Nitrifying bacteria carrier as well as preparation method and application thereof

Abstract

The invention discloses a nitrobacteria carrier as well as a preparation method and application thereof, and belongs to the technical field of microbial preparations. The preparation method of the nitrobacteria carrier comprises the following steps: (1) carbonizing a biomass material to obtain biochar; the biomass material comprises at least one of pine, walnut shell, peanut shell and corn straw; (2) treating the biochar with alkali liquor to obtain modified biochar; and (3) mixing the modified charcoal with sandy soil to obtain the nitrobacteria carrier. The bacterial carrier is prepared by combining the modified biochar with the sandy soil, nitrobacteria are adsorbed, certain survival conditions are provided for growth of the nitrobacteria, the nitrification capacity of the nitrobacteria can be improved, and the bacterial carrier has the potential of developing preparations such as nitrobacteria fertilizers, livestock manure leavening agents or waste water treating agents.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial preparations, and in particular to a nitrifying bacteria carrier and a preparation method and application thereof. Background Art

[0002] Livestock and poultry manure is a high-quality organic resource, rich in nitrogen, phosphorus, potassium and other nutrients required by plants. Through composting, not only can livestock and poultry manure be rendered harmless, but it can also be converted into high-efficiency organic fertilizer for fertilizing farmland, improving soil fertility and crop yields. The nitrogen in livestock and poultry manure is one of the important nutrients required for crop growth and plays an important role in the growth and development of crops. However, there is nitrogen fixation and release in the process of livestock and poultry composting. At the same time, there is also a large amount of nitrogen loss, which mainly includes NH3, NO X The volatilization of gaseous nitrogen and water-soluble nitrogen are lost with the leachate.

[0003] Nitrogen conversion in aerobic composting of livestock and poultry manure occurs primarily through the action of microorganisms such as ammonifying bacteria, nitrifying bacteria, and denitrifying bacteria. In the initial stages of composting, a large amount of organic nitrogen is converted into inorganic ammonium nitrogen through the ammonification action of ammonifying bacteria. This inorganic ammonium nitrogen is further converted into NH3 by the ammonifying bacteria. Ammonium nitrogen is then converted into nitrite nitrogen by the action of nitrite-forming bacteria, and nitrite nitrogen is converted into nitrate nitrogen by the nitrification action of nitrifying bacteria. This nitrogen is then assimilated and absorbed by organisms. The nitrification process inhibits the production of NH3 and reduces nitrogen loss in the form of NH3. Throughout the composting process, as the ammonium nitrogen generated in the early stages of composting is further converted into NH3 and nitrate nitrogen, the ammonia nitrogen content generally increases and then decreases, while the nitrate nitrogen content gradually increases. The higher the activity of ammonifying and denitrifying bacteria, the more likely nitrogen is to be volatilized and lost as NH3, N2, and N2O. The higher the activity of nitrifying bacteria, the more likely nitrogen is converted to nitrate nitrogen and fixed for absorption and utilization by plants. Nitrogen loss is closely related to the activity of these microorganisms. Furthermore, the nitrification process is also susceptible to environmental factors. Therefore, providing a new carrier that can provide ample growth space for nitrifying bacteria and enhance bacterial activity is of great significance for nitrogen conversion in nature. Summary of the Invention

[0004] The present invention aims to provide a nitrifying bacteria carrier, its preparation method, and its application to address the aforementioned problems in the prior art. The present invention prepares a bacterial carrier by combining modified biochar with sandy soil, which adsorbs nitrifying bacteria and provides certain living conditions for their growth, thereby improving their nitrification capacity. The carrier has the potential to be developed into a nitrifying bacteria fertilizer, livestock manure fermentation agent, or wastewater treatment agent.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for preparing a nitrifying bacteria carrier, comprising the following steps:

[0007] (1) carbonizing a biomass material to obtain biochar; the biomass material comprises at least one of pine wood, walnut shells, peanut shells, and corn stalks;

[0008] (2) treating the biochar with an alkali solution to obtain modified biochar;

[0009] (3) Mixing the modified biochar with sand to obtain the nitrifying bacteria carrier.

[0010] Optionally, the temperature of the carbonization treatment is 300-700° C., and the time is 1.5-3 h; the heating rate during the carbonization treatment is 10° C. / min.

[0011] Optionally, the alkali solution includes a NaOH solution; the concentration of the NaOH solution is 1 mol / L; and the mass ratio of the biochar to the NaOH solution is 1:15-25.

[0012] Optionally, the mass ratio of the modified biochar to the sand is 1:1.5-3.

[0013] The present invention also provides a nitrifying bacteria carrier prepared by the above preparation method.

[0014] The present invention also provides use of the nitrifying bacteria carrier in preparing a microbial preparation or a microbial fertilizer.

[0015] The present invention also provides a method for preparing a nitrifying bacteria preparation, comprising the following steps:

[0016] The nitrifying bacteria are activated and cultured, and then centrifuged to obtain wet bacteria bodies; the nitrifying bacteria carrier is mixed with the wet bacteria bodies, and adsorption treatment is performed in a buffer solution to obtain a nitrifying bacteria preparation.

[0017] Optionally, the OD of the nitrifying bacteria after activation culture is 600 =1.0±0.2; the mass volume ratio of the nitrifying bacteria carrier to the wet bacteria is 0.5-2g:0.5-2mL; the temperature of the adsorption treatment is 25-30°C, the time is 1-3h, and the rotation speed is 100-200rpm.

[0018] The present invention also provides a nitrifying bacteria preparation prepared by the above preparation method.

[0019] The present invention also provides the use of the nitrifying bacteria preparation in aerobic composting of livestock and poultry manure, improving soil environment or treating wastewater.

[0020] The present invention discloses the following technical effects:

[0021] The present invention uses common agricultural waste as the main raw material to prepare alkali-modified biochar, adopts mechanochemical action, and mixes biochar with sand by mechanical grinding and magnetic stirring to prepare a nitrifying bacteria carrier, which has the advantages of low cost, low energy consumption and good controllability. Nitrifying bacteria can adhere to and grow on the carrier, forming a microbial film on the carrier surface. The porous structure and breathable properties of the carrier provide a good habitat for nitrifying bacteria, which can promote the growth and metabolism of nitrifying bacteria. The nitrifying bacteria preparation prepared using the nitrifying bacteria carrier can effectively and quickly remove ammonium ions, and has the potential to develop preparations such as nitrifying bacteria fertilizers, livestock and poultry feces fermentation agents or wastewater treatment agents, and provides new resources for natural nitrogen conversion, with a wide range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a photo of the nitrifying bacteria carrier obtained by combining 500°C carbonized modified pine biochar with sand in Example 1;

[0024] Figure 2 The adsorption performance test results of different nitrifying bacteria carriers prepared in Example 1;

[0025] Figure 3 The changes in ammonium ion content under the action of different nitrifying bacteria preparations prepared in Example 1;

[0026] Figure 4 The removal rate of ammonium ions under the action of different nitrifying bacteria preparations prepared in Example 1. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The technical solutions of the present invention are as follows:

[0033] The present invention grinds waste materials such as pine wood, walnut shells, peanut shells, and corn stalks in a ball mill, passes through a 30-mesh sieve, wraps a crucible with tin foil, and burns them into biochar at different temperatures in a muffle furnace. The biochar is then alkali-modified using a 1 mol / L NaOH solution, and finally the modified biochar is combined with sand by magnetic stirring to prepare a novel nitrifying bacteria carrier.

[0034] The preparation method of the nitrifying bacteria carrier of the present invention specifically comprises the following steps:

[0035] (1) Preparation of modified biochar: Pine wood, walnut shells, peanut shells, and corn stalks were cleaned, dried, ground, and passed through a 30-mesh sieve. They were placed in a muffle furnace and calcined at different temperatures of 300°C, 500°C, and 700°C for 2 h. The mixtures were naturally cooled, cleaned, and dried in an oven at 80°C to constant weight to prepare biochar. Biochar was mixed with 1 mol / L NaOH in a ratio of 1:20 (w:w), allowed to stand for 24 h, and the biochar was modified. The mixtures were washed until neutral, filtered, and dried in an oven at 80°C to constant weight.

[0036] (2) Sand pretreatment: Grind the sand, pass it through a 30-mesh sieve, wash it, and dry it in an oven at 80°C to constant weight for later use;

[0037] (3) The modified biochar and sand were mixed evenly in a ratio of 1:2, washed, and dried in an oven at 80°C to a constant weight to obtain a nitrifying bacteria carrier.

[0038] The method for using the nitrifying bacteria carrier of the present invention specifically comprises the following steps:

[0039] The nitrifying bacteria were activated and cultured in a specific culture medium at 30°C, enriched, centrifuged, and the supernatant was discarded to obtain wet bacteria for later use; 0.5 g of nitrifying bacteria carrier was weighed, 0.5 mL of wet bacteria were aspirated, the carrier and wet bacteria were added to 50 mL of PBS buffer, and adsorbed on a shaker at 30°C and 150 rpm for 2 hours, centrifuged, and the supernatant was discarded to obtain a nitrifying bacteria preparation.

[0040] The technical principles of the present invention are as follows:

[0041] Biochar, with its porous structure and large surface area, can attract nitrifying bacteria, providing them with a favorable habitat and promoting their growth and activity. Nitrifying bacteria are aerobic and require ample oxygen to survive. Biochar loading provides spaces for these bacteria to thrive. Nitrifying bacteria prefer to grow in a slightly alkaline environment, with a pH range of approximately 7.5 to 8.2. Therefore, NaOH provides a slightly alkaline environment, favoring their growth. Sandy soil, with its large particles and good aeration, attracts nitrifying bacteria. This property facilitates aerobic respiration, especially for aerobic microorganisms, where ample oxygen facilitates their growth and metabolism. Sandy soil is predominant in arid and semi-arid regions, where most plants are grown. Loading bacteria onto sandy soils allows them to better adapt to the soil environment, providing a resource for crop cultivation and agricultural development in arid and water-scarce regions.

[0042] In addition, biochar can be used to adsorb and fix nutrients in fertilizers, such as nitrogen, phosphorus, and potassium, creating a slow-release effect. This helps reduce nutrient loss, improves fertilizer utilization, and enables crops to absorb nutrients continuously and stably. The porous structure of biochar can increase soil porosity, improving soil aeration and water permeability. Nitrifying bacteria preparations prepared using nitrifying bacteria carriers can not only utilize the nitrifying ability of nitrifying bacteria to change soil pH, aeration, and moisture content, but also utilize the characteristics of the carrier itself to improve soil structure and nutrient distribution, thereby promoting plant growth and development.

[0043] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0044] Example 1

[0045] This embodiment provides a method for preparing a nitrifying bacteria carrier and its application method. The specific process is as follows:

[0046] (1) Preparation of modified biochar: Pine wood, walnut shells, peanut shells, and corn stalks were cleaned, ground through a 30-mesh sieve, cleaned, and dried in an 80°C oven to constant weight. The dried pine wood, walnut shells, peanut shells, and corn stalks were placed in a muffle furnace and carbonized at different temperatures (300°C, 500°C, and 700°C) for 2 h, with a heating rate of 10°C / min. The samples were washed and dried to obtain biochar. A solid NaOH sample was dissolved in 1000 mL of pure water and diluted to a volume in a 1000 mL volumetric flask to prepare a 1 mol / L NaOH solution. The biochar and NaOH solution were mixed at a ratio of 1:20 (w:w) at room temperature for 24 h, washed until neutral, and dried in an 80°C oven to constant weight to obtain modified biochars carbonized from different biomass materials at different temperatures.

[0047] (2) Preparation of sand-modified biochar: Grind the sand through a 30-mesh sieve, wash it, and dry it. Mix the modified biochar obtained in step (1) with the sand in a ratio of 1:2, add 50 mL of pure water, stir it with a magnetic stirrer for 24 h, mix it evenly, wash it, and dry it in an oven at 80°C to constant weight to obtain nitrifying bacteria carriers prepared from different biomass materials. Among them, the phenotype of the nitrifying bacteria carrier obtained by combining 500°C carbonized modified pine biochar with sand is as follows: Figure 1 shown.

[0048] (3) Application of nitrifying bacteria carrier: Nitrifying bacteria were activated and cultured in a specific culture medium (0.382 g (NH4)2SO4, 2 g sodium citrate, 0.05 g MgSO4·7H2O, 0.2 g K2HSO4, 0.12 g NaCl, 0.01 g MnSO4·H2O, 0.01 g FeSO4, 1 L water, pH = 7.2) at 30 °C for 24 h until OD 600 =1.0±0.2, centrifuge, discard the supernatant to obtain wet bacteria. Add 0.5 g of nitrifying bacteria carrier and 0.5 mL of wet bacteria to 50 mL of PBS buffer, shake at 30°C, 150 rpm for 2 h, centrifuge at 4000 rpm for 10 min, and discard the supernatant to obtain the nitrifying bacteria preparation.

[0049] Example 2

[0050] This example studies the adsorption performance of the nitrifying bacteria carrier prepared in Example 1 on bacteria. The specific process is as follows:

[0051] The nitrifying bacteria preparation prepared in Example 1 was added to 50 mL of PBS buffer at a 10% addition amount, shaken at 160 rpm for 2 h, desorbed, diluted, and 0.1 mL was aspirated and cultured on a culture medium for 24 h. The number of viable bacteria was determined to evaluate the adsorption performance of the nitrifying bacteria carrier.

[0052] The results are as follows Figure 2 shown. Figure 2 The types of vectors corresponding to the serial numbers are shown in Table 1. Figure 2 It can be seen that the number of live bacteria adsorbed by the nitrifying bacteria carriers obtained by combining the modified pine biochar and walnut biochar obtained by carbonization at 500℃ with sand is the largest, and the number of live bacteria adsorbed by the nitrifying bacteria carrier obtained by combining the modified corn straw biochar obtained by carbonization at 300℃ with sand is second.

[0053] Table 1 Correspondence between vector numbers and vector types

[0054]

[0055] Example 3

[0056] This example studies the nitrifying ability of the nitrifying bacteria preparation prepared in Example 1. The specific process is as follows:

[0057] Prepare heterotrophic ammoniation culture medium (0.382g NH4Cl, 2g sodium acetate, 0.05g MgSO4·7H2O, 0.2g K2HSO4, 0.12g NaCl, 0.01g MnSO4·H2O, 0.01g FeSO4, 1L pure water, pH = 7.2), take the nitrifying bacteria preparation prepared in Example 1 and add it to 100mL heterotrophic ammoniation culture medium at a rate of 1%, culture at 30°C, and refer to "Water Quality - Determination of Ammonia Nitrogen - Nessler's Reagent Spectrophotometry (HJ 535-2009)" to detect the ammonium ion (NH4 + ) content changes and calculated the ammonium ion removal rate to evaluate the nitrification ability of nitrifying bacteria preparations prepared with different carriers.

[0058] The results are as follows Figure 3 and Figure 4 shown. Figure 3 and Figure 4 The types of carriers corresponding to the serial numbers are shown in Table 1. Overall, the nitrifying bacteria preparations prepared by 1-4 have a good removal effect on ammonium ions, and the removal rate continues to increase with time. On the 7th day, the ammonium ion concentration has dropped to about 38 mg / L. The nitrifying bacteria preparations prepared by 5-11 have a high removal rate of ammonium ions in the early stage, and the ammonium ion concentration on the 1st to 2nd day is significantly lower than that of other nitrifying bacteria preparations, but the degree of change in the ammonium ion concentration in the later stage is small, and the removal rate is significantly slowed down. The nitrifying bacteria preparation prepared by 12 has a poor removal effect on ammonium ions in the early stage, but after the 4th day, the ammonium ion concentration has dropped significantly and the removal rate has increased, indicating that the nitrifying bacteria preparation prepared by 12 has a better sustained-release effect. In the process of removing ammonium ions, nitrifying bacteria preferentially convert NH4 + Oxidized to NO2- 、NO3 - , resulting in NH4 + The concentration decreases rapidly. But high concentration NH4 + or NO2 - It may temporarily inhibit the activity of nitrifying bacteria, resulting in NH4 + Consumption slows down; pH or dissolved oxygen changes may also inhibit nitrifying bacteria, causing NH4 + Reaccumulation, NH4 appears + The concentration of NH4 increases temporarily. However, if the activity of nitrifying bacteria increases, it may promote the + and NO2 - Completely oxidized to nitrate.

[0059] In summary, the nitrifying bacteria preparation prepared by the present invention has a strong nitrifying ability, indicating that the nitrifying bacteria carrier of the present invention and the nitrifying bacteria preparation prepared therefrom can be used to prepare fertilizers to improve the soil environment, or to prepare livestock manure fermentation agents to improve the utilization rate of nitrogen in livestock manure and accelerate nitrogen conversion, or to prepare wastewater treatment agents to purify wastewater. It has a wide range of applications and high application value.

[0060] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a nitrifying bacteria carrier, characterized in that: The steps include: (1) carbonizing a biomass material to obtain biochar; the biomass material comprises at least one of pine wood, walnut shells, peanut shells, and corn stalks; (2) treating the biochar with an alkali solution to obtain modified biochar; (3) Mixing the modified biochar with sand to obtain the nitrifying bacteria carrier.

2. The preparation method according to claim 1, characterized in that The temperature of the carbonization treatment is 300-700° C., and the time is 1.5-3 hours; the heating rate during the carbonization treatment is 10° C. / min.

3. The preparation method according to claim 1, characterized in that The alkali solution includes a NaOH solution; the concentration of the NaOH solution is 1 mol / L; the mass ratio of the biochar to the NaOH solution is 1:15-25.

4. The preparation method according to claim 1, characterized in that The mass ratio of the modified biochar to the sand is 1:1.5-3.

5. A nitrifying bacteria carrier prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the nitrifying bacteria carrier according to claim 5 in the preparation of microbial preparations or microbial fertilizers.

7. A method for preparing a nitrifying bacteria preparation, characterized in that: The steps include: The nitrifying bacteria are activated and cultured, and then centrifuged to obtain wet bacteria bodies; the nitrifying bacteria carrier according to claim 5 is mixed with the wet bacteria bodies, and adsorption treatment is performed in a buffer solution to obtain a nitrifying bacteria preparation.

8. The preparation method according to claim 7, characterized in that The OD of the nitrifying bacteria after activation culture 600 =1.0±0.2; the mass volume ratio of the nitrifying bacteria carrier to the wet bacteria is 0.5-2g:0.5-2mL; the temperature of the adsorption treatment is 25-30°C, the time is 1-3h, and the rotation speed is 100-200rpm.

9. A nitrifying bacteria preparation prepared by the preparation method according to claim 7 or 8.

10. Use of the nitrifying bacteria preparation according to claim 9 in aerobic composting of livestock and poultry manure, improving soil environment or treating wastewater.

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