Microbial fertilizer as well as preparation method and application thereof

Abstract

The invention discloses a microbial fertilizer and a preparation method and application thereof.The microbial fertilizer comprises a biochar carrier and non-symbiotic nitrogen-fixing bacteria attached to the biochar carrier, and the non-symbiotic nitrogen-fixing bacteria are Klebsiella pneumoniae sp. The straw biochar has the advantage that the effect of improving the activity of nitrogenase is more remarkable according to the particle size of 0.25-2 mm. The biochar with the particle size of 0.25-2mm provides a good habitat for strain cells and can better preserve water and fertilizer, so that growth and reproduction of strains are guaranteed, and the biochar can be put into soil for use. The loading effect of the corn straw biochar is superior to that of wood biochar, the C: N value of the corn straw biochar is remarkably lower and ranges from 61 to 84, and growth of nitrogen-fixing bacteria is facilitated. The content of total nitrogen and microbial biomass nitrogen in soil is effectively improved, and the corn stalk carbon has a more obvious effect on increasing the microbial biomass nitrogen in the soil and is closely related to the activity of soil microorganisms.

Description

Technical Field

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

[0002] In nutrient-deficient and barren environments, non-symbiotic nitrogen-fixing microorganisms can fix molecular nitrogen in the air and secrete growth hormones. Non-symbiotic nitrogen fixation has few restrictions and is widely distributed, but its importance is often overlooked due to its low nitrogen fixation efficiency. In farmland systems without fertilizers and with no or few legumes, non-symbiotic nitrogen fixation may be the main source of nitrogen and plays a key role in the balance of nitrogen cycle in global ecosystems. Faced with a series of negative impacts such as decreased soil fertility, non-point source pollution, and increased greenhouse gas emissions caused by the long-term and large-scale use of chemical fertilizers, the enhancement of non-symbiotic nitrogen fixation and its development and utilization are even more important for the sustainable development of agriculture.

[0003] In agronomic production, non-symbiotic nitrogen-fixing bacteria are often used to make fertilizers to increase the biological nitrogen fixation potential of the soil and improve soil fertility. However, due to the low activity and poor environmental adaptability of exogenously added free nitrogen-fixing bacteria, their survival and reproduction in the soil are greatly restricted, making it difficult to promote and apply on a large scale. Biochar is a type of solid material with characteristics such as porosity, high specific surface area and high carbon content produced by high-temperature pyrolysis of biological residues under anaerobic conditions. The rich pore structure of biochar has a protective and rapid colonization effect on microorganisms. The active carbon components and mineral nutrients can provide nutrition for microorganisms. Material conditions such as high carbon and low nitrogen are conducive to promoting non-symbiotic nitrogen fixation, and it is expected to become an ideal carrier for non-symbiotic nitrogen-fixing microorganisms. At present, there are many studies on the non-symbiotic nitrogen fixation mechanism and strengthening mechanism, and there is still a blank in the selection of non-symbiotic nitrogen fixation carrier materials, which also limits the further development of this technology. The pores and particle sizes of biochar carriers prepared from different raw materials are different, and their water and fertilizer retention capabilities are significantly different. The loading effect of nitrogen-fixing bacteria also changes accordingly, which ultimately affects the activity and nitrogen fixation ability of nitrogen-fixing bacteria.

[0004] Therefore, a microbial fertilizer and a preparation method and application thereof are provided to solve the above-mentioned technical problems. Summary of the invention

[0005] The purpose of the present invention is to provide a microbial fertilizer and a preparation method and application thereof. The microbial fertilizer uses a biochar carrier as an adsorption matrix, and adsorbs non-symbiotic nitrogen-fixing bacteria on the biochar carrier, and screens out the optimal particle size of the biochar carrier, which can protect and quickly colonize the nitrogen-fixing bacteria, thereby improving the survival rate and activity of the nitrogen-fixing bacteria. The biochar carrier and the nitrogen-fixing bacteria produce a certain interaction as the culture time increases, and a culture time period for the biochar carrier to maintain the nitrogen-fixing ability of the nitrogen-fixing bacteria is proposed. The microbial fertilizer improves the soil structure, and promotes the rapid fertilization of poor soil by increasing carbon and fixing nitrogen.

[0006] The object of the present invention is achieved in that:

[0007] A microbial fertilizer comprises a biochar carrier and non-symbiotic nitrogen-fixing bacteria attached to the biochar carrier. The non-symbiotic nitrogen-fixing bacteria is Klebsiella pneumoniae, and the preservation number of Klebsiella pneumoniae is CGMCC No.32396.

[0008] The biochar carrier is a corn straw biochar carrier, and the particle size of the corn straw biochar carrier is 0.25-2 mm.

[0009] The corn straw biochar is prepared by oxygen-limited pyrolysis at 500°C.

[0010] A method for preparing microbial fertilizer comprises the following steps:

[0011] Step 1, picking a ring of non-symbiotic nitrogen-fixing bacterial colonies from a nitrogen-free solid culture medium, inoculating them into LB liquid culture medium for activation, and culturing them in a constant temperature shaker until the growth logarithmic phase to obtain a bacterial liquid;

[0012] Step 2, taking out the bacterial solution of step 1 and performing centrifugation operation, pouring out the supernatant to obtain the strain precipitate, washing and resuspending the strain precipitate with a sterile phosphate buffer solution PBS, adjusting the optical density of the suspension at 600 nm to 0.7-0.8, and obtaining a non-symbiotic nitrogen-fixing bacteria suspension;

[0013] Step 3, the sterile biochar carrier and the non-symbiotic nitrogen-fixing bacteria suspension are fully mixed at a ratio of 10% (W / V) to obtain a solid-liquid mixture, and cultured in a constant temperature shaker for 24 hours until the non-symbiotic nitrogen-fixing bacteria are adsorbed onto the surface and pores of the biochar carrier;

[0014] Step 4: centrifuge the solid-liquid mixture of step 3, remove the supernatant, obtain the biochar-loaded non-symbiotic nitrogen-fixing bacteria material, place it in a 25°C incubator and culture it for 91 days, and use a pure biochar carrier as a control treatment; each treatment is repeated 24 times for later destructive sampling; and sterile operation is used throughout the process;

[0015] Step 5, collect biochar-non-symbiotic nitrogen-fixing bacteria composite samples and pure biochar samples at culturing time 0d, 7d, 14d, 21d, 35d, 49d, 70d, and 91d respectively from the biochar-loaded non-symbiotic nitrogen-fixing bacteria material and the pure biochar carrier in step 4; store the biochar-non-symbiotic nitrogen-fixing bacteria composite samples in a dark and low temperature environment, with a shelf life of no more than one year.

[0016] In step 1, the rotation speed of the constant temperature shaker is 180 r / min and the temperature is 28°C. In step 3, the rotation speed of the constant temperature shaker is 180 r / min and the temperature is 28°C.

[0017] In step 2, the bacterial liquid is centrifuged at a speed of 4000 r / min and the centrifugation time is 10 min.

[0018] In step 4, the mixed solution of step 3 is centrifuged at a speed of 4000 r / min and the centrifugation time is 10 min.

[0019] The invention discloses an application of a microbial fertilizer, wherein the microbial fertilizer is used for quickly fertilizing poor soil.

[0020] The beneficial effects of the present invention are: 1. The corn straw biochar of the present invention has a more significant effect on improving the nitrogenase activity with a particle size of 0.25-2mm. The 0.25-2mm particle size biochar provides a good habitat for bacterial strain cells, and can better retain water and fertilizer, thereby ensuring the growth and reproduction of the strain, and can be put into the soil for use. The loading effect of corn straw biochar is better than that of wood biochar, and its C:N value is significantly lower, ranging from 61 to 84, which is more conducive to the growth of nitrogen-fixing bacteria. The application of microbial fertilizer effectively increases the total nitrogen and microbial biomass nitrogen content in the soil. Corn straw charcoal is more effective in increasing soil microbial biomass nitrogen, which is closely related to the activity of soil microorganisms.

[0021] 2. The Klebsiella pneumoniae isolated and screened in paddy soil by the present invention plays a role in biological nitrogen fixation in a farmland system without fertilizer application and without or with few legumes. The combination of biochar and nitrogen-fixing bacteria can improve the survival rate of nitrogen-fixing bacteria, strengthen the non-symbiotic nitrogen-fixing function, help reduce the application of nitrogen fertilizer, and alleviate the dilemma of nitrogen fertilizer deficiency in poor soil. This provides an important idea for promoting rapid fertilization of poor soil by increasing carbon and fixing nitrogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The electron microscope scanning images of corn straw biochar with different particle sizes prepared at 500° C. of the present invention, Sa: Straw biochar particle size 2-5 mm; Sb: Straw biochar particle size 0.25-2 mm; Sc: Straw biochar particle size <0.25 mm;

[0023] Figure 2 The electron microscope scanning images of different particle sizes of dead branch basswood biochar prepared at 500℃, Ta: wood biochar particle size 2-5mm; Tb: wood biochar particle size 0.25-2mm; Tc: wood biochar particle size <0.25mm;

[0024] Figure 3 for Figure 1 Electron microscopic scanning image of corn straw biochar loaded with bacterial strains;

[0025] Figure 4 for Figure 2 Scanning electron microscope image of deadwood biochar loaded with bacterial strains;

[0026] Figure 5 This is the trend diagram of the number of nitrogen-fixing bacteria changing with culture time after biochar loading;

[0027] Figure 6 The isothermal nitrogen adsorption-desorption curve of corn straw carbon of the present invention;

[0028] Figure 7 is the isothermal nitrogen adsorption-desorption curve of wood charcoal;

[0029] Figure 8 is the X-ray diffraction pattern of biochar;

[0030] Fig. 9 is the Fourier infrared spectrum of biochar;

[0031] Fig.10 This is a graph showing the change trend of nitrogenase activity on biochar with culture time;

[0032] Fig.11 This is the trend diagram of the change of microbial biomass carbon content after biochar loading strains;

[0033] Fig.12 This is the trend diagram of the change of nitrogen content in microbial biomass after biochar loading strains;

[0034] Fig.13 This is the trend diagram of biochar element content changing with cultivation time;

[0035] Fig.14 This is the trend diagram of biochar carbon-nitrogen ratio changing with cultivation time;

[0036] Fig.15 This is the trend diagram of soil microbial biomass nitrogen content changing with culture time under different treatments;

[0037] Fig.16 This is a graph showing the changing trend of soil total nitrogen content with incubation time under different treatments. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0039] A microbial fertilizer comprises a biochar carrier and non-symbiotic nitrogen-fixing bacteria attached to the biochar carrier, wherein the classification and name of the non-symbiotic nitrogen-fixing bacteria is Klebsiella pneumoniae, and the preservation number of Klebsiella pneumoniae is CGMCC No. 32396. The preservation date is October 28, 2024, and the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration.

[0040] The biochar carrier is a corn straw biochar carrier, and the particle size of the corn straw biochar carrier is 0.25-2 mm.

[0041] The corn straw biochar is prepared by oxygen-limited pyrolysis at 500°C.

[0042] A method for preparing microbial fertilizer comprises the following steps:

[0043] Step 1: Pick a ring of non-symbiotic nitrogen-fixing colonies from a nitrogen-free solid culture medium, inoculate them into LB liquid culture medium for activation, and culture them in a constant temperature shaker until the logarithmic growth phase to obtain a bacterial solution; the speed of the constant temperature shaker is 180 r / min and the temperature is 28°C.

[0044] Step 2, taking out the bacterial solution of step 1 and centrifuging it, pouring out the supernatant to obtain the strain precipitate, washing and resuspending the strain precipitate with sterile phosphate buffer solution PBS, adjusting the optical density of the suspension at 600nm to 0.7-0.8, and obtaining a non-symbiotic nitrogen-fixing bacteria suspension; wherein the bacterial solution centrifugal speed is 4000r / min, and the centrifugation time is 10min.

[0045] Step 3: The sterile biochar carrier and the non-symbiotic nitrogen-fixing bacteria suspension are fully mixed at a ratio of 10% (W / V) to obtain a solid-liquid mixture, and cultured in a constant temperature shaker for 24 hours until the non-symbiotic nitrogen-fixing bacteria are adsorbed to the surface and pores of the biochar carrier; the rotation speed of the constant temperature shaker is 180 r / min and the temperature is 28°C.

[0046] Step 4: Centrifuge the solid-liquid mixture of step 3, remove the supernatant, obtain biochar-loaded non-symbiotic nitrogen-fixing bacteria material, and culture it in a constant temperature box at 25°C for 91 days. At the same time, use pure biochar carrier as a control treatment; each treatment is repeated 24 times for later destructive sampling, and sterile operation is adopted throughout the process; the solid-liquid mixture of step 3 is centrifuged at a speed of 4000r / min, and the centrifugation time is 10min.

[0047] Step 5, collect biochar-non-symbiotic nitrogen-fixing bacteria composite samples and pure biochar samples at culturing time 0d, 7d, 14d, 21d, 35d, 49d, 70d, and 91d respectively from the biochar-loaded non-symbiotic nitrogen-fixing bacteria material and the pure biochar carrier in step 4; store the biochar-non-symbiotic nitrogen-fixing bacteria composite samples in a dark and low temperature environment, with a shelf life of no more than one year.

[0048] Example 1

[0049] 1. The biochar carrier described in this embodiment is made of corn straw as raw material and is prepared by oxygen-limited pyrolysis at 500°C. Three particle sizes are prepared by sieving: 2-5mm (a, coarse), 0.25-2mm (b, medium) and <0.25mm (c, fine). The biochar carrier is corn straw charcoal with a particle size of 0.25-2mm and a cultivation time of 14 days.

[0050] 2. To illustrate that the production of biochar by oxygen-limited pyrolysis method has the best effect of loading strains with corn straw charcoal of 0.25-2 mm particle size, the following comparative test is used to illustrate that wood charcoal is used as the material for comparison, and 2-5 mm and <0.25 mm are selected as the particle size comparison.

[0051] 3. Corn stalks (S) and deadwood (T) were selected as raw materials and prepared by oxygen-limited pyrolysis at 500°C. Three particle sizes were prepared by sieving: 2-5 mm (a, coarse), 0.25-2 mm (b, medium) and <0.25 mm (c, fine). Each portion of biochar was washed three times with distilled water to remove free cells and impurities, and then sterilized at 121°C with high temperature and high pressure.

[0052] 4. Scanning electron microscope image of corn straw biochar Figure 1 , Sa represents corn straw charcoal with a particle size of 2-5 mm, Sb represents corn straw charcoal with a particle size of 0.25-2 mm, and Sc represents corn straw charcoal with a particle size of <0.25 mm.

[0053] 5. Scanning electron microscope image of dead branch basswood biochar Figure 2 , Ta represents wood charcoal with a particle size of 2-5 mm, Tb represents wood charcoal with a particle size of 0.25-2 mm, and Tc represents wood charcoal with a particle size of <0.25 mm.

[0054] 6. Klebsiella pneumoniae was accessed into LB liquid culture medium for activation, washed with sterile phosphate buffered solution (PBS) and resuspended with nitrogen-fixing bacteria precipitation, sterile biochar and bacterial suspension were fully mixed at a ratio of 10% (W / V), shaken, and centrifuged to obtain biochar-loaded non-symbiotic nitrogen-fixing bacteria materials SBC-N and TBC-N, and a portion was frozen for storage. The carbon-bacteria composite material can be used as microbial fertilizer SBC-N and TBC-N. The other part was tested for the number of live bacteria on the biochar by dilution plate method during the culture period (91 days in total) to evaluate the loading rate.

[0055] 7. Data processing

[0056] EXCEL2016 and SPSS22.0 were used for experimental data processing, one-way ANOVA was used for variance analysis, and LSD and SNK significance tests (α=0.05) were used. MDI Jade9 was used for XRD analysis, and Avantage5.9 was used for peak fitting in XPS analysis. Origin2024 was used for graphics drawing.

[0057] 8. Scanning electron microscope image of corn stalk charcoal loaded with Klebsiella pneumoniae. Figure 3 , where S represents corn straw charcoal, a, b, and c represent particle sizes of 2-5 mm, 0.25-2 mm, and <0.25 mm, and P represents Klebsiella pneumoniae. The number of nitrogen-fixing bacteria loaded in Sa-P and Sb-P was significantly higher than that in Sc-P treatment. The strains in Sa-P treatment were mainly distributed in the pores of biochar, while the strains in Sb-P treatment were distributed in the pores and surface of biochar. After being loaded into biochar, the strains secreted and formed a large number of biofilms, building a good microbial community.

[0058] 9. Scanning electron microscope image of wood charcoal loaded with Klebsiella pneumoniae. Figure 4 , where T represents wood charcoal, a, b, and c represent particle sizes of 2-5 mm, 0.25-2 mm, and <0.25 mm, and P represents Klebsiella pneumoniae. The number of nitrogen-fixing bacteria loaded in the Ta-P treatment was significantly the highest, and its biochar had high pore richness and large specific surface area, providing more attachment points for strains. The strains in the Tb-P treatment were mainly distributed in the pores of the biochar, which had good water retention and air permeability, providing a stable shelter for the growth and reproduction of the strains.

[0059] 10. The results of the dilution plate method are as follows Figure 5As shown in the figure, non-symbiotic nitrogen-fixing bacteria (Klebsiella pneumoniae) can survive in the pure biochar system for a period of time, indicating that biochar provides essential nutrients and breeding sites for the growth of nitrogen-fixing bacteria. After the strain was loaded with corn straw charcoal, the number of nitrogen-fixing bacteria decreased significantly within 7 days of cultivation, and tended to stabilize after 35 days, showing a trend of Sb≥Sa>Sc. After the strain was loaded with wood charcoal, the number of nitrogen-fixing bacteria decreased significantly after 14 days of cultivation, and tended to stabilize after 35 days, showing a trend of Tb>Ta>Tc; among them, the Tc treatment had the largest decrease, indicating that the particle size of biochar was not conducive to maintaining the growth of strain reproduction.

[0060] 11. Scanning electron microscopy (SEM) (see Figures 1 to 4 ) Observation of the surface morphology of biochar materials found that the number of strains loaded on biochar with a particle size of >0.25mm was significantly higher than that on biochar with a particle size of <0.25mm, and its good pore structure can provide sufficient space for strain growth. Biochar with a particle size of 0.25-2mm has better water retention and air permeability, which is conducive to regulating environmental humidity. Compared with wood charcoal, corn straw biochar has a honeycomb structure with high porosity and large specific surface area, but it is also prone to collapse, while wood charcoal has a higher structural stability.

[0061] 12. The porosity and specific surface area of ​​biochar were determined by BET-N2 adsorption method (Micromeritics 3Flex, USA) (see Figure 6 and Figure 7 ), the specific surface areas of Sa, Sb and Sc materials are 28.69m 2 / g, 40.85m 2 / g and 46.11m 2 / g, and the average pore diameters are 2.98nm, 2.89nm and 3.35nm respectively. Among them, the Sc material has more debris and less honeycomb pore structure, resulting in a larger pore diameter. The specific surface areas of Ta, Tb and Tc materials are 24.77m 2 / g, 29.86m 2 / g and 40.70m 2 / g, and the average pore diameters are 3.53nm, 3.35nm and 3.04nm, respectively. It can be seen that the Sb material is in a moderate position in terms of both specific surface area and porosity, which neither adsorbs excessive strains to cause nutrient competition, nor can it maintain a sufficient number of strains for growth and reproduction.

[0062] 13. X-ray diffractometer (XRD, Rigaku Ultima IV, Japan) was used to determine the crystal structure of biochar ( Figure 8), the spectral peaks of SiO2 and MoO3 were clearly detected in corn straw charcoal, as well as the presence of FeO(OH) and CaCO3 substances. The spectral peaks of CaCO3, FeO, SiO2 and MoS2 were present in the X-ray spectrum of wood charcoal. These mineral components support the growth of non-symbiotic nitrogen-fixing bacteria on biochar and continue to perform nitrogen fixation functions.

[0063] 14. The surface functional groups of biochar were determined by Fourier transform infrared spectrometer (FT-IR, Thermo Fisher iS5, USA). Fig. 9 ), wood charcoal contains more -OH and -CH2 bonds, and corn straw charcoal contains more COC and metal bonds (Mg-O and Fe-O). The more surface functional groups there are, the more negative charges on the surface of biochar increase, thereby increasing the adsorption capacity of biochar to strains.

[0064] 15. The study found that the biochar with a particle size of 0.25-2 mm obtained by screening can provide some mineral nutrients for non-symbiotic nitrogen-fixing bacteria, has good water retention and air permeability, and has the best loading effect on Klebsiella pneumoniae in this embodiment, among which corn straw charcoal is better than wood charcoal.

[0065] Example 2

[0066] 1. The biochar carrier described in this embodiment is made of corn stalks (S) and dead linden (T) as raw materials, and is prepared by oxygen-limited pyrolysis at 500°C. Three particle sizes are prepared by sieving: 2-5mm (a, coarse), 0.25-2mm (b, medium) and <0.25mm (c, fine). Each portion of biochar is washed three times with distilled water to remove free cells and impurities, and then sterilized at 121°C with high temperature and high pressure.

[0067] 2. Klebsiella pneumoniae was inoculated into LB liquid culture medium for activation, washed with sterile phosphate buffer solution (PBS) and the nitrogen-fixing bacteria precipitate was resuspended, the sterile biochar and the bacterial suspension were fully mixed at a ratio of 10% (W / V), shaken, and centrifuged to obtain biochar-loaded non-symbiotic nitrogen-fixing bacteria materials SBC-N and TBC-N, and a portion was frozen for storage. The carbon-bacteria composite material can be used as microbial fertilizers SBC-N and TBC-N.

[0068] 3. Another part of the carbon / bacteria composite material was sampled at set time intervals (0d, 7d, 14d, 21d, 35d, 49d, 70d, 91d), and the changing trend of nitrogenase activity with culture time was analyzed by gas chromatography. Specific operation: 1.0g of fresh moist biochar was placed in a 110ml sterilized serum bottle, plugged with an isobutyl rubber stopper and sealed. The serum bottle was flushed with high-purity nitrogen for 10s (blowing out the rest of the gas), 11ml of acetylene gas was added with a syringe (the acetylene concentration in the serum bottle was 10%), and cultured at 28℃ for 48h. At the same time, an ethylene standard curve was made. Nitrogenase can reduce acetylene to ethylene, and the ethylene peak area was measured by gas chromatograph (Agilent GC 7890B, USA) to calculate the nitrogenase activity.

[0069] 4. Fig.10 The trend of nitrogenase activity of non-symbiotic nitrogen-fixing bacteria with culture time after biochar was loaded. It shows that within a certain period of time, biochar supports the growth of non-symbiotic nitrogen-fixing bacteria and continues to play a nitrogen-fixing role. After the strain was loaded with corn straw charcoal, the nitrogenase activity continued to increase within 14 days of culture, with the highest value of 1276.42nmol / g / h (Sb material). After 21 days, the nitrogenase activity decreased significantly and stabilized at 35 days, showing the law of Sb>Sa>Sc. After the strain was loaded with wood charcoal, the nitrogenase activity showed a downward trend within 7 days of culture and remained stable after 21 days. Biochars of different particle sizes showed the law of Tb>Tc≈Ta. Compared with Ta, the nitrogenase activity of Tc material was higher in the early stage (within 21 days), and was obviously insufficient in the middle and late stages, indicating that the Tc material was not sustainable in supporting the function of nitrogen-fixing bacteria. In addition, the nitrogenase activity of corn straw charcoal was significantly higher than that of wood charcoal.

[0070] 5. Fig.11 This is the trend of the change of microbial biomass carbon content with culture time after non-symbiotic nitrogen-fixing bacteria are loaded with biochar. Chloroform fumigation-total organic carbon analyzer (Elementar, vario TOC select, Germany) was used for determination. After the strain was loaded with corn straw charcoal, the microbial biomass carbon content fluctuated greatly within 21 days of culture and tended to be stable after 35 days. Among them, the 0.25-2mm particle size biochar was significantly higher than the other embodiments, showing Sb>Sa>Sc. After the strain was loaded with wood charcoal, the microbial biomass carbon content decreased with the extension of culture time within 0 to 21 days of culture, which to a certain extent indicated that the microbial activity gradually decreased on wood charcoal; as the culture time continued to extend, the microbial biomass carbon content increased and remained stable, which may be that the microbial residues were reused and the carbon source was released. Therefore, the microbial biomass carbon content increased sharply after 35 days of culture, and the 0.25-2mm particle size was still more conducive to microbial growth, and finally showed the rule of Tb>Ta>Tc.

[0071] 6. Fig.12This is the trend of the change of microbial biomass nitrogen content with culture time after the non-symbiotic nitrogen-fixing bacteria were loaded with biochar. The chloroform fumigation-total organic carbon analyzer (Elementar, vario TOC select, Germany) was used for determination. After the strain was loaded with corn straw charcoal and wood charcoal, the microbial biomass nitrogen content first increased and then decreased within 14 days of culture, and entered a stable period after 35 days of culture, with Sb and Tb treatments being significantly higher.

[0072] 7. In order to further compare the differences in elemental composition between corn straw charcoal and wood charcoal, charcoal bacteria composite samples were collected at the early, middle and late stages of cultivation (0d, 21d, 35d, 91d). The four elements of carbon (C), hydrogen (H), nitrogen (N) and sulfur (S) were analyzed using an organic element analyzer (Elemantar: Varia ELcube, Germany), and the changing trend of biochar element content with cultivation time was plotted.

[0073] 8. The trend of element content (C, N, H and S) in biochar with cultivation time is as follows: Fig.13 As shown, biochar carbon and nitrogen Fig.14 As shown. Compared with wood charcoal, corn straw charcoal contains higher N content and lower C content, thus reducing the carbon-nitrogen ratio of biochar, which may be more conducive to bacterial growth. It is worth noting that the carbon-nitrogen ratio of charcoal-bacteria composite materials decreases with the extension of culture time, indicating that the strain load has a significant effect on the elemental composition of the biochar surface, which provides an effective idea for improving the properties of biochar.

[0074] 9. In a further embodiment, corn straw biochar and wood biochar have a more significant effect on improving nitrogenase activity with a particle size of 0.25-2 mm. Biochar with a particle size of 0.25-2 mm provides a good habitat for bacterial cells and can better retain water and fertilizer, thereby ensuring the growth and reproduction of the strain. Compared with wood charcoal, the carbon-nitrogen ratio in corn straw charcoal is significantly lower, which is more conducive to bacterial growth.

[0075] Example 3

[0076] 1. The biochar carrier described in this embodiment is made of corn straw (S) and dead linden (T) as raw materials, and is prepared by oxygen-limited pyrolysis at 500°C, with a screening particle size of 0.25-2 mm. Each portion of biochar is washed three times with distilled water to remove free cells and impurities, and then sterilized at 121°C with high temperature and high pressure.

[0077] 2. In this embodiment, calcareous purple soil is used as the test object to illustrate the technical effects of the present invention.

[0078] 3. Preparation of microbial fertilizer

[0079] Klebsiella pneumoniae was inoculated into LB liquid culture medium for activation, washed with sterile phosphate buffer solution (PBS) and the nitrogen-fixing bacteria precipitate was resuspended, the sterile biochar and the bacterial suspension were fully mixed at a ratio of 10% (W / V), shaken, and centrifuged to obtain biochar-loaded non-symbiotic nitrogen-fixing bacteria materials SBC-N and TBC-N, which were stored in the dark and at low temperature for use.

[0080] 4. Soil culture test design

[0081] Collect nitrogen-poor soil (calcareous purple soil) samples from the 0-20cm soil layer and bring them back to the laboratory. After natural air drying, pass through a 2mm sieve. Mix the prepared organic multi-microbial fertilizer and soil samples at a ratio of 1:100 (mass ratio). Keep the soil moisture content at about 20%. At the same time, set up no biochar treatment (CK), pure biochar mixed soil treatment (S0 and T0) and pure strain mixed soil (B0) as controls. Each treatment was repeated three times. Place in a 500ml tissue culture bottle, make the bottle cap breathable, and incubate in a 25℃ artificial climate incubator for 91 days.

[0082] 5. Data processing

[0083] EXCEL2016 and SPSS22.0 were used to process the experimental data, and one-way ANOVA was used for variance analysis, and LSD and SNK significance tests were used (α=0.05).

[0084] 6. Soil microbial biomass nitrogen content

[0085] Different treatments significantly affected soil microbial biomass nitrogen content ( Fig.15 ), the soil microbial biomass nitrogen content of the B0 treatment was significantly higher than that of the other treatments within 0 to 7 days, but decreased with the extension of the culture time, indicating that the added free strains were limited in survival in the soil and difficult to maintain activity; a second small peak appeared at 49 days of culture, which may be due to the microbial residues injecting rich nutrients into the soil, and the soil microbial activity increased accordingly. Overall, compared with S0 and CK, the SBC-N treatment increased the soil microbial biomass nitrogen content from 0 to 14 days and after 49 days of culture, indicating that corn straw loading not only increased the number of nitrogen-fixing bacteria, but also increased soil microbial activity. Compared with T0, the TBC-N treatment increased the soil microbial biomass nitrogen content from 14 to 70 days of culture. Whether it has a long-term effect requires further study in long-term field experiments.

[0086] 7. Total nitrogen content of soil

[0087] The total nitrogen content of the soil was determined by concentrated sulfuric acid digestion-Kjeldahl method. The trend of soil total nitrogen content with incubation time (91d) is as follows Fig.16As shown, the B0 treatment is significantly lower than the other treatments. It may be that the addition of a large number of free strains accelerates the competition of soil microorganisms for nitrogen sources, thereby reducing the total nitrogen content in the soil. Compared with CK, the T0 treatment reduced the total nitrogen content in the soil, which may be related to the low nitrogen content in the wood charcoal; the TBC-N treatment significantly increased the total nitrogen content in the soil during the entire culture period, indicating that the wood charcoal-loaded non-symbiotic nitrogen-fixing bacteria has a long-term effect on promoting soil biological nitrogen fixation, and this effect can be continuously exerted, and has a good stimulating effect on the long-term nitrogen supply potential of the soil. Compared with the S0 treatment, SBC-N reduced the total nitrogen content of the soil within 35 days of culture, but increased the total nitrogen content of the soil after 91 days of culture. Combined with Examples 1 and 2, the reason may be that SBC-N increased the activity of soil microorganisms and promoted the soil nitrogen cycle, thereby maintaining the nitrogen balance of the soil. The characteristics of the soil microbial community structure can be further determined in the later stage, or long-term field experiments can be carried out to verify this hypothesis.

[0088] 8. The Klebsiella pneumoniae Klebsiella pneumoniae screened from paddy soil by the present invention has non-symbiotic nitrogen fixation function, and is applicable to poor calcareous purple soil. Biochar-loaded nitrogen-fixing bacteria are effective in improving soil total nitrogen and microbial biomass nitrogen content, and have development potential, providing good bacterial species resources for developing environmentally friendly organic polymicrobial fertilizers in poor calcareous purple soil regions. The present invention utilizes the excellent properties of biochar to improve non-symbiotic nitrogen fixation efficiency, and provides a scientific basis for increasing poor soil nitrogen reserves and coordinating soil carbon-nitrogen balance, which is of great significance for promoting biochar agricultural applications and agricultural green development.

Claims

1. A microbial fertilizer, characterized in that: The invention comprises a biochar carrier and non-symbiotic nitrogen-fixing bacteria attached to the biochar carrier. The non-symbiotic nitrogen-fixing bacteria is Klebsiella pneumoniae, and the preservation number of Klebsiella pneumoniae is CGMCC NO.32396.

2. The microbial fertilizer according to claim 1, characterized in that: The biochar carrier is a corn straw biochar carrier, and the particle size of the corn straw biochar carrier is 0.25-2 mm.

3. The microbial fertilizer according to claim 1, characterized in that: The corn straw biochar is prepared by oxygen-limited pyrolysis at 500°C.

4. A method for preparing the microbial fertilizer according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1, picking a ring of non-symbiotic nitrogen-fixing bacterial colonies from a nitrogen-free solid culture medium, inoculating them into LB liquid culture medium for activation, and culturing them in a constant temperature shaker until the growth logarithmic phase to obtain a bacterial liquid; Step 2, taking out the bacterial solution of step 1 and performing centrifugation operation, pouring out the supernatant to obtain the strain precipitate, washing and resuspending the strain precipitate with a sterile phosphate buffer solution PBS, adjusting the optical density of the suspension at 600 nm to 0.7-0.8, and obtaining a non-symbiotic nitrogen-fixing bacteria suspension; Step 3, the sterile biochar carrier and the non-symbiotic nitrogen-fixing bacteria suspension are fully mixed at a ratio of 10% (W / V) to obtain a solid-liquid mixture, and cultured in a constant temperature shaker for 24 hours until the non-symbiotic nitrogen-fixing bacteria are adsorbed onto the surface and pores of the biochar carrier; Step 4: centrifuge the solid-liquid mixture of step 3, remove the supernatant, obtain the biochar-loaded non-symbiotic nitrogen-fixing bacteria material, place it in a 25°C incubator and culture it for 91 days, and use a pure biochar carrier as a control treatment; each treatment is repeated 24 times for later destructive sampling, and sterile operation is used throughout the process; Step 5, collect biochar-non-symbiotic nitrogen-fixing bacteria composite samples and pure biochar samples at culturing time 0d, 7d, 14d, 21d, 35d, 49d, 70d, and 91d respectively from the biochar-loaded non-symbiotic nitrogen-fixing bacteria material and the pure biochar carrier in step 4; store the biochar-non-symbiotic nitrogen-fixing bacteria composite samples in a dark and low temperature environment, with a shelf life of no more than one year.

5. The method for preparing the microbial fertilizer according to claim 3, characterized in that: In step 1, the rotation speed of the constant temperature shaker is 180 r / min and the temperature is 28°C. In step 3, the rotation speed of the constant temperature shaker is 180 r / min and the temperature is 28°C.

6. The method for preparing the microbial fertilizer according to claim 3, characterized in that: In step 2, the bacterial liquid is centrifuged at a speed of 4000 r / min and the centrifugation time is 10 min.

7. The method for preparing the microbial fertilizer according to claim 3, characterized in that: In step 4, the mixed solution of step 3 is centrifuged at a speed of 4000 r / min and the centrifugation time is 10 min.

8. An application of the microbial fertilizer according to any one of claims 1 to 3, characterized in that: Microbial fertilizer is used to quickly fertilize poor soil.

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