Candida sp. XHZG06-95a3 and use thereof in improving acidified soil

Abstract

The present invention relates to the field of microorganisms. Disclosed are Candida sp. XHZG06-95A3 and a use thereof in improving acidified soil. The Candida sp. XHZG06-95A3 belongs to Candida palmioleophila in taxonomy, and the preservation number is CGMCC No. 29215. The provided Candida sp. XHZG06-95A3 has the capability to improve the environment pH and solubilize phosphate, can reduce the acidity of acidified soil and improve the effective phosphorus content therein, and can adapt to a wide environment temperature and pH range. This is the first time that a strain capable of improving the environment pH and having the capability to solubilize phosphate has been found in Candida palmioleophila.

Description

A Candida strain XHZG06-95A3 and its application in improving acidified soil Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a Candida sp. XHZG06-95A3 and application thereof in improving acidified soil. Background Art

[0002] The pH of soil is determined by the chemical balance between acidic and alkaline substances. When this balance is disrupted, resulting in an excess of acidic substances and a deficiency of alkaline substances, soil acidification occurs. In recent years, soil acidification has become increasingly serious due to human activities such as the overuse of acidic fertilizers and continuous cropping, posing a growing threat to the ecological environment and agricultural production. Acidification increases the activity of heavy metals in the soil, reducing soil fertility and the absorption rate of nutrients. For example, in acidified soil, phosphate ions easily bind to iron and aluminum ions, becoming immobilized and losing their fertilizing properties, making them difficult for plants to absorb and utilize.

[0003] Soil acidification can be alleviated by applying soil conditioners. The acidifying soil conditioners currently used are mainly chemical conditioners such as lime, which have the hidden dangers of soil compaction and decreased fertility. In comparison, microbial conditioners have less adverse effects on the soil. Therefore, in recent years, the methods of microbial improvement of acidified soil and the development of related bacterial resources have received increasing attention. Acidifying soil improving bacteria are required to have good stress resistance, be able to adapt to the acidified soil environment, and be able to utilize acidic substances in the soil or be able to secrete alkaline substances. At present, due to the limitations of bacterial resources and the stress resistance and adaptability of microorganisms to acidified soils, how to improve the remediation effect of microbial conditioners on acidified soils remains a core issue of concern to researchers.

[0004] Candida palmioleophila is a species within the genus Candida, which currently lacks a unified Chinese translation. Current research on this species primarily focuses on wastewater treatment. Some strains within this species have been found to remove ammonia nitrogen, TN, and COD from wastewater (e.g., strain WW7 in patent CN112961790B) or to perform heterotrophic nitrification and aerobic denitrification (e.g., strain NOB-1 in patent CN114292762B). However, no strains within this species have been reported to improve acidified soils.

[0005] Summary of the Invention

[0006] To address the technical issues of limited bacterial resources and poor adaptability to acidified soils, the present invention provides a strain of Candida species, XHZG06-95A3. This strain has the ability to increase environmental pH and solubilize phosphate, reducing the acidity of acidified soils and increasing their available phosphorus content. It also adapts to a wide range of environmental temperatures and pH levels.

[0007] The present invention also provides a method for preparing an acidified soil conditioner based on microbial fermentation. The acidified soil conditioner prepared by this method can quickly increase the pH of acidified soil and simultaneously increase the soluble phosphorus content in the soil, thereby achieving a good acidified soil remediation effect.

[0008] The specific technical solutions of the present invention are:

[0009] In a first aspect, the present invention provides a Candida sp. XHZG06-95A3, which belongs to Candida palmioleophila in taxonomy and has a deposit number of CGMCC No. 29215.

[0010] The Candida XHZG06-95A3 of the present invention belongs to Candida palmioleophila (this species currently has no unified Chinese translation), and is deposited in the General Microbiology Center of the China Culture Collection Administration Committee. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is December 4, 2023.

[0011] The strain of the present invention has the ability to increase environmental pH, possibly by utilizing acidic substances in the environment to reduce environmental acidity and producing amines to neutralize hydrogen ions. Furthermore, the strain has the ability to solubilize phosphate, converting insoluble phosphorus into soluble phosphorus. Due to these effects, the strain of the present invention can be used to improve acidified soils. When applied to acidified soils, it can reduce soil acidity while increasing the available phosphorus content in the soil that can be absorbed and utilized by plants.

[0012] Furthermore, the strain of the present invention has a high adaptability to different environmental pH and temperatures. Tests have shown that the strain can grow well in an environmental pH range of 3 to 8 and an environmental temperature range of 15 to 35°C. It also effectively increases the environmental pH and effectively dissolves phosphate in an environment with a pH of 4 to 7 and a temperature range of 15 to 35°C.

[0013] In a second aspect, the present invention provides a method for preparing an acidified soil conditioner based on microbial fermentation, comprising the following steps:

[0014] (1) inoculating an acidified soil improving bacterium into a fermentation culture medium to perform a first fermentation to obtain a primary fermentation broth; the acidified soil improving bacterium is Candida XHZG06-95A3, which belongs to Candida palmioleophila in taxonomy and has a preservation number of CGMCC No. 29215;

[0015] (2) adding mussel shell powder to the primary fermentation liquid to perform a second fermentation to obtain a secondary fermentation liquid;

[0016] (3) Separate the products from the secondary fermentation liquid to obtain an acidified soil conditioner.

[0017] In the present invention, an acidified soil improver (Candida XHZG06-95A3) with a phosphate-solubilizing effect is used for the first fermentation, which can rapidly expand the strain and secrete a substance that can increase the pH of the soil. Mussel shell powder is then added for a second fermentation. The phosphate-solubilizing effect of the acidified soil improver can convert the insoluble phosphorus in the mussel shell powder into soluble phosphorus, thereby enabling the acidified soil improver of the present invention to 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 acidified soil, the acidified soil improver and the substance that can increase the pH of the soil produced during the first fermentation, as well as the mussel shell powder, can be used to quickly increase the pH of the soil. At the same time, the acidified soil improver can also convert the insoluble phosphorus in the soil into soluble phosphorus by using the acidified soil improver, and the soluble phosphorus released from the mussel shell powder during the second fermentation process can be used to quickly increase the available phosphorus content in the soil.

[0018] Preferably, 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, Mg2SO4·7H2O 0.5-1 g / L, KCl 0-0.3 g / L, the solvent is water, and the pH is 6-7.

[0019] Preferably, in step (1), the OD of the primary fermentation broth is 600 It is 0.7 to 1.2.

[0020] Preferably, in step (2), the mass-to-volume ratio of the mussel shell powder to the first fermentation liquid is 2-8 g:100 mL.

[0021] 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.

[0022] By adding a carbon source, the strain XHZG06-95A3 can be encouraged to perform its phosphate-solubilizing function during the second fermentation, converting the insoluble phosphorus in the mussel shell powder into soluble phosphorus that can be absorbed and utilized by plants. Furthermore, the strain XHZG06-95A3 used in the present invention is more effective in promoting its phosphate-solubilizing function when using one or more of glucose, galactose, sucrose, and starch as a carbon source.

[0023] Furthermore, the mass-to-volume ratio of the carbon source to the primary fermentation liquid is 0.5-2.0 g:100 mL.

[0024] 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.

[0025] By calcining the 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.

[0026] Preferably, in step (1), the temperature of the first fermentation is 20-30°C.

[0027] Preferably, in step (2), the temperature of the second fermentation is 20-30° C., and the time is 1-5 days.

[0028] Preferably, in step (1), the specific process of inoculating the acidifying soil improving bacteria into the fermentation culture medium includes the following steps: inoculating the acidifying 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.

[0029] Furthermore, the activation culture medium is LB solid culture medium.

[0030] Preferably, in step (2), the particle size of the mussel shell powder is less than or equal to 10 μm.

[0031] In a third aspect, the present invention provides an acidified soil conditioner prepared by the method.

[0032] In a fourth aspect, the present invention provides use of the Candida sp. XHZG06-95A3 or the acidified soil conditioner in improving acidified soil.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] (1) The Candida species XHZG06-95A3 provided by the present invention has the ability to increase environmental pH and solubilize phosphate, can reduce the acidity of acidified soil, and increase the available phosphorus content therein, and can adapt to a wide range of environmental temperatures and pH values. This is the first time that a strain capable of increasing environmental pH and solubilizing phosphate has been discovered within the species Candida palmioleophila.

[0035] (2) The present invention uses the Candida albicans XHZG06-95A3 with a phosphate-solubilizing effect, and combines the first fermentation with the second fermentation process after adding mussel shell powder to produce an acidified soil conditioner that can quickly increase the pH of the acidified soil and increase the soluble phosphorus content in the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 shows the improvement effect of strain XHZG06-95A3 on acidified soil.

[0037] Figure 2 is a colony photograph of strain XHZG06-95A3.

[0038] FIG3 shows the effect of initial pH on the growth of strain XHZG06-95A3.

[0039] FIG4 shows the effect of the initial pH on the pH-raising effect of strain XHZG06-95A3.

[0040] FIG5 shows the effect of temperature on the growth of strain XHZG06-95A3.

[0041] Figure 6 shows the effect of temperature on the pH-raising effect of strain XHZG06-95A3.

[0042] Figure 7 shows the effect of temperature on the phosphate solubilization ability of strain XHZG06-95A3.

[0043] FIG8 shows the effect of initial pH on the phosphate solubilization ability of strain XHZG06-95A3.

[0044] FIG9 shows the change of soluble phosphorus concentration in the fermentation liquid during the second fermentation process. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, any changes and advantages that may occur to a person skilled in the art are intended to be included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0046] The “strain XHZG06-95A3” described in the following examples refers to the strain deposited in the General Microbiology Center of the China Culture Collection Administration on December 4, 2023, with the deposit number CGMCC No. 29215, and its microbial classification name is Candida palmioleophila.

[0047] Example 1: Isolation and identification of strain XHZG06-95A3

[0048] Strain XHZG06-95A3 was isolated from seaside soil. Its morphological, physiological and biochemical characteristics and 16S rDNA sequence are as follows:

[0049] (1) Morphological and physiological and biochemical characteristics:

[0050] The colony morphology of strain XHZG06-95A3 is shown in Figure 2 .

[0051] (2) 16S rDNA sequence:

[0052] The 16S rDNA sequence of strain XHZG06-95A3 is 600 bp long, as shown in SEQ ID NO: 1, and is as follows:

[0053] After identification, strain XHZG06-95A3 belongs to Candida palmioleophila (there is currently no unified Chinese translation for this species).

[0054] Example 2: pH tolerance and ability to reduce environmental acidity of strain XHZG06-95A3 In order to explore the adaptability of strain XHZG06-95A3 to different pH environments, this example tested the growth of strain XHZG06-95A3 at different starting pH values ​​and its ability to increase environmental pH. The specific process and test results are as follows:

[0055] 2.1 Preparation of culture medium

[0056] Prepare LB solid medium and culture solution according to the following formula:

[0057] (1) LB solid medium:

[0058] 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 dilute to 1 L with distilled water. Adjust the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0059] (2) Culture medium:

[0060] Dissolve 10 g / L yeast extract, 15 g / L glucose, 20 g / L peptone, 0.5 g / L K2HPO4, 5 g / L (NH4)2HPO4, 0.5 g / L Mg2SO4·7H2O, and 0.1 g / L KCl in 950 mL of distilled water, then add distilled water to make up to 1 L. Adjust the pH to the desired value with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0061] 2.2 Effect of initial pH on the growth of strain XHZG06-95A3 and the ability to improve environmental pH The strain XHZG06-95A3 was inoculated on LB solid medium and cultured at 25°C until a single colony grew. A single colony was picked and inoculated into culture solutions with pH values ​​of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively. The culture was shaken at 25°C and 180 rpm. Samples were taken at regular intervals to detect the absorbance OD value of the culture solution at 600 nm. 600 Table 1, Table 2, Figure 3 and Figure 4 show the OD of the culture solution at different initial pH values. 600 Changes of pH value over time.

[0062] Table 1 Effect of initial pH on the growth of strain XHZG06-95A3

[0063] Table 2 Effect of initial pH on pH raising effect of strain XHZG06-95A3

[0064] Analysis and conclusion of the test results: It can be seen from Table 1, Table 2, Figure 3 and Figure 4 that strain XHZG06-95A3 has good acid resistance and can survive at a pH of 1 to 8. Among them, it can grow well in an environment of pH 3 to 8, and can also effectively increase the pH of the culture medium.

[0065] Example 3: Temperature tolerance and ability to reduce environmental acidity of strain XHZG06-95A3 In order to explore the adaptability of strain XHZG06-95A3 to different temperature environments, this example tested the growth of strain XHZG06-95A3 at different temperatures and its ability to increase environmental pH. The specific process and test results are as follows:

[0066] 3.1 Preparation of culture medium

[0067] Prepare LB solid medium and culture solution according to the following formula:

[0068] (1) LB solid medium:

[0069] 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 dilute to 1 L with distilled water. Adjust the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0070] (2) Culture medium:

[0071] Dissolve 10 g / L yeast extract, 15 g / L glucose, 20 g / L peptone, 0.5 g / L K2HPO4, 5 g / L (NH4)2HPO4, 0.5 g / L Mg2SO4·7H2O, and 0.1 g / L KCl in 950 mL of distilled water, then add distilled water to make up to 1 L. Adjust the pH to the desired value with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0072] 3.3 Effect of temperature on the growth of strain XHZG06-95A3 and its ability to increase environmental pH The strain XHZG06-95A3 was inoculated on LB solid medium and cultured at 25°C until a single colony grew. A single colony was picked and inoculated into a culture medium with a pH of 5.0. The culture temperature was set to 15°C, 20°C, 25°C, 30°C, and 35°C, respectively. The culture was shaken at 180 r / min. Samples were taken at regular intervals to detect the absorbance OD value of the culture medium at 600 nm. 600 Table 3, Table 4, Figure 5 and Figure 6 show the OD of the culture solution at different temperatures. 600 Changes of pH value over time.

[0073] Table 3 Effect of temperature on the growth of strain XHZG06-95A3

[0074] Table 4 Effect of temperature on pH improvement of strain XHZG06-95A3

[0075] Analysis and conclusion of the test results: It can be seen from Table 3, Table 4, Figure 5 and Figure 6 that when the temperature is 15-35°C, the strain XHZG06-95A3 can grow well and increase the pH of the culture solution. Therefore, this strain can function under most ambient temperatures.

[0076] Example 4: Effect of Temperature on Phosphate Solubilization Ability of Strain XHZG06-95A3 In order to explore the effect of temperature on the phosphate solubilization ability of strain XHZG06-95A3, this example used a phosphate solubilization culture solution containing phosphate rock powder to test the phosphate solubilization ability of strain XHZG06-95A3 at different temperatures. The specific process and test results are as follows:

[0077] 4.1 Preparation of culture medium

[0078] Prepare LB solid medium and phosphate-solubilizing culture medium according to the following formula:

[0079] (1) LB solid medium:

[0080] 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 dilute to 1 L with distilled water. Adjust the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0081] (2) Phosphate-dissolving culture medium:

[0082] Dissolve 10 g / L yeast extract, 15 g / L glucose, 20 g / L peptone, 0.5 g / L Mg2SO4·7H2O, and 0.1 g / L KCl in 950 mL of distilled water, then add 5 g / L phosphate rock powder and disperse evenly. Add distilled water to make up to 1 L. Adjust the pH to the desired value with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0083] 4.2 Effect of temperature on the phosphate solubilization ability of strain XHZG06-95A3

[0084] Experimental group (inoculated strain): strain XHZG06-95A3 was inoculated onto LB solid medium and cultured at 25°C until a single colony grew. A single colony was picked and inoculated into a phosphate-dissolving culture medium with a pH of 6.0. The culture temperature was set to 15°C, 20°C, 25°C, 30°C, and 35°C, respectively, and the culture was shaken at 180 rpm. Samples were taken at regular intervals, and after centrifugation, the supernatant was taken and the soluble phosphorus concentration in the supernatant was detected by ammonium molybdate colorimetry.

[0085] Control group (no inoculation of strain): Take phosphate-dissolving culture medium with a pH of 6.0, set the temperature to 15℃, 20℃, 25℃, 30℃, and 35℃ respectively, and shake at 180r / min. Take samples at regular intervals, centrifuge, take the supernatant, and use ammonium molybdate colorimetry to detect the soluble phosphorus concentration in the supernatant.

[0086] Based on the soluble phosphorus concentrations measured in the experimental and control groups, the amount of phosphate solubilized was calculated using the following formula: phosphate solubilized = soluble phosphorus concentration in the supernatant of the experimental group - soluble phosphorus concentration in the supernatant of the control group. Table 5 and Figure 7 show the change in phosphate solubilized over time at different temperatures.

[0087] Table 5 Effect of temperature on the phosphate solubilization ability of strain XHZG06-95A3

[0088] Analysis and conclusion of the test results: As can be seen from Table 5 and Figure 7, strain XHZG06-95A3 has good adaptability to temperature and can effectively solubilize phosphate at temperatures between 15 and 35°C. Among them, the solubilization efficiency is relatively high at 20 to 30°C.

[0089] Example 5: Effect of Initial pH on Phosphate Solubilization Ability of Strain XHZG06-95A3 In order to explore the effect of initial environmental pH on the phosphate solubilization ability of strain XHZG06-95A3, this example used a phosphate solubilization culture solution containing phosphate rock powder to test the phosphate solubilization ability of strain XHZG06-95A3 at different initial pH values. The specific process and test results are as follows:

[0090] 5.1 Preparation of culture medium

[0091] Prepare LB solid medium and phosphate-solubilizing culture medium according to the following formula:

[0092] (1) LB solid medium:

[0093] 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 dilute to 1 L with distilled water. Adjust the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0094] (2) Phosphate-dissolving culture medium:

[0095] Dissolve 10 g / L yeast extract, 15 g / L glucose, 20 g / L peptone, 0.5 g / L Mg2SO4·7H2O, and 0.1 g / L KCl in 950 mL of distilled water, then add 5 g / L phosphate rock powder and disperse evenly. Add distilled water to make up to 1 L. Adjust the pH to the desired value with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0096] 5.2 Effect of initial pH on the phosphate solubilization ability of strain XHZG06-95A3

[0097] Experimental Group (Inoculated Strain): Strain XHZG06-95A3 was inoculated onto LB solid medium and cultured at 25°C until a single colony grew. The colony was then picked and inoculated into phosphate-solubilizing medium at pH values ​​of 4.0, 5.0, 6.0, or 7.0. The culture was shaken at 25°C and 180 rpm. Samples were taken at regular intervals and centrifuged. The supernatant was then assayed for soluble phosphorus concentration using the ammonium molybdate colorimetric method. A phosphate-solubilizing medium without inoculation of strain XHZG06-95A3 served as a control.

[0098] Control group (no inoculation of strain): Take phosphate-dissolving culture medium with pH of 4.0, 5.0, 6.0, and 7.0, shake on a shaker at 25°C and 180 r / min, take samples at regular intervals, centrifuge, take the supernatant, and use ammonium molybdate colorimetry to detect the soluble phosphorus concentration in the supernatant.

[0099] Based on the soluble phosphorus concentrations measured in the experimental and control groups, the amount of phosphate solubilized was calculated using the following formula: phosphate solubilized = soluble phosphorus concentration in the supernatant of the experimental group - soluble phosphorus concentration in the supernatant of the control group. Table 6 and Figure 8 show the change in phosphate solubilized over time at different initial pH values.

[0100] Table 6 Effect of initial pH on phosphate solubilization ability of strain XHZG06-95A3

[0101] Analysis and conclusion of the test results: As can be seen from Table 6 and Figure 8, strain XHZG06-95A3 has a high tolerance to acidic environments and can effectively solubilize phosphate when the environmental pH is 4.0-7.0.

[0102] Example 6: Effect of strain XHZG06-95A3 on improving acidified soil In order to explore the effect of strain XHZG06-95A3 on improving acidified soil, this example tested the changes in soil pH and available phosphorus content after applying the strain. The specific process and test results are as follows:

[0103] 6.1 Soil Source

[0104] The soil sample was taken from paddy soil after multiple rounds of planting. The pH was 4.9 (soil pH was measured using a water-immersed glass electrode method) and the available phosphorus content was 22.56 mg / kg (soil available phosphorus content was measured using the Olsen method).

[0105] 6.2 Preparation of culture medium

[0106] Prepare LB solid medium and culture solution according to the following formula:

[0107] (1) LB solid medium:

[0108] 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 dilute to 1 L with distilled water. Adjust the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0109] (2) Expansion culture medium:

[0110] Dissolve 10 g / L yeast extract, 15 g / L glucose, 20 g / L peptone, 0.5 g / L K2HPO4, 5 g / L (NH4)2HPO4, 0.5 g / L Mg2SO4·7H2O, and 0.1 g / L KCl 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 by high-pressure steam at 121°C for 20 min.

[0111] 6.3 Expansion of strain XHZG06-95A3

[0112] The strain XHZG06-95A3 was inoculated onto LB solid culture medium and cultured at 25°C until a single colony grew. The single colony was picked and inoculated into an expansion culture medium, and cultured in a shaking incubator at 20°C and 180 rpm for 12 h to obtain an XHZG06-95A3 bacterial solution.

[0113] 6.4 Acidified soil improvement test

[0114] In a flowerpot with an inner bottom diameter of 21 cm, an inner opening diameter of 30 cm, and a height of 25 cm, a soil sample was placed at 8 kg per pot. The XHZG06-95A3 bacterial solution was sprayed into the flowerpot containing the soil sample at a spray rate of 8 mL per pot, stirring evenly. Samples were taken at regular intervals to test the pH value and available phosphorus content of the soil. Table 7 and Figure 1 show the changes in soil pH and available phosphorus content over time.

[0115] Table 7 Effect of strain XHZG06-95A3 on improving acidified soil

[0116] Analysis and conclusion of the test results: As can be seen from Table 7 and Figure 1, after being applied to acidified soil, strain XHZG06-95A3 can effectively increase the pH of the soil and increase the available phosphorus content in the soil, thus having a good improvement effect on acidified soil.

[0117] Example 7: Phosphorus-dissolving effect of mussel shell powder in the second fermentation

[0118] 7.1 Preparation of culture medium

[0119] Prepare LB solid medium and fermentation broth according to the following formula:

[0120] (1) LB solid medium:

[0121] 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 dilute to 1 L with distilled water. Adjust the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0122] (2) Fermentation broth:

[0123] Dissolve 10 g / L yeast extract, 15 g / L glucose, 20 g / L peptone, 0.5 g / L K2HPO4, 5 g / L (NH4)2HPO4, 0.5 g / L Mg2SO4·7H2O, and 0.1 g / L KCl 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 by high-pressure steam at 121°C for 20 min.

[0124] 7.2 Pretreatment of mussel shell powder

[0125] 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.

[0126] 7.3 First fermentation

[0127] The strain XHZG06-95A3 was inoculated onto LB solid medium and cultured at 25°C until a single colony grew. A single colony was picked and inoculated into the fermentation medium. The culture was shaken at 25°C and 180 rpm until the OD 600 is 0.8, and a fermentation liquid is obtained.

[0128] 7.4 Second Fermentation: Calcined mussel shell powder was added to the first fermentation broth at a rate of 50 g / L. Glucose, galactose, sucrose, and starch were then added as carbon sources at a rate of 5 g / L. A control containing no carbon source was used. The mixture was stirred at 40 rpm and 25°C. The available phosphorus concentration in the fermentation broth was measured at regular intervals using the ammonium molybdate colorimetric method. The results are shown in Figure 9.

[0129] A blank control, without inoculation of strain XHZG06-95A3, was established: 50 g / L of calcined mussel shell powder was added to the fermentation broth. The culture was stirred at 100 rpm and 25°C. Samples were taken at regular intervals and centrifuged. Soluble phosphorus concentration in the supernatant was measured using the ammonium molybdate colorimetric method. The results are shown in Figure 9.

[0130] Analysis and conclusion of the experimental results: As can be seen from Figure 9, 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.

[0131] Example 8: Preparation of Acidified Soil Conditioner (Sample #1)

[0132] 8.1 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 dilute to 1 L with distilled water. Adjust the pH to 6.5 with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0136] (2) Fermentation broth:

[0137] Dissolve 10 g / L yeast extract, 15 g / L glucose, 20 g / L peptone, 0.5 g / L K2HPO4, 5 g / L (NH4)2HPO4, 0.5 g / L Mg2SO4·7H2O, and 0.1 g / L KCl 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 by high-pressure steam at 121°C for 20 min.

[0138] 8.2 Pretreatment of mussel shell powder

[0139] 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.

[0140] 8.3 First Fermentation

[0141] The strain XHZG06-95A3 was inoculated onto LB solid medium and cultured at 25°C until a single colony grew. A single colony was picked and inoculated into the fermentation medium. The culture was shaken at 25°C and 180 rpm until the OD 600 is 0.8, and a fermentation liquid is obtained.

[0142] 8.4 Second Fermentation: 50 g / L of calcined mussel shell powder and 5 g / L of glucose were added to the first fermentation broth. The mixture was stirred at 40 rpm at 25°C for 3 days to obtain a second fermentation broth. The second fermentation broth was concentrated under reduced pressure and freeze-dried to obtain an acidified soil conditioner.

[0143] Example 9: Preparation of Acidifying Soil Conditioner (Sample #2)

[0144] 9.1 Preparation of culture medium

[0145] Prepare LB solid medium and fermentation broth according to the following formula:

[0146] (1) LB solid medium:

[0147] 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 dilute to 1 L with distilled water. Adjust the pH to 6.5 with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0148] (2) Fermentation broth:

[0149] Dissolve 10 g / L yeast extract, 15 g / L glucose, 20 g / L peptone, 0.5 g / L K2HPO4, 5 g / L (NH4)2HPO4, 0.5 g / L Mg2SO4·7H2O, and 0.1 g / L KCl in 950 mL of distilled water, then add distilled water to make up to 1 L. Adjust the pH to the desired value with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0150] 9.2 First Fermentation

[0151] The strain XHZG06-95A3 was inoculated onto LB solid medium and cultured at 25°C until a single colony grew. A single colony was picked and inoculated into the fermentation medium. The culture was shaken at 25°C and 180 rpm until the OD 600 is 0.8, and a fermentation liquid is obtained.

[0152] 9.3 Second Fermentation: 50 g / L of mussel shell powder with a particle size of 5-10 μm was added to the first fermentation liquid. Glucose was then added at 5 g / L. The mixture was stirred at 40 rpm at 25°C for 3 days to obtain a second fermentation liquid. The second fermentation liquid was concentrated under reduced pressure and freeze-dried to obtain an acidified soil conditioner.

[0153] Example 10: Preparation of Acidifying Soil Conditioner (Sample #3)

[0154] 10.1 Preparation of culture medium

[0155] Prepare LB solid medium and fermentation broth according to the following formula:

[0156] (1) LB solid medium:

[0157] 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 dilute to 1 L with distilled water. Adjust the pH to 6.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0158] (2) Fermentation broth:

[0159] Dissolve 5 g / L yeast extract, 20 g / L glucose, 20 g / L peptone, 1 g / L K2HPO4, 3 g / L (NH4)2HPO4, 0.5 g / L Mg2SO4·7H2O, and 0.3 g / L KCl 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 by high-pressure steam at 121°C for 20 min.

[0160] 10.2 Pretreatment of mussel shell powder

[0161] 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.

[0162] 10.3 First Fermentation

[0163] The strain XHZG06-95A3 was inoculated onto LB solid medium and cultured at 25°C until a single colony grew. A single colony was picked and inoculated into the fermentation medium. The culture was shaken at 25°C and 180 rpm until the OD 600 is 1.0, and a fermentation liquid is obtained.

[0164] 10.4 Second Fermentation: Add 20 g / L of calcined mussel shell powder and 20 g / L of glucose to the first fermentation broth. Stir at 40 rpm at 25°C for 1 day to obtain a second fermentation broth. Concentrate the second fermentation broth under reduced pressure and freeze-dry to obtain an acidified soil conditioner.

[0165] Example 11: Preparation of Acidifying Soil Conditioner (Sample #4)

[0166] 11.1 Preparation of culture medium

[0167] Prepare LB solid medium and fermentation broth according to the following formula:

[0168] (1) LB solid medium:

[0169] 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 dilute to 1 L with distilled water. Adjust the pH to 7.0 with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0170] (2) Fermentation broth:

[0171] Dissolve 10 g / L yeast extract, 20 g / L glucose, 10 g / L peptone, 0.5 g / L K2HPO4, 6 g / L (NH4)2HPO4, and 1 g / L Mg2SO4·7H2O in 950 mL of distilled water, then dilute to 1 L with distilled water. Adjust the pH to the desired value with dilute sulfuric acid and sodium hydroxide solution, and sterilize by high-pressure steam at 121°C for 20 min.

[0172] 11.2 Pretreatment of mussel shell powder

[0173] 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.

[0174] 11.3 First Fermentation

[0175] The strain XHZG06-95A3 was inoculated onto LB solid medium and cultured at 25°C until a single colony grew. A single colony was picked and inoculated into the fermentation medium. The culture was shaken at 25°C and 180 rpm until the OD 600 is 0.7, and a fermentation liquid is obtained.

[0176] 11.4 Second Fermentation: Add 80 g / L of calcined mussel shell powder to the first fermentation broth. Stir at 40 rpm and 25°C for 5 days to obtain a second fermentation broth. Concentrate the second fermentation broth under reduced pressure and freeze-dry to obtain an acidified soil conditioner.

[0177] Example 12: Effect of Acidified Soil Conditioner on the Restoration of Acidified Soil

[0178] 12.1 Soil Source

[0179] The soil sample was taken from paddy soil after multiple rounds of planting. The pH was 4.9 (soil pH was measured using a water-immersed glass electrode method) and the available phosphorus content was 22.56 mg / kg (soil available phosphorus content was measured using the Olsen method).

[0180] 12.2 Treatment of acidified soil conditioners

[0181] A flowerpot with an inner bottom diameter of 21 cm, an inner opening diameter of 30 cm, and a height of 25 cm was filled with soil samples at a rate of 8 kg per pot. The acidified soil conditioners (samples #1 to #4) prepared in Examples 4 to 7 were taken, along with mussel shell powder and a primary fermentation broth of strain XHZG06-95A3 (prepared according to the method in Example 4) as controls. These were added to the flowerpot containing the soil samples. The acidified soil conditioner and mussel shell powder were added at a rate of 0.3 kg per pot, and the primary fermentation broth was added at a rate of 10 mL per pot. The mixture was stirred evenly. After 15 and 30 days, samples were taken and the soil pH and available phosphorus content were tested. The results are shown in Table 8.

[0182] Table 8 Acidified soil improvement effect

[0183] Test results analysis and conclusions:

[0184] (1) As can be seen from Table 8, 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 phosphate-solubilizing effect, which can convert insoluble phosphorus in the soil into soluble phosphorus that can be absorbed and utilized by plants.

[0185] (2) As can be seen from Table 8, 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.

[0186] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used herein are conventional in the art and can be obtained from conventional commercial sources. The methods used herein are conventional in the art, unless otherwise specified.

[0187] 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 fall within the scope of protection of the technical solution of the present invention.

Claims

1. A Candida strain XHZG06-95A3, characterized in that: The Candida XHZG06-95A3 belongs to Candida palmioleophila in taxonomy, and its preservation number is CGMCC No.29215.

2. 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 into a fermentation culture medium to perform a first fermentation to obtain a primary fermentation broth; the acidified soil improving bacterium is Candida XHZG06-95A3, which belongs to Candida palmioleophila in taxonomy and has a preservation number of CGMCC No. 29215; (2) adding mussel shell powder to the primary fermentation liquid to perform a second fermentation to obtain a secondary fermentation liquid; (3) Separate the products from the secondary fermentation liquid to obtain an acidified soil conditioner.

3. The method according to claim 2, 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, Mg2SO4·7H2O 0.5-1 g / L, KCl 0-0.3 g / L, the solvent is water, and the pH is 6-7.

4. The method according to claim 2, characterized in that In step (1), the OD of the primary fermentation broth is 600 It is 0.7 to 1.

2.

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

6. The method according to claim 2, 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.

7. The method according to claim 2, 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 hours.

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

9. An acidified soil conditioner prepared by the method according to any one of claims 2 to 8.

10. Use of the Candida sp. XHZG06-95A3 according to claim 1 or the acidified soil conditioner according to claim 9 in improving acidified soil.

Images