Screening method of phosphate solubilizing bacteria and nitrogen-fixing bacteria loading material and preparation method of microbial inoculum

Abstract

The invention belongs to the field of microbial agents, particularly discloses screening of a phosphate solubilizing bacteria and nitrogen-fixing bacteria loading material, and also provides a preparation method of bacterial strain loaded biomass charcoal, which comprises the following steps: bacterial strain activation and amplification culture to obtain a bacterial suspension; performing high-temperature and high-pressure sterilization on the biomass charcoal material, and drying to obtain a sterilized and dried carrier; and adding the bacterial suspension into a sterilized and dried carrier, carrying out shake culture at a set culture temperature, and filtering to obtain a filter cake which is bacterial strain-loaded biomass charcoal (bacterial agent). According to the invention, a loading material which is most suitable for nitrogen-fixing bacteria and phosphate-solubilizing bacteria is screened from four kinds of biomass charcoal, namely rice straw charcoal, bamboo charcoal, pig manure charcoal and mushroom dreg charcoal, and bacillus cereus or azospirillum brazil is loaded on the charcoal material, so that the survival time of strains is prolonged, and the content of available nitrogen and phosphorus in soil organisms is increased.

Description

Technical Field

[0001] The invention belongs to the field of microbial agents, and specifically relates to a biological fertilizer prepared from biochar, nitrogen-fixing bacteria and phosphate-solubilizing bacteria, and a preparation method thereof. Background Art

[0002] Organic fertilizers have long played an important role in improving soil fertility. However, the application of slow-release organic fertilizers alone cannot meet the growth needs of crops (Chen Fenglin, 2024). Therefore, a method is needed to improve the functional effects of biochar.

[0003] The addition of biochar will affect the diversity of microbial communities, community composition and soil fungal symbiotic networks, thereby affecting nitrogen and phosphorus cycles. Biochar can increase the availability of resources for fertile organisms, thereby rapidly increasing the abundance, activity and diversity of soil nitrogen and phosphorus-related microorganisms. At the same time, it can accommodate diverse microbial communities and provide a place for the fusion of microorganisms from different communities, thereby achieving the effect of changing soil functions. However, the survival and growth of fused communities and their effects on soil are still understudied (Martin Hartman, 2023).

[0004] Biochar can also be used to improve soil physical and chemical properties due to its loose structure and high carbon content, effectively increase soil porosity and carbon source, and is conducive to the survival of microorganisms related to nitrogen and phosphorus cycles (Mao Yilian, 2023). It is an ideal carrier in microbial agents. However, the diverse raw materials lead to different effects of different biochars on the microbial environment (Zhang Jun, 2017), so it is urgent to screen out a suitable biochar loading material for the preparation of microbial agents. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for screening phosphate-solubilizing bacteria and nitrogen-fixing bacteria loading materials and preparing a bacterial agent.

[0006] In order to solve the above problems, the present invention provides a screening of phosphate-solubilizing bacteria and nitrogen-fixing bacteria loading materials: the most suitable loading materials for nitrogen-fixing bacteria and phosphate-solubilizing bacteria are screened out from four types of biomass charcoal, namely, rice straw charcoal, bamboo charcoal, pig manure charcoal and fungus residue charcoal.

[0007] Improvement in the screening of the phosphate-solubilizing bacteria and nitrogen-fixing bacteria loading materials of the present invention: the most suitable loading material for nitrogen-fixing bacteria and phosphate-solubilizing bacteria is bacterial residue charcoal.

[0008] The present invention also provides a method for preparing biochar loaded with bacterial strains, comprising the following steps:

[0009] 1) Strain activation and expansion culture:

[0010] Activating the strain to obtain an activated bacterial suspension; and then using the activated bacterial suspension to prepare a bacterial suspension with an OD600 value of 0.65 to 0.75 (preferably 0.7);

[0011] The strain is a phosphate-solubilizing bacterium or a nitrogen-fixing bacterium;

[0012] 2) Preparation of carrier materials:

[0013] The biomass charcoal material is sterilized (sterilized at high temperature and high pressure) and then dried to obtain a sterilized and dried carrier;

[0014] 3) Preparation of carrier materials:

[0015] The bacterial suspension obtained in step 1) is added to the sterilized and dried carrier obtained in step 2), and the culture is shaken at 135±20 rpm for 24±1 h at the set culture temperature, and filtered. The filter cake obtained is the biomass charcoal (bacterial agent) loaded with the strain;

[0016] The carrier after sterilization and drying: bacterial suspension = (3 ± 0.2 g) / 100 ml.

[0017] As an improvement of the biomass charcoal loaded with bacterial strains of the present invention: the biomass charcoal is rice straw charcoal, bamboo charcoal, pig manure charcoal and fungus residue charcoal (preferred).

[0018] Specifically: the mushroom residue charcoal is prepared from the mushroom residue obtained from the culture medium of Pleurotus eryngii.

[0019] As a further improvement of the biochar loaded with strains of the present invention, the step 1) is: inoculating the strain into a sterilized LB liquid culture medium at 1% (v / v), shaking at 135±20 rpm for 16±0.5 h at a set culture temperature, to obtain a strain activated bacterial suspension;

[0020] The activated bacterial suspension of the strain was cultured by streaking on a sterilized LB solid medium and cultured at the set culture temperature for 24±0.5h. A single colony (a small piece of a single colony) was inoculated into a sterilized LB liquid medium. After sealing, the culture was shaken at 135±20rpm at the set culture temperature (the culture time was about 24±1 hour) until the OD600 value of the bacteria in the obtained bacterial suspension was 0.65-0.75.

[0021] Note: Small single colony: LB liquid culture medium = (0.01±0.002g) / 100ml.

[0022] Further improvement of the biochar loaded with bacterial strains of the present invention:

[0023] The strain is Bacillus cereus (bio-61125Bacillus cereus), the culture temperature is set at 37±0.5°C, and the obtained biochar loaded with the strain is named phosphate-solubilizing bacteria-biochar agent;

[0024] The strain is Azospirillum brasilense, the culture temperature is set at 30±0.5°C, and the obtained biochar loaded with the strain is named nitrogen-fixing bacteria-biochar agent.

[0025] As a further improvement of the biochar loaded with strains of the present invention: the sterilization in step 2) is conventional high temperature and high pressure sterilization, that is, sterilization in a high pressure steam sterilizer at 120° C. and 108 kPa for 30 minutes;

[0026] The drying is performed at 55±5° C. to a constant weight.

[0027] When the bacterial agent (biomass charcoal loaded with bacterial strains) of the present invention is actually used, 5% (g / g) of the weight of the soil is added to the soil and mixed and cultured.

[0028] The present invention selects the most suitable loading material for nitrogen-fixing bacteria and phosphate-solubilizing bacteria from four types of biochars, namely, rice straw charcoal, bamboo charcoal, pig manure charcoal and fungus residue charcoal, and loads Bacillus cereus or Azospirillum brasilense onto the biochar material to prolong the survival time of the strain and increase the biologically effective nitrogen and phosphorus content in the soil. Biochar is widely used to improve soil fertility due to its diversity, easy availability and good effect.

[0029] The beneficial effects of the present invention are mainly reflected in:

[0030] 1) Low cost and simple production:

[0031] The present invention uses rice straw charcoal, bamboo charcoal, pig manure charcoal and fungus residue charcoal as loading materials, which are sourced from agricultural and forestry wastes.

[0032] 2) The screening effect is obvious:

[0033] The loading is achieved by adding the carrier to the bacterial suspension and oscillating it. The operation process is simple and the loading effect is obvious, so as to better screen the suitable carrier.

[0034] In summary, the present invention screens a carrier that can provide survival and growth conditions for nitrogen-fixing bacteria and phosphate-solubilizing bacteria strains while improving the biological effectiveness of soil nitrogen and phosphorus, and prepares a low-cost and easily available strain-biomass charcoal carrier (microbial agent) biofertilizer, which is of great significance for studying the relationship between soil nitrogen and phosphorus cycles and microorganisms and improving the utilization of nitrogen and phosphorus in farmland. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0036] Figure 1 The surface SEM observations of the four bacterial agents obtained after the bio-61125 Bacillus cereus strain was loaded onto four types of biochar materials (using an electron microscope, magnified to 5K);

[0037] The four types of biomass charcoal materials are rice straw charcoal, bamboo charcoal, pig manure charcoal, and fungus residue charcoal;

[0038] Figure 2 The surface SEM observation images of the four bacterial agents obtained after the Azospirillum brasilense strain was loaded onto four types of biomass carbon materials (using an electron microscope, magnified to 5K times);

[0039] The four types of biomass charcoal materials are rice straw charcoal, bamboo charcoal, pig manure charcoal, and fungus residue charcoal;

[0040] Figure 3 The contents of nitrate nitrogen and ammonia nitrogen in soil after incubation of four bacterial agents and CK on days 0, 7 and 14. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0042] In the present invention, the preparation methods of biomass carbon materials are all conventional techniques, for example, as follows:

[0043] The preparation method of rice straw charcoal is as follows: roasting rice straw at 500-600°C for 3-4 hours;

[0044] The preparation method of bamboo charcoal is as follows: roasting bamboo stems at 500-600°C for 3-4 hours;

[0045] The preparation method of pig manure charcoal is as follows: roasting pig manure at 500-600°C for 3-4 hours;

[0046] The preparation method of mushroom residue charcoal is as follows: roasting the mushroom residue at 500-600°C for 3-4 hours, wherein the mushroom residue is obtained from the culture matrix of Pleurotus eryngii.

[0047] Phosphate-solubilizing bacteria-Bacillus cereus (bio-61125 Bacillus cereus standard strain) was from China Industrial Microbiological Culture Collection Center (No. bio-61125);

[0048] The nitrogen-fixing bacteria, Azospirillum brasilense, was obtained from Guangdong Microbial Culture Collection Center (GDMCC No. 1.1899).

[0049] Embodiment 1, the preparation method of biomass charcoal-phosphate-solubilizing bacteria agent, carries out the following steps in sequence:

[0050] 1) Preparation of bacterial suspension

[0051] The bio-61125 Bacillus cereus standard strain (glycerol storage) was inoculated into the sterilized LB liquid medium at 1% (v / v), sealed with a high-temperature resistant tissue culture sealing film (the sealing method mentioned in the article was used), and then shaken at 37°C and 135 rpm for 16 hours to obtain a strain activated bacterial suspension;

[0052] The activated bacterial suspension of the strain was streaked on the surface of a sterile plate (LB solid culture medium), sealed and cultured in a 37°C incubator for 24 hours, a small piece of a single colony was inoculated into the sterilized LB liquid culture medium, sealed again, and shaken at 37°C 135 rpm for about 24 hours, and the OD600 value was determined using a microplate reader to prepare a phosphate-solubilizing bacteria suspension for subsequent use.

[0053] Small piece of single colony: LB liquid medium = (0.01±0.002g) / 100ml.

[0054] Preparation of LB liquid medium: add 25g of LB broth (containing 10g of peptone, 5g of yeast extract, and 10g of NaCl) to each liter of water, adjust the pH to 7.4-7.6 with NaOH solution, and then seal and sterilize at conventional high temperature and high pressure (120°C 108 kPa sterilization for 30 minutes). For LB solid medium, add 15g of agar to each liter of liquid medium and perform conventional high temperature and high pressure sterilization.

[0055] 2) Preparation of carrier materials:

[0056] The biochar was sterilized at high temperature and high pressure (120° C., 108 kPa for 30 minutes), dried (dried at 55° C. to constant weight) and sealed for later use.

[0057] Application Example 1: Referring to Example 1, the following steps are specifically performed:

[0058] 1) Take four 50 mL portions of sterilized LB liquid culture medium into 250 mL conical flasks, inoculate bio-61125 Bacillus cereus single colony, shake at 37°C 135 rpm for about 24 hours, and make the average OD600 of the bacterial suspension 0.7.

[0059] 2) Sterilized and dried rice straw charcoal, bamboo charcoal, pig manure charcoal and fungus residue charcoal are used as four carrier raw materials, and each carrier raw material is subjected to the following operations respectively:

[0060] Weigh 1.50 g of carrier raw material and add it to the conical flask in step 1) and mix well.

[0061] 3) Place the conical flask obtained in step 2) in a constant temperature shaking box at 37°C and 135rpm for culturing for 24 hours, so that the strain is loaded on the biochar to obtain bio-61125 Bacillus cereus-biochar bacterial agent.

[0062] The bacterial agents obtained by using rice straw charcoal, bamboo charcoal, pig manure charcoal and fungus residue charcoal as four carrier raw materials are as follows:

[0063] bio-61125Bacillus cereus-rice straw charcoal agent, bio-61125Bacillus cereus-bamboo charcoal agent, bio-61125Bacillus cereus-pig manure charcoal agent, bio-61125Bacillus cereus-mushroom residue charcoal agent.

[0064] 4) Add the bacterial agent obtained in step 3) at 5% (g / g) of the soil mass, mix and culture, and take samples on the 7th and 14th days for storage.

[0065] Blank (CK) control: the addition of the bacterial agent in step 3) was omitted, and the rest of the operations were the same.

[0066] The bio-61125Bacillus cereus-biomass charcoal agent obtained in step 3) was treated and its surface was observed using an electron microscope. By comparison, it was found that the number of bacteria on the surface of the agent was significantly denser when the charcoal was used as a carrier, and the loading effect was the best ( Figure 1 ).

[0067] 2 mm air-dried soil samples were taken at 7 and 14 days of cultivation, and the available phosphorus content in the soil was measured by the sodium bicarbonate method (Bao Shidan, 1999). On the 7th and 14th days, the available phosphorus content of the soil when bio-61125Bacillus cereus-mushroom residue charcoal was added was significantly higher than that when other bacterial agents were added (Table 1). The results showed that mushroom residue charcoal is a biomass charcoal suitable for bio-61125Bacillus cereus loading and can effectively increase the available phosphorus content in the soil.

[0068] Table 1 Available phosphorus content of four biochar-phosphate-solubilizing bacteria cultured at 0, 7 and 14 days

[0069]

[0070] Different lowercase letters indicate significant differences among different treatments within the same culture time (P<0.05).

[0071] Rice straw charcoal represents the treatment with the addition of bio-61125Bacillus cereus-rice straw charcoal inoculant;

[0072] Bamboo charcoal represents the treatment with the addition of bio-61125Bacillus cereus-bamboo charcoal bacteria agent;

[0073] Pig manure charcoal represents the treatment with the addition of bio-61125Bacillus cereus-pig manure charcoal bacteria;

[0074] The fungus residue charcoal represents the treatment with the addition of bio-61125Bacillus cereus-fugus residue charcoal inoculant;

[0075] The blank control represents the treatment without the addition of bacterial agent.

[0076] Example 2: A method for preparing a biochar-nitrogen-fixing bacteria agent, comprising the following steps:

[0077] 1) Preparation of bacterial suspension:

[0078] The Azospirillum brasilense standard strain (glycerol storage) was inoculated into the sterilized LB liquid culture medium at 1% (v / v), sealed, and shaken at 30°C and 135 rpm for 16 h to obtain the activated bacterial suspension.

[0079] The activated bacterial suspension of the strain was streaked on the surface of a sterile plate (LB solid culture medium), sealed and cultured in a 30°C incubator for 24 hours, a small piece of a single colony was inoculated into the sterilized LB liquid culture medium, sealed again and shaken at 30°C 135 rpm for 24 hours, the OD600 value was measured using a microplate reader, and a nitrogen-fixing bacterial suspension was prepared for subsequent use.

[0080] Small piece of single colony: LB liquid medium = (0.01±0.002g) / 100ml.

[0081] 2) Preparation of carrier materials:

[0082] The biochar is sterilized at high temperature and high pressure, dried (dried at 55°C to constant weight) and sealed for later use.

[0083] Application Example 2: Referring to Example 1, the following steps are specifically performed:

[0084] 1) Take four 50 mL portions of sterilized LB liquid culture medium into 250 mL conical flasks, inoculate a single colony of Azospirillum brasilense, and shake at 30°C and 135 rpm for 24 h until the average OD600 of the bacterial suspension is 0.7.

[0085] 2) Sterilized and dried rice straw charcoal, bamboo charcoal, pig manure charcoal and fungus residue charcoal are used as four carrier raw materials, and each carrier raw material is subjected to the following operations respectively:

[0086] Weigh 1.50 g of carrier raw material and add it to the conical flask in step 1) and mix well.

[0087] 3) placing the conical flask in step 2) in a constant temperature shaking box at 30° C. and 135 rpm for culturing for 24 hours, so that the strain is loaded on the biochar to obtain the Azospirillum brasilense-biochar bacteria agent.

[0088] The bacterial agents obtained by using rice straw charcoal, bamboo charcoal, pig manure charcoal and fungus residue charcoal as four carrier raw materials are as follows:

[0089] Azospirillum brasilense-rice straw charcoal agent, Azospirillum brasilense-bamboo charcoal agent, Azospirillum brasilense-pig manure charcoal agent, Azospirillum brasilense-mushroom residue charcoal agent.

[0090] 4) Add the bacterial agent obtained in step 3) at 5% (g / g) of the soil mass, mix and culture, and collect samples on the 7th and 14th days for storage.

[0091] Blank (CK) control: the addition of the bacterial agent in step 3) was omitted, and the rest of the operations were the same.

[0092] The surface of the Azospirillum brasilense-biomass charcoal inoculant obtained in step 3) was observed using an electron microscope after treatment. Figure 2 ), and found that pig manure charcoal and fungus residue charcoal were more suitable as carriers of nitrogen-fixing bacteria than rice straw charcoal and bamboo charcoal, with significantly more bacteria attached to the surface. At the same time, SEM showed that Azospirillum brasilense was most densely distributed on the surface of fungus residue charcoal, with a higher survival rate.

[0093] The nitrate nitrogen and ammonium nitrogen contents at 0, 7 and 14 days of cultivation were as follows Figure 3The nitrate nitrogen content is shown in Table 2. After 7 days of cultivation, the nitrate nitrogen content of each treatment increased significantly, among which the inorganic nitrogen (nitrate nitrogen and ammonium nitrogen) content of the soil treated with Azospirillum brasilense-mushroom residue charcoal was significantly higher than that of other treatments; from 7 days to 14 days, the ammonium nitrogen content of the soil increased significantly, and there was no significant difference in the ammonium nitrogen content among the four treatments, but the nitrate nitrogen content of the soil treated with Azospirillum brasilense-mushroom residue charcoal was the highest. After 7 days, the addition of the four bacterial agents could significantly increase the nitrate nitrogen and ammonium nitrogen content in the soil, and the nitrate nitrogen content showed the rule of bacterial residue charcoal> pig manure charcoal> bamboo charcoal> rice straw charcoal. This shows that the bacterial agent made of Azospirillum brasilense loaded with bacterial residue charcoal as a carrier can significantly increase the ammonium nitrogen content in the soil while significantly increasing the total nitrogen content in the soil, which is beneficial to the growth and development of crops.

[0094] From the dynamic changes of alkaline nitrogen content, it generally showed an upward trend from 0 to 7 days and a downward trend from 7 to 14 days (Table 2). Among them, the alkaline nitrogen content in the soil treated with Azospirillum brasilense-slag charcoal was significantly higher than that in other treatments, and it was suitable as a carrier material for loading Azospirillum brasilense.

[0095] Table 2 Alkaline nitrogen content of four biochar-nitrogen-fixing bacteria cultured for 7 and 14 days

[0096]

[0097]

[0098] Different lowercase letters indicate significant differences among different treatments within the same culture time (P<0.05).

[0099] Rice straw charcoal represents the treatment with the addition of Azospirillum brasilense-rice straw charcoal inoculant;

[0100] Bamboo charcoal represents the treatment with the addition of Azospirillum brasilense-bamboo charcoal inoculant;

[0101] Pig manure charcoal represents the treatment with the addition of Azospirillum brasilense-pig manure charcoal bacteria;

[0102] Mushroom residue charcoal represents the treatment with the addition of Azospirillum brasilense-mushroom residue charcoal inoculant;

[0103] Blank control: treatment without added bacterial agent.

[0104] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Screening of phosphate-solubilizing bacteria and nitrogen-fixing bacteria loading materials, characterized in that: From four types of biomass charcoal, namely rice straw charcoal, bamboo charcoal, pig manure charcoal and mushroom residue charcoal, the most suitable ones as loading materials for nitrogen-fixing bacteria and phosphate-solubilizing bacteria were screened out.

2. The screening of phosphate-solubilizing bacteria and nitrogen-fixing bacteria loading materials according to claim 1, characterized in that: The most suitable loading material for nitrogen-fixing bacteria and phosphate-solubilizing bacteria is bacterial residue charcoal.

3. A method for preparing biochar loaded with bacterial strains, characterized in that The following steps are involved: 1) Strain activation and expansion culture: Activating the strain to obtain an activated bacterial suspension; and then using the activated bacterial suspension to prepare a bacterial suspension with an OD600 value of 0.65 to 0.75; 2) Preparation of carrier materials: The biomass charcoal material is sterilized and then dried to obtain a sterilized and dried carrier; 3) Take the bacterial suspension obtained in step 1) and add it to the sterilized and dried carrier obtained in step 2) at the set culture temperature. Oscillate at 135±20 rpm for 24±1 h, filter, and the resulting filter cake is the biochar loaded with the strain; The carrier after sterilization and drying: bacterial suspension = (3 ± 0.2 g) / 100 ml.

4. The method for preparing biochar loaded with bacterial strains according to claim 3, characterized in that: The biomass charcoal is rice straw charcoal, bamboo charcoal, pig manure charcoal and fungus residue charcoal.

5. The method for preparing biochar loaded with bacterial strains according to claim 4, characterized in that: The step 1) is: inoculating the strain into a sterilized LB liquid culture medium at 1% (v / v), shaking at 135±20 rpm for 16±0.5 h at a set culture temperature, to obtain an activated bacterial suspension of the strain; The activated bacterial suspension of the strain was cultured by streaking on a sterilized LB solid medium and cultured at the set culture temperature for 24±0.5h. A single colony was inoculated into a sterilized LB liquid medium, which was sealed and cultured with shaking at 135±20rpm at the set culture temperature until the OD600 value of the bacteria in the obtained bacterial suspension was 0.65-0.

75.

6. The method for preparing biochar loaded with bacterial strains according to claim 5, characterized in that: The strain is Bacillus cereus (bio-61125Bacillus cereus), the culture temperature is set at 37±0.5°C, and the obtained biochar loaded with the strain is phosphate-solubilizing bacteria-biochar agent; The strain is Azospirillum brasilense, the culture temperature is set at 30±0.5°C, and the obtained biochar loaded with the strain is a nitrogen-fixing bacteria-biochar agent.

7. The method for preparing biochar loaded with bacterial strains according to any one of claims 3 to 6, characterized in that: The sterilization in step 2) is conventional high temperature and high pressure sterilization; The drying is performed at 55±5° C. to a constant weight.

8. Use of the biochar loaded with bacterial strains prepared by any one of the methods of claims 3 to 7, characterized in that: Add to the soil as a bacterial agent and mix well; increase the content of biologically available nitrogen and phosphorus in the soil.

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