Method for deeply degrading penicillin V through combined circulating cascade of paracoccus and aspergillus niger and application of method

Abstract

The invention provides a method for deeply degrading penicillin V through combined circulating cascade of paracoccus and aspergillus niger and application of the method, and belongs to the technical field of antibiotic pollutant degradation, paracoccus is paracoccus sp KDSPL-02, aspergillus niger is Aspergillus niger, and the method is used for deeply mineralizing and degrading penicillin V to dissolve possible pollution caused by the paracoccus sp KDSPL-02 and the aspergillus niger is Aspergillus niger. Waste hyphae generated in the production process are subjected to harmless treatment.

Description

Technical Field

[0001] The present invention relates to a method for deep degradation of penicillin V, in particular to a method for deep degradation of penicillin V by combined cyclic cascade of Paracoccus and Aspergillus niger, belonging to the technical field of degradation of antibiotic pollutants. Background Art

[0002] Solid residues from the microbial and fermentation industries (such as spent mycelium from antibiotic fermentation) have garnered widespread attention as important biological resources. Global production of bulk fermentation products, such as antibiotics, generates nearly 10 million tons of fermentation residues annually. These residues are rich in organic matter (30-52% crude protein, 2-20% fat, and approximately 30-65% cellulose), making them valuable as a resource. However, residual antibiotics can cause environmental pollution, posing a threat to ecological safety and human health. Consequently, they have been listed on the National List of Hazardous Wastes since 2008, requiring their disposal to comply with hazardous waste treatment standards. Developing a harmless disposal and resource-based approach to antibiotic fermentation residues has become a research hotspot and a challenging issue.

[0003] Existing treatment methods use strategies that destroy the entire mycelium, such as landfilling, incineration and other traditional simple technologies. However, these extensive treatment methods have the problem of secondary pollution. Landfilling will cause the antibiotics contained in it to seep into the ground with rainwater, causing groundwater pollution. Incineration will produce large amounts of carbon oxides, nitrogen oxides and sulfur oxides, causing air pollution and acid rain. At the same time, these solid wastes are derived from food, so destructive treatment is almost equivalent to wasting food. In recent years, there has been a trend to develop new biosafety and environmentally friendly treatment strategies. The technology of selective degradation of antibiotics by whole-cell catalysis has made progress, but there is a risk of incomplete degradation of antibiotics, and the intermediates may become new pollutants, thereby threatening ecological safety.

[0004] Penicillin V (phenoxymethylpenicillin), a broad-spectrum antibiotic with superior acid stability compared to penicillin G, is industrially produced through fermentation and biosynthesis by industrial strains of Penicillium chrysogenum using phenoxyacetic acid as a precursor. However, the stability of this molecule in the environment makes its degradation more difficult, especially the mineralization and degradation of the key intermediate phenoxyacetic acid, which remains a technical challenge. Summary of the Invention

[0005] In response to the above problems, the present invention provides a method for deep degradation of penicillin V by combining Paracoccus and Aspergillus niger in a cyclic cascade manner and its application, which are used to achieve mineralization degradation of penicillin V.

[0006] To achieve the above object, the technical solution of the present invention is: a method for deep degradation of penicillin V, wherein the method utilizes a combined cyclic cascade of Paracoccus and Aspergillus niger to deeply degrade penicillin V.

[0007] Furthermore, the Paracoccus sp. is Paracoccus sp. KDSPL-02, and the Paracoccus sp. in the method includes whole cells or immobilized whole cells of the bacterium.

[0008] Furthermore, the Aspergillus niger is Aspergillus niger, with a preservation number of CGMCC No. 3.3928; the Aspergillus niger in the method includes whole cells or immobilized whole cells of the fungus.

[0009] Furthermore, the method includes the following steps: first, using whole cells of Paracoccus sp. to perform a primary degradation of penicillin V to obtain intermediate products 6-APA and phenoxyacetic acid; then, using whole cells of Aspergillus niger to perform a secondary degradation of the intermediate product phenoxyacetic acid to obtain catechol; finally, returning to the whole cells of Paracoccus sp. to perform a tertiary degradation of the intermediate catechol and 6-APA, ultimately achieving complete mineralization degradation of penicillin V.

[0010] Furthermore, the immobilized whole cells of Paracoccus are prepared using chitosan, sodium alginate, and biochar as carriers and an embedding immobilization technology.

[0011] Furthermore, the immobilized whole cells of Aspergillus niger are prepared using sodium alginate, PVA, and biochar as carriers and an embedding immobilization technology.

[0012] Furthermore, the device used to implement the method is a combination of a fixed bed reactor and an expanded bed reactor; the Paracoccus is placed in the fixed bed reactor, and the Aspergillus niger is placed in the expanded bed reactor.

[0013] The application of a method for deep degradation of penicillin V, coupled with membrane technology, converts the treatment of solid hazardous waste into wastewater treatment.

[0014] Furthermore, it is coupled with membrane technology to treat mycelial solid waste generated in penicillin V production.

[0015] Furthermore, it is coupled with membrane technology to be used for the harmless treatment of mycelial solid waste generated in the production of β-lactam antibiotics.

[0016] The beneficial effects of the method for deep degradation of penicillin V by combining Paracoccus and Aspergillus niger in a cyclic cascade manner and its application are as follows: The present invention combines the discovered Paracoccus sp. KDSPL-02 strain (deposit number: CGMCC No. 19330), which can degrade penicillin V into 6-aminopenicillanic acid (6-APA) and phenoxyacetic acid, with the Aspergillus niger strain (deposit number: CGMCC No. 3.3928), which has the activity of catalyzing the conversion of phenoxyacetic acid into catechol. Through cyclic cascade catalysis, deep mineralization degradation of penicillin V is achieved.

[0017] Based on the separate immobilization of two bacterial whole cells, the present invention is technically implemented in a combination of a fixed bed reactor and an expanded bed reactor (FBR-EBB) system, which can efficiently and harmlessly treat wastewater and waste gas generated in the production of penicillin V.

[0018] The more outstanding innovation of the present invention lies in that, by coupling with membrane technology, a technical route for converting the treatment of solid hazardous waste into wastewater treatment is constructed. Penicillin V in waste mycelium is transferred to the aqueous phase through water extraction-membrane separation, and then degraded through two-stage independent immobilized cell catalytic beds to form a harmless treatment method for antibiotic waste mycelium and waste solid waste, providing an environmentally friendly technical solution for the clean production of antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Figure 1 is the HPLC chromatogram of penicillin V degradation by Paracoccus sp. KDSPL-02; Figure 2 This is the HPLC chromatogram of phenoxyacetic acid degradation by Aspergillus niger; Figure 3 This is the HPLC chromatogram of the mineralization and degradation of catechol by Paracoccus; Figure 4 Schematic diagram of the cyclic cascade degradation process of penicillin V. DETAILED DESCRIPTION

[0021] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] The present invention provides a method in which whole cells of Paracoccus sp. KDSPL-02 catalyze the decomposition of penicillin V into 6-aminopenicillanic acid (6-APA) and phenoxyacetic acid. Aspergillus niger CGMCC No. 3.3928 is then used to degrade phenoxyacetic acid into catechol. Finally, 6-APA and catechol are degraded by Paracoccus sp. KDSPL-02 to achieve mineralization and degradation of penicillin V. This constructed cascade cyclic degradation system for penicillin V can be used to treat the corresponding wastewater and waste gas using a fluidized bed reactor or coupled with membrane technology. Penicillin V in the waste mycelium is transferred to an aqueous phase through water extraction and membrane separation. The waste mycelium is then rendered harmless using immobilized whole cells of KDSPL-02 placed in a fixed-bed reactor and whole cells of CGMCC No. 3.3928 placed in an expanded-bed reactor.

[0023] Example 1 Degradation of Penicillin V by Paracoccus Inoculate 5 ml of LB liquid medium (1% inoculum) with Paracoccus sp. KDSPL-02 and shake at 28°C and 200 rpm for 48 hours to obtain a seed culture. Transfer the culture to 50 ml of LB medium and culture under the same conditions for an additional 48 hours. Collect whole Paracoccus cells by centrifugation at 6000 rpm for 5 minutes.

[0024] Resuspend the harvested whole cells in 50 mM sodium phosphate buffer (pH 7.0) to a wet cell concentration of 15 g / L. Transfer the suspension to 100 ml of dilution water containing 0.2 g / L penicillin V and shake at 160 rpm at 30°C. A penicillin V solution without cell suspension was prepared under the same conditions as a control. Residual penicillin V concentrations were determined by high-performance liquid chromatography. All experiments were performed in triplicate to ensure reproducibility.

[0025] Depend on Figure 1 HPLC analysis revealed that the penicillin V peak area decreased and a product peak appeared, with the retention time of the product corresponding to that of the phenoxyacetic acid standard, indicating that Paracoccus can degrade penicillin V into phenoxyacetic acid.

[0026] The HPLC conditions for penicillin V were as follows: separation using a C18 reverse phase column, a mobile phase of water:methanol = 5:5, a column temperature of 35°C, an ultraviolet detection wavelength of 225 nm, a flow rate of 1 mL / min, and an injection volume of 20 μL.

[0027] Example 2 Degradation of phenoxyacetic acid by Aspergillus niger A 1 cm² slant agar block containing refrigerated Aspergillus niger hyphae (stored at 4°C) was thoroughly minced and inoculated into 20 mL of PDA liquid medium. A seed culture was obtained by shaking the culture at 28°C, 200 rpm, for 48 hours. The seed culture was then transferred to 200 mL of PDA liquid medium and incubated under the same conditions to promote fungal growth. After 48 hours of incubation, whole Aspergillus niger cells were collected by centrifugation at 3000 rpm for 5 minutes.

[0028] 0.5 g of whole cells (wet weight) were resuspended in 20 mL of sodium phosphate buffer (pH 7.0). Phenoxyacetic acid (150 mg / L) was added to the cell suspension to a final concentration of 0.1 mg / mL. Biocatalytic degradation was initiated by shaking at 200 rpm and 28°C. After 48 hours, the residual phenoxyacetic acid concentration was determined by high-performance liquid chromatography (HPLC) using the same conditions as for penicillin V. All experiments were performed in triplicate to ensure reproducibility.

[0029] Depend on Figure 2 As can be seen from the HPLC analysis, the peak area of ​​phenoxyacetic acid decreased, and a product peak appeared. The retention time of the product corresponded to that of the catechol standard, indicating that Aspergillus niger can degrade phenoxyacetic acid into catechol.

[0030] Example 3 Degradation of phenoxyacetic acid by Aspergillus niger As in Example 2, the reaction temperature was changed to 20-40°C (e.g., 20°C, 25°C, 30°C, 35°C, 40°C), the pH was controlled at 7.0, and the substrate concentration was 150 mg / L. The degradation of phenoxyacetic acid by Aspergillus niger whole cells was carried out, and the same effect as in Example 2 was achieved within 24-30 hours.

[0031] Example 4 Degradation of phenoxyacetic acid by Aspergillus niger As in Example 2, the reaction temperature was controlled at 30 ° C. and the pH was 7. The substrate concentration was changed from 100 to 300 mg / L (e.g., 100 mg / L, 120 mg / L, 140 mg / L, 160 mg / L, 180 mg / L, 200 mg / L, 220 mg / L, 240 mg / L, 260 mg / L, 280 mg / L, and 300 mg / L) to degrade phenoxyacetic acid by Aspergillus niger whole cells. The same effect as in Example 2 was achieved within 24-28 hours.

[0032] Example 5 Degradation of phenoxyacetic acid by Aspergillus niger As in Example 2, the reaction temperature was controlled at 30°C, the substrate concentration was 200 mg / L, and the pH was varied from 4 to 9 (e.g., pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.5, 8.0, 8.5, 9.0) to degrade phenoxyacetic acid by Aspergillus niger whole cells. The same effect as in Example 2 was achieved within 24 to 26 hours.

[0033] Example 6 Paracoccus catalyzes the degradation of catechol KDSPL-02 whole cells, obtained using the same method as in Example 1, were adjusted to a wet weight concentration of 15 g / L and transferred to 100 ml of distilled water containing 0.2 g / L catechol. The suspension was shaken at 30°C and 160 rpm for a catalytic reaction. The catechol concentration in the samples was analyzed hourly by high-performance liquid chromatography (HPLC) using the same conditions as the penicillin V assay to assess degradation activity. Each experiment was performed in triplicate, with a control group under the same conditions for comparison.

[0034] Depend on Figure 3 It can be seen that the peak area of ​​catechol became smaller and no new peak appeared after HPLC detection, indicating that Paracoccus achieved mineralization degradation of catechol.

[0035] Example 7 Degradation of catechol catalyzed by Paracoccus Similar to Example 6, the reaction temperature was varied from 20°C to 40°C (e.g., 20°C, 25°C, 35°C, and 40°C), the pH was controlled at 7.0, and the substrate concentration was 150 mg / L. Paracoccus catalyzed the degradation of catechol, achieving the same effect as in Example 6 within 36-48 hours.

[0036] Example 8 Degradation of catechol catalyzed by Paracoccus As in Example 6, the reaction temperature was controlled at 30°C and the pH was 7. The substrate concentration was varied from 100 to 300 mg / L (e.g., 100 mg / L, 120 mg / L, 140 mg / L, 160 mg / L, 180 mg / L, 200 mg / L, 220 mg / L, 240 mg / L, 260 mg / L, 280 mg / L, and 300 mg / L). Paracoccus catalyzed the degradation of catechol, achieving the same effect as in Example 6 within 35 to 40 hours.

[0037] Example 9: Degradation of catechol by Paracoccus catalyzed by As in Example 6, the reaction temperature was controlled at 30°C, the substrate concentration was 200 mg / L, and the pH was adjusted to 5-8 (e.g., pH 5.0, 5.5, 6.0, 6.5, 7.5, 8.0). Paracoccus catalyzed degradation of catechol was carried out, and the same effect as in Example 6 was achieved within 36-38 hours.

[0038] Example 10 Cascade catalysis of penicillin V degradation by KDSPL-02 whole cells and CGMCC No. 3.3928 whole cells Combine Figure 4 As shown, 3 g (wet weight) of KDSPL-02 whole cells were prepared according to the method of Example 1 and resuspended in 200 mL of pH 7.0 phosphate buffer and placed in reaction tank 1 (i.e., storage tank 1 in the figure); 5 g of CGMCC No. 3.3928 whole cells were prepared according to the method of Example 2 and resuspended in 200 mL of pH 7.0 phosphate buffer and placed in reaction tank 2 (i.e., storage tank 2 in the figure).

[0039] First, penicillin V was added to reaction tank 1 at a final concentration of 0.2 g / L and allowed to react for 36 hours at 28°C and 200 rpm in a shaker. After the reaction, cells were removed by filtration, and the resulting filtrate was transferred to reaction tank 2, where the reaction continued for 24 hours under the same conditions (28°C and 200 rpm). Subsequently, cells were removed from reaction tank 2 by filtration, and the filtrate was returned to reaction tank 1 for an additional 24 hours at 28°C and 200 rpm, forming a cascade degradation system. High-performance liquid chromatography (HPLC) was used to monitor the concentrations of penicillin V, phenoxyacetic acid, and catechol during the degradation process. Ultimately, mineralization of penicillin V was achieved, and the presence of substrates and any intermediates could not be detected by HPLC.

[0040] Example 11 Immobilization of whole cells of Aspergillus niger Sodium alginate was used as the main support material, and polyvinyl alcohol (PVA) and biochar were used as auxiliary materials to achieve the immobilization of Aspergillus niger whole cells. The specific preparation steps are as follows: Weigh 100 mL of deionized water into a beaker and place it in a thermostatic water bath set to 60°C. Then, add sodium alginate at various mass ratios (e.g., 0.5%, 1%, 1.5%, and 2%) to the beaker. Maintain the temperature while stirring continuously until it is completely dissolved. Remove from heat and set aside for later use. Separately, weigh 100 mL of deionized water into a beaker and place it in a thermostatic water bath set to 90°C. Add polyvinyl alcohol (PVA) at various mass ratios (e.g., 0.05%, 0.1%, 0.5%, and 1%) (add slowly, or it will dissolve poorly) and biochar at various mass ratios (e.g., 0.05%, 0.1%, 0.5%, and 1%). Maintain the temperature while stirring continuously until the PVA forms a gel. Mix the two solutions thoroughly and cool to room temperature. Add 1.0 g of whole-cell Aspergillus niger to each beaker and stir continuously to thoroughly mix the cells with the mixed solution. A peristaltic pump was used to drip the bacteria-containing mixed solution into a uniformly stirred CaCl₂ solution to form sodium alginate gel microspheres with a diameter of 0.2–0.5 cm. After complete incorporation, the prepared particles were soaked in the CaCl₂ solution for 1 hour, sealed with sterile gauze, and placed in a refrigerator at 4°C to cure and crosslink for 12 hours. The crosslinked particles were then rinsed two to three times with deionized water to obtain immobilized gel microspheres of sodium alginate and polyvinyl alcohol biochar.

[0041] Example 12 Immobilization of whole cells of Paracoccus sp. KDSPL-02 A mixture of sodium alginate, chitosan, and biochar was used as the immobilization material, and sodium alginate and calcium chloride were used as the outer coating material for the spheres to achieve the immobilization of whole cells of Paracoccus KDSPL-02. The specific preparation steps are as follows: Weigh 200 mL of deionized water into a beaker and place the beaker in a constant-temperature water bath set to 60°C. Then, add sodium alginate at various mass ratios (e.g., 0.5%, 1%, 1.5%, and 2%) to the beaker. Maintain the temperature and stir continuously until it is completely dissolved. Remove from heat, cool to approximately 30°C, then add chitosan at various mass ratios (e.g., 0.1%, 0.2%, 0.5%, and 1%) and biochar at various mass ratios (e.g., 0.05%, 0.1%, 0.5%, and 1%). Once fully dissolved, cool to room temperature. Add 1.0 g of whole-cell Paracoccus strain (OD600 = 3.2) to each beaker and stir continuously to thoroughly mix the bacteria with the mixed solution. Use a peristaltic pump to drip the mixed solution containing the bacteria into the uniformly stirred CaCl2 solution to form sodium alginate gel microspheres. The immobilized particles range in diameter from 0.2 to 0.5 cm. After the solution is completely added, the prepared particles are soaked in the CaCl2 solution for 1 hour, sealed with sterile gauze, and placed in a 4°C refrigerator for curing and crosslinking for 12 hours. The fixed and cross-linked particles are rinsed with deionized water 2-3 times to obtain the prepared sodium alginate and chitosan biochar immobilized gel particle microspheres.

[0042] Example 13 Immobilized whole cells catalyze the degradation of penicillin V in a FBR-EBB reactor cascade The wastewater containing penicillin V was pumped into a fixed bed of 300 g of microspheres containing 30 g (wet weight) of KDSPL-02 cells immobilized therein. Figure 4 The first stage of degradation of penicillin V was catalyzed in a fixed column. The primary degradation solution was then injected from the bottom into an expanded bed ( Figure 4 The intermediate phenoxyacetic acid is catalytically degraded in the expansion column. Finally, the secondary degradation solution is pumped back into the fixed bed, where it is completely mineralized and degraded by KDSPL-02 immobilized cells. The system operates in a loop until no antibiotic residues or intermediates are detectable by HPLC. The treated water is collected and reused for washing the mycelium. The system run time depends on the initial substrate concentration; complete mineralization degradation can be achieved within 36 hours at a penicillin V concentration of 100-200 mg / L.

[0043] Example 14: Coupling of a membrane system and an FBR-EBB reactor system to harmlessly treat waste mycelium from penicillin V fermentation Combine Figure 4As shown, 1000 g of waste mycelium from penicillin V production was washed six times with 8 liters of water in a ceramic membrane system (Ceramic Membrane Filtration, Figure 1). The antibiotic-containing wastewater was collected as the feed liquid for the fixed-bed reactor. The waste wet mycelium was collected as a harmless solid. Subsequent treatment of the penicillin V-containing wastewater was the same as in Example 13. The catalytic degradation process was monitored using high-performance liquid chromatography. Test results showed that penicillin V in water was completely mineralized and degraded after 16 hours.

[0044] Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

Claims

1. A method for deep degradation of penicillin V, characterized in that: The method utilizes Paracoccus and Aspergillus niger to deeply degrade penicillin V through a combined cyclic cascade.

2. The method for deep degradation of penicillin V according to claim 1, characterized in that: The Paracoccus sp. is Paracoccus sp. KDSPL-02, and the Paracoccus sp. in the method includes whole cells or immobilized whole cells of the bacterium.

3. The method for deep degradation of penicillin V according to claim 1, characterized in that: The Aspergillus niger is Aspergillus niger, and its preservation number is CGMCC No. 3.3928. The Aspergillus niger in the method includes whole cells or immobilized whole cells of the fungus.

4. The method for deep degradation of penicillin V according to claim 1, characterized in that: The method comprises the following steps: firstly, using whole cells of Paracoccus sp. to perform a primary degradation of penicillin V to obtain intermediate products 6-APA and phenoxyacetic acid; then, using whole cells of Aspergillus niger to perform a secondary degradation of the intermediate product phenoxyacetic acid to obtain catechol; and finally, returning to the whole cells of Paracoccus sp. to perform a tertiary degradation of the intermediate catechol and 6-APA, thereby ultimately achieving complete mineralization degradation of penicillin V.

5. The method for deep degradation of penicillin V according to claim 2, characterized in that: The immobilized whole cells of Paracoccus are prepared by using chitosan, sodium alginate and biochar as carriers and adopting the embedding immobilization technology.

6. The method for deep degradation of penicillin V according to claim 3, characterized in that: The immobilized whole cells of Aspergillus niger are prepared by using sodium alginate, PVA and biochar as carriers and adopting the embedding immobilization technology.

7. The method for deep degradation of penicillin V according to claim 1, characterized in that: The device used for implementing the method is a combination of a fixed bed reactor and an expanded bed reactor; the Paracoccus is placed in the fixed bed reactor, and the Aspergillus niger is placed in the expanded bed reactor.

8. Use of the method for deep degradation of penicillin V according to any one of claims 1 to 7, characterized in that: Coupled with membrane technology, the treatment of solid hazardous waste is transformed into wastewater treatment.

9. The use according to claim 8, characterized in that Coupled with membrane technology, it is used to treat mycelial solid waste generated in penicillin V production.

10. The use according to claim 8, characterized in that Coupled with membrane technology, it is used for the harmless treatment of mycelial solid waste generated in the production of β-lactam antibiotics.

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