function into a fermentation culture medium, performing a first fermentation, and obtaining a primary fermentation liquid; the strain name of the acidified soil-improving bacterium is XHZG06-95A3, and its classification is named Candida palmioleophila , the deposit number is CGMCC No.29215; (2) adding mussel shell powder to the primary fermentation liquid to carry out a second fermentation to obtain a secondary fermentation liquid; (3) The secondary fermentation liquid is concentrated under reduced pressure and then freeze-dried to obtain an acidified soil conditioner.

2. The method according to claim 1, characterized in that In step (1), the fermentation broth comprises the following components in concentrations: yeast extract 5-10 g / L, glucose 10-20 g / L, peptone 10-20 g / L, K2HPO4 0.5-1 g / L, (NH4)2HPO4 3-6 g / L, MgSO4·7H2O 0.5-1 g / L, KCl 0-0.3 g / L, the solvent is water, and the pH value is 6-7.

3. The method according to claim 1, characterized in that In step (1), the OD of the primary fermentation broth is 600 It is 0.7~1.

2.

4. The method according to claim 1 or 3, characterized in that In step (2), the mass volume ratio between the mussel shell powder and the first fermentation liquid is 2-8 g: 100 mL.

5. The method according to claim 1, characterized in that In step (2), before the second fermentation, a carbon source is added to the first fermentation broth; the carbon source is one or more of glucose, galactose, sucrose and starch.

6. The method according to claim 1, characterized in that In step (2), before adding the mussel shell powder to the primary fermentation liquid, the mussel shell powder is first calcined at 550-650° C. for 3-4 h.

7. The method according to claim 1 or 6, characterized in that In step (2), the particle size of the mussel shell powder is less than or equal to 10 μm.

8. An acidified soil conditioner prepared by the method according to any one of claims 1 to 7.

9. Use of the acidified soil conditioner according to claim 8 in improving acidified soil.

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