Method for converting phosphorus in bone black into soluble phosphorus by adopting penicillium fimbriatum

Abstract

The invention discloses a method for converting phosphorus in bone black into soluble phosphorus by adopting penicillium fimbriatum, and belongs to the technical field of waste conversion and utilization. According to the method disclosed by the invention, the insoluble phosphorus in the bone black is efficiently and stably converted into the soluble phosphorus by utilizing the specifically preserved Penicillium seckii, the phosphorus dissolving amount within 10 days is up to 1616.71 mg / L, the process is environment-friendly, the raw material adaptability is high, and a feasible microbial conversion way is provided for high-valued utilization of bone black phosphorus resources.

Description

Technical Field

[0001] This invention belongs to the field of waste conversion and utilization technology, specifically a method for converting phosphorus in bone char into soluble phosphorus using Penicillium fimbriae. Background Technology

[0002] Phosphorus is an essential nutrient for plant growth and plays an irreplaceable role in agricultural production. However, global phosphate rock resources are limited and unevenly distributed, and the production and application of phosphate fertilizers are accompanied by resource consumption and environmental pollution. Therefore, developing new and sustainable ways to utilize phosphorus resources has become a research hotspot in the fields of agriculture and the environment.

[0003] Bone char, a waste-derived material produced by the thermal cracking and carbonization of animal bones, is rich in phosphorus. However, most of its phosphorus exists in an insoluble form, making it difficult for plants to directly absorb and utilize. Traditional chemical phosphorus solubilization methods often involve high energy consumption and secondary pollution, while microbial phosphorus solubilization technology has attracted widespread attention due to its environmental friendliness and sustainability. Currently, various phosphorus-solubilizing microorganisms have been reported, but their conversion efficiency for insoluble phosphorus in bone char is generally low, and the adaptability and stability of the strains need to be improved.

[0004] Therefore, there is an urgent need to develop an efficient, stable, and environmentally friendly microbial phosphorus solubilization method to achieve the effective conversion and recycling of phosphorus resources in bone char, alleviate the phosphorus resource shortage, and promote the high-value utilization of agricultural waste. Summary of the Invention

[0005] In view of this, the present invention aims to provide a method for converting phosphorus in bone char into soluble phosphorus using Penicillium chrysogenum. The present invention utilizes a specifically preserved Penicillium chrysogenum (… Penicillium steckii This method efficiently and stably converts insoluble phosphorus in bone char into soluble phosphorus, providing a feasible microbial transformation pathway for the high-value utilization of bone char phosphorus resources.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for converting phosphorus in bone char into soluble phosphorus using Penicillium fimbriae, comprising the following steps: Bone char powder and Penicillium fibrillosa were placed together in a reaction medium and cultured to convert phosphorus in bone char into soluble phosphorus. The Penicillium fissure ( Penicillium steckii It was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 41749.

[0007] Preferably, the mass-to-volume ratio of the bone char powder to the activated *Penicillium fibrillum* is (0.25~0.5) g:4.5 mL; the activated *Penicillium fibrillum* has a spore count of 1 × 10⁻⁶.8 per mL.

[0008] Preferably, the culture conditions are: 30 ℃, 180 r / min shaker culture for 15 days.

[0009] Preferably, the reaction medium is NBRIP-P medium: glucose 10 g / L, MgCl2·6H2O 5 g / L, MgSO4·7H2O 0.25 g / L, FeSO4·7H2O 0.01 g / L, KCl 0.2 g / L, (NH4)2SO4 0.1 g / L, pH 7.0~7.4.

[0010] According to the method of claim 1, the bone char powder is obtained by pulverizing animal bones after thermal decomposition and carbonization; the animal bones are derived from pigs, cattle, sheep, and chickens.

[0011] It contains at least the following beneficial technical effects: This invention utilizes a specifically preserved Penicillium fimbriae ( Penicillium steckii This method efficiently and stably converts insoluble phosphorus in bone char into soluble phosphorus, achieving a phosphorus soluble content of up to 1616.71 mg / L within 10 days. Furthermore, the process is environmentally friendly and adaptable to raw materials, providing a feasible microbial transformation pathway for the high-value utilization of phosphorus resources in bone char. Attached Figure Description

[0012] Figure 1 This is a SEM image of Penicillium fibrillosa.

[0013] Figure 2 The effective phosphorus content of Penicillium frenulum and bone char at different sampling times; Figure 3 Dehydrogenase activity of Penicillium frenulum reacting with bone char at different sampling times; Figure 4 This is a SEM image of the interaction between Penicillium fimbriae and bone char. Detailed Implementation

[0014] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the invention in any way.

[0015] Example 1 A method for converting phosphorus in bone char into soluble phosphorus using Penicillium fimbriae includes the following steps: 1. Activate Penicillium fimbriae Take 150 mL of PDB medium (1 L formula: 200.0 g peeled potatoes, cut into small pieces, add 1000 mL of water and boil for 30 min, filter with double-layer gauze, add 20 g of glucose to the filtrate, heat over low heat and stir constantly with a glass rod, and add distilled water to make up to 1000 mL, pH 5.0) into a 250 mL Erlenmeyer flask, add 1 mL of Penicillium chrysogenum inoculum to the activation medium, and culture at 30 ℃ and 180 r / min for 4 days with shaking.

[0016] 2. Preparation of spore suspension The activated *Penicillium fibrillosa* was inoculated onto PDA medium and incubated at 30 °C for 4 days. After the spores matured, they were washed off the plates with sterile water and counted using a hemocytometer. The spore concentration was obtained by dilution to 1 × 10⁻⁶. 8 A spore suspension of 1 spore per mL.

[0017] 3. Bone char preparation Pig bones were cleaned and freeze-dried at -55 °C for 2 days. Then, they were thermally pyrolyzed at 400 °C for 120 min under limited oxygen conditions. After cooling, large bone char pieces were obtained. These char pieces were then ball-milled at 450 r / min for 5 h with a zirconium oxide particle to large bone char weight ratio of 15:1 and ethanol as a grinding aid to obtain micron- and nano-sized multi-level bone char. The micron- and nano-sized multi-level bone char was sterilized by irradiation at a dose of 10.0 kGy.

[0018] 4. Experiment on the conversion of insoluble phosphorus in bone char Take a 100 mL Erlenmeyer flask, add 50 mL of reaction medium, and inoculate 4.5 mL of spore suspension into the reaction medium at a 9 v / v% inoculum rate. Use a control without inoculation. NBRIP-P + 9 v / v% inoculum of *Penicillium fimbriae* served as the blank control; NBRIP-P + (0.25 g, 0.5 g) bone char + 9 v / v% inoculum of *Penicillium fimbriae* served as the experimental group, with three replicates for each group. Incubate at 30 ℃ and 180 r / min with shaking for 15 days, and take samples on days 7, 10, and 15.

[0019] The NBRIP medium (1 L formulation) consists of: 10 g glucose, 5 g MgCl2·6H2O, 0.25 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 0.2 g KCl, 0.1 g (NH4)2SO4, and 5.0 g Ca3(PO4)2, with a pH of 7.0~7.4. The NBRIP-P medium is the NBRIP medium without the addition of Ca3(PO4)2.

[0020] 5. Determination of dehydrogenase activity Transfer 2 mL of *Penicillium fibrillaria* culture from each treatment group to a centrifuge tube and centrifuge at 4000 r / min for 15 min at 4 °C. Discard the supernatant, resuspend the precipitate in 2 mL of deionized water, and add 2 mL of 0.2% (w / v) triphenyltetrazolium chloride (TTC) to the suspension. Incubate the mixture overnight at 37 °C. Extract the resulting red formazan with 4 mL of acetone and measure the absorbance at 484 nm using a UV-Vis spectrophotometer.

[0021] 6. Determination of available phosphorus content Phosphorus standard solutions of 0 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, and 10 mg / L were prepared. The dissolved phosphorus amount was determined using the molybdenum-antimony colorimetric method. A phosphorus standard curve was plotted with phosphorus concentration on the x-axis and absorbance on the y-axis.

[0022] 7. SEM characterization of phosphate-solubilizing bacteria and bone char phosphate solubilization The culture of *Penicillium fimbriatum* was transferred to a centrifuge tube and centrifuged at 2000 r / min for 15 min at 4 ℃. The supernatant was discarded, and the precipitate was collected. The precipitate was then treated with 15% formaldehyde (prepared with 0.1 mol / L, pH 7.4 phosphate buffer) for 2 h. After rinsing with the buffer, the precipitate was fixed with 6% glutaraldehyde (prepared with 0.1 mol / L, pH 7.4 phosphate buffer) and incubated overnight at 4 ℃. The surface morphology was then observed using a scanning electron microscope.

[0023] 8. Statistical Analysis Statistical analysis was performed using IBM SPSS 26.0 software. One-way ANOVA and Duncan's test were used to analyze the data. All samples were replicated in triplicate. Data are expressed as mean ± standard error. p <  0.05 The significance level is indicated by the graph. Graphpad Prism version 8 software was used to draw the graph.

[0024] Results and Discussion Depend on Figure 1 It is evident that after treatment with 9% *Penicillium fimbriae* and 0.5% bone char, the phosphorus solubility reached 1616.71 mg / L on day 10 and 1293.95 mg / L on day 15. After treatment with 9% *Penicillium fimbriae* and 1% bone char, the phosphorus solubility was 1521.70 mg / L on day 10 and 1228.31 mg / L on day 15. Considering cost and phosphorus solubility efficiency, the co-application ratio of 9% *Penicillium fimbriae* and 0.5% bone char is optimal.

[0025] Bacterial dehydrogenase activity reflects the metabolic activity of microorganisms. Figure 2It is evident that the metabolic activity of *Penicillium fibrillati* is dynamic. Initially, the presence of a certain amount of insoluble phosphorus in the environment stimulates *Penicillium fibrillati* to increase dehydrogenase activity, enhancing its metabolic capacity to secrete more phosphate-solubilizing substances to dissolve the insoluble phosphorus, thereby increasing the available phosphorus content. This increase in available phosphorus provides more energy and material for phosphate-solubilizing bacteria, further promoting *Penicillium fibrillati* growth and increasing dehydrogenase activity, forming a positive feedback loop. By day 10, the phosphate solubility reaches its peak. Excessively high available phosphorus content in the bacterial culture may alter intracellular osmotic pressure, affecting normal cellular physiological functions, and thus triggering negative feedback regulation, reducing the phosphate-solubilizing capacity and metabolic activity of *Penicillium fibrillati*, thereby decreasing dehydrogenase activity. Furthermore… Figure 4 SEM characterization visually verified that *Penicillium fimbriatum* can interact with bone char. In summary, using *Penicillium fimbriatum* to convert phosphorus in bone char into soluble phosphorus is an efficient and stable method.

[0026] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for converting phosphorus in bone char to soluble phosphorus using Penicillium crustosum, characterized by, The method comprises the following steps: The bone charcoal powder and the Penicillium steckii are cultured in a reaction medium, and phosphorus in the bone charcoal is converted into soluble phosphorus; The Penicillium fissure ( Penicillium steckii It was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 41749.

2. The method of claim 1, wherein, The mass-volume ratio of the bone charcoal powder and the activated Penicillium crustosum is (0.25-0.5) g:4.5 mL; the spore number of the activated Penicillium crustosum is 1 × 10 8 spores / mL.

3. The method of claim 1, wherein, The culture condition is that the culture is carried out at 30 DEG C and 180 r / min for 15 days.

4. The method of claim 1, wherein, The reaction medium is NBRIP-P medium: glucose 10 g / L, MgCl2·6H2O 5 g / L, MgSO4·7H2O 0.25 g / L, FeSO4·7H2O 0.01 g / L, KCl 0.2 g / L, (NH4)2SO4 0.1 g / L, and pH is 7.0-7.

4.

5. The method of claim 1, wherein, The bone charcoal powder is obtained by crushing after pyrolysis and carbonization of animal bone; and the animal bone is derived from pigs, cattle, sheep and chickens.

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