Method for deep degradation of penicillin V by combination of a micrococcus and aspergillus niger in a cyclic cascade and its application

Abstract

The application provides a method for deep degradation of penicillin V by combining Paracoccus sp KDSPL-02 and Aspergillus niger in a cyclic cascade, and application thereof, and belongs to the technical field of degradation of antibiotic pollutants. The Paracoccus sp KDSPL-02 is Paracoccus sp, and the Aspergillus niger is Aspergillus niger. The application is used for deep mineralization and degradation of penicillin V to resolve the pollution possibly caused by penicillin V, and harmless treatment of waste mycelia generated in the production process.

Description

Technical Field

[0001] This invention relates to a method for the deep degradation of penicillin V, and more particularly to a method for the deep degradation of penicillin V by a combined cyclic cascade of Paracoccus and Aspergillus niger, belonging to the field of antibiotic pollutant degradation technology. Background Technology

[0002] Microbial and fermentation industrial solid residues (such as antibiotic fermentation mycelium) have received widespread attention as important biological resources. Globally, the production of major fermentation products, such as antibiotics, generates nearly ten million tons of fermentation residue annually. This residue contains abundant organic matter (30-52% crude protein, 2-20% fat, and approximately 30-65% cellulose), possessing resource utilization value. However, residual antibiotics can cause environmental pollution, threatening ecological security and human health. Therefore, since 2008, it has been included in the "National Hazardous Waste List," and its disposal must comply with hazardous waste treatment standards. How to achieve the harmless treatment and resource utilization of antibiotic fermentation residues has become a research hotspot and a challenging problem.

[0003] Current treatment methods employ a strategy of destroying the entire mycelium, such as landfilling and incineration—traditional and simple technologies. However, these extensive methods pose secondary pollution problems. Landfilling can cause antibiotics to seep into the groundwater with rainwater, polluting groundwater. Incineration produces large amounts of carbon oxides, nitrogen oxides, and sulfur oxides, causing air pollution and acid rain. Furthermore, since this solid waste originates from food, destructive disposal is almost equivalent to wasting food. In recent years, there has been a trend towards developing new biosafety and environmentally friendly treatment strategies. Technology using whole-cell catalytic selective degradation of antibiotics has made progress, but incomplete antibiotic degradation exists, and intermediate products may become new pollutants, thus threatening ecological security.

[0004] Penicillin V (phenoxymethylpenicillin), a broad-spectrum antibiotic, exhibits better acid stability than penicillin G. Industrially, it is produced through fermentation and biosynthesis using industrial strains of *Penicillium chrysogenum* as a precursor. However, the molecule's stability in the environment makes its degradation more difficult, particularly the mineralization and degradation of the key intermediate phenoxyacetic acid, which remains a significant technical challenge. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for the deep degradation of penicillin V by a combined cyclic cascade of Paracoccus and Aspergillus niger, and its application, for achieving the mineralization and degradation of penicillin V.

[0006] To achieve the above objectives, 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 is Paracoccus sp. KDSPL-02, and the paracoccus in the method includes the whole cell or immobilized whole cell of this bacterium.

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

[0009] Furthermore, the method includes the following steps: firstly, using whole cells of Paracoccus to degrade penicillin V once to obtain intermediate products 6-APA and phenoxyacetic acid; then, using whole cells of Aspergillus niger to degrade the intermediate product phenoxyacetic acid a second time to obtain catechol; finally, returning to whole cells of Paracoccus to degrade the intermediate catechol and 6-APA a third time, ultimately achieving complete mineralization and degradation of penicillin V.

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

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

[0012] Furthermore, the apparatus 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 the deep degradation of penicillin V, coupled with membrane technology, transforms the treatment of solid hazardous waste into wastewater treatment.

[0014] Furthermore, it can be coupled with membrane technology for the treatment of mycelial solid waste generated during penicillin V production.

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

[0016] The beneficial effects of the present invention, which describes a method for the deep degradation of penicillin V by a combined cyclic cascade of *Paragonimus* and *Aspergillus niger*, and its application are as follows: This invention combines the discovered Paracoccus sp. KDSPL-02 (accession number: CGMCC No. 12494), a Paracoccus strain capable of degrading penicillin V into 6-aminopenicillanic acid (6-APA) and phenoxyacetic acid, with a Aspergillus niger strain (accession number: CGMCC No. 3.3928), which has the activity of catalytically converting phenoxyacetic acid into catechol. Through cyclic cascade catalysis, deep mineralization and degradation of penicillin V are achieved.

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

[0018] The most significant innovation of this invention lies in its coupling with membrane technology, which constructs a technical route for the treatment of solid hazardous waste into wastewater treatment. Penicillin V in waste mycelium is transferred to the aqueous phase through water extraction-membrane separation, and then degraded by two-stage independent immobilized cell catalytic beds to form a harmless treatment method for antibiotic waste mycelium waste solid waste, providing an environmentally friendly technical solution for the clean production of antibiotics. Attached Figure Description

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

[0020] Figure 1 This is a high-performance liquid chromatogram of penicillin V degradation in Paracoccus KDSPL-02; Figure 2 This is a high-performance liquid chromatogram of phenoxyacetic acid degradation by Aspergillus niger; Figure 3 This is a high-performance liquid chromatogram of the mineralization degradation of catechol by Paracoccus; Figure 4 This is a schematic diagram of the cyclic cascade degradation process of penicillin V. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] This invention provides a cascaded degradation system for penicillin V using Paracoccus sp. KDSPL-02, which catalyzes the decomposition of penicillin V into 6-aminopenicillanic acid (6-APA) and phenoxyacetic acid via whole-cell catalysis. The phenoxyacetic acid is then degraded into catechol by Aspergillus niger CGMCC No. 3.3928, and finally, 6-APA and catechol are further degraded by Paracoccus sp. KDSPL-02 to achieve the mineralization and degradation of penicillin V. This constructed system can be used for the treatment of its corresponding wastewater and waste gas using a fluidized bed reactor, or it can be coupled with membrane technology to transfer penicillin V from waste mycelium to the aqueous phase via water extraction-membrane separation. The waste mycelium is then treated harmlessly using whole-cell immobilized by KDSPL-02 in a fixed-bed reactor and whole-cell immobilized by CGMCC No. 3.3928 in an expanded-bed reactor.

[0023] Example 1: Degradation of Penicillin V by Paracoccus Paracoccus KDSPL-02 was inoculated into 5 ml LB liquid medium with an inoculum volume of 1% and cultured at 28°C and 200 rpm for 48 h with shaking to obtain seed culture. The seed culture was then transferred to 50 ml LB medium and cultured under the same conditions for another 48 h. The whole cells of Paracoccus were collected by centrifugation at 6000 rpm for 5 minutes.

[0024] The collected whole cells were resuspended in 50 mM pH 7.0 sodium phosphate buffer to adjust the cell wet weight concentration to 15 g / L. The suspension was transferred to 100 ml of diluent containing 0.2 g / L penicillin V and reacted at 30°C and 160 rpm. A penicillin V solution without cell slurry was prepared as a control under the same conditions. The concentration of residual penicillin V was determined by high performance liquid chromatography. All experiments were performed in triplicate to ensure reproducibility.

[0025] Depend on Figure 1 As can be seen from the high-performance liquid chromatography (HPLC) analysis, the peak area of ​​penicillin V decreased, and a product peak appeared. The retention time of the product corresponded to that of the phenoxyacetic acid standard. This indicates that *Paragonimus westermani* can degrade penicillin V into phenoxyacetic acid.

[0026] The high-performance liquid chromatography (HPLC) conditions for penicillin V were as follows: separation was performed using a C18 reverse-phase column, the mobile phase was water:methanol = 5:5, the column temperature was 35℃, the UV detection wavelength was 225nm, the flow rate was 1mL / min, and the injection volume was 20μL.

[0027] Example 2: Degradation of phenoxyacetic acid by Aspergillus niger A slant agar block (1 cm²) containing refrigerated Aspergillus niger hyphae (stored at 4°C) was thoroughly chopped and inoculated into 20 mL of PDA liquid medium. The culture was incubated at 28°C and 200 rpm with shaking for 48 hours to obtain a seed culture. 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, and the mixture was shaken at 28 °C and 200 rpm to initiate biocatalytic degradation. After 48 h, the concentration of residual phenoxyacetic acid was determined by high-performance liquid chromatography (HPLC) under the same detection conditions as penicillin V. All experiments were performed in triplicate to ensure reproducibility.

[0029] Depend on Figure 2 As can be seen from the high-performance liquid chromatography (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. This indicates that Aspergillus niger can degrade phenoxyacetic acid into catechol.

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

[0031] Example 4 Degradation of phenoxyacetic acid by Aspergillus niger Similar to Example 2, the reaction temperature was controlled at 30 °C and the pH at 7. The substrate concentration was varied by 100-300 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, 300 mg / L) to degrade phenoxyacetic acid by whole cells of Aspergillus niger. The same effect as in Example 2 was achieved within 24-28 hours.

[0032] Example 5 Degradation of phenoxyacetic acid by Aspergillus niger Similar to Example 2, the reaction temperature was controlled at 30 °C and the substrate concentration at 200 mg / L. 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 carry out the degradation of phenoxyacetic acid by whole cells of Aspergillus niger. The same effect as in Example 2 was achieved within 24-26 hours.

[0033] Example 6: Degradation of catechol catalyzed by Paracoccus 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 subjected to a catalytic reaction in a shaker at 30°C and 160 rpm. The concentration of catechol in the sample was analyzed hourly by high-performance liquid chromatography (HPLC) under the same conditions as for penicillin V to assess degradation activity. Each experiment was performed in triplicate, with a control group included for comparison under the same conditions.

[0034] Depend on Figure 3 As can be seen from the high performance liquid chromatography (HPLC) analysis, the peak area of ​​catechol decreased and no new peaks appeared, indicating that Paracoccus achieved mineralization degradation of catechol.

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

[0036] Example 8: Paracoccus-catalyzed degradation of catechol Similar to Example 6, the reaction temperature was controlled at 30 °C and the pH at 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, 300 mg / L) to carry out the degradation of catechol by Paracoccus catalysis, achieving the same effect as Example 6 within 35-40 hours.

[0037] Example 9: Degradation of catechol catalyzed by Paracoccus Similar to Example 6, the reaction temperature was controlled at 30 °C, the substrate concentration at 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) to carry out the degradation of catechol by Paracoccus catalysis, achieving the same effect as Example 6 within 36-38 hours.

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

[0039] First, penicillin V at a final concentration of 0.2 g / L was added to reaction tank 1 and catalytically reacted at 28°C and 200 rpm in a shaker for 36 hours. 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, 200 rpm). Subsequently, cells in reaction tank 2 were removed by filtration, and the filtrate was returned to reaction tank 1, where it was reacted again at 28°C and 200 rpm for 24 hours, thus forming a cascade degradation system. The concentrations of penicillin V, phenoxyacetic acid, and catechol during the degradation process were monitored using high-performance liquid chromatography (HPLC). Ultimately, the mineralization degradation of penicillin V was achieved, and the presence of substrates and any intermediates was undetectable by HPLC.

[0040] Example 11: Whole-cell immobilization of Aspergillus niger Sodium alginate was used as the main supporting material, supplemented by polyvinyl alcohol (PVA) and biochar as auxiliary materials, to achieve the immobilization of whole cells of Aspergillus niger. The specific preparation steps are as follows: Weigh 100 mL of deionized water into a beaker and place it in a constant temperature water bath at 60 °C. Then add sodium alginate in different mass ratios (e.g., 0.5%, 1%, 1.5%, 2%) to the beaker, maintaining the temperature and stirring continuously until it is completely dissolved. Stop heating and set aside for later use. Separately, weigh 100 mL of deionized water into a beaker and place it in a constant temperature water bath at 90 °C. Add polyvinyl alcohol in different mass ratios (e.g., 0.05%, 0.1%, 0.5%, 1%) (add slowly, otherwise it will be difficult to dissolve) and biochar in different mass ratios (e.g., 0.05%, 0.1%, 0.5%, 1%), maintaining the temperature and stirring continuously until the PVA reaches a gel-like state. 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 ensure the bacteria are fully mixed with the solution. A peristaltic pump was used to drip the bacterial mixture into a uniformly stirred CaCl2 solution to prepare sodium alginate gel microspheres with an immobilized particle diameter between 0.2 and 0.5 cm. After all the particles were added, they were immersed in the CaCl2 solution for 1 hour, sealed with sterile gauze, and placed in a 4 °C refrigerator for 12 hours to cure and crosslink. The immobilized and crosslinked particles were rinsed with deionized water 2-3 times to obtain the prepared sodium alginate and polyvinyl alcohol biochar immobilized gel microspheres.

[0041] Example 12 Whole-cell immobilization of Paracoccus 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 spheroidization. This method achieved whole-cell immobilization of Paracoccus KDSPL-02. The specific preparation steps are as follows: Weigh 200 mL of deionized water into a beaker and place it in a constant temperature water bath at 60 °C. Then, add sodium alginate in different mass ratios (e.g., 0.5%, 1%, 1.5%, 2%) to the beaker, maintaining the temperature and stirring continuously until it is completely dissolved. Stop heating and cool to approximately 30 °C. Then, add chitosan in different mass ratios (e.g., 0.1%, 0.2%, 0.5%, 1%) and biochar in different mass ratios (e.g., 0.05%, 0.1%, 0.5%, 1%). After they are fully miscible, cool to room temperature. Add 1.0 g of whole-cell paracoccus strain (OD600=3.2) to each beaker and stir continuously to ensure the bacteria are fully mixed with the solution. Use a peristaltic pump to dropwise add the bacterial solution into a uniformly stirred CaCl2 solution to form sodium alginate gel microspheres with an immobilized particle diameter between 0.2 and 0.5 cm. After all the particles have been added, the prepared particles are immersed in CaCl2 solution for 1 hour, sealed with sterile gauze, and placed in a 4°C refrigerator for 12 hours to cure and crosslink. The fixed and crosslinked particles are rinsed with deionized water 2-3 times to finally obtain the prepared immobilized gel microspheres of sodium alginate and chitosan biochar.

[0042] Example 13 Immobilized whole cells in an FBR-FBB reactor cascade catalytic degradation of penicillin V Wastewater containing penicillin V was pumped into a fixed bed containing 300 g microspheres with 30 g (wet weight) KDSPL-02 cells fixed. Figure 4 In the fixed column, the first-stage degradation of penicillin V is catalyzed. Then, the primary degradation solution is injected from the bottom into an expanded bed containing 100 g of solid microspheres containing 10 g (wet weight) Aspergillus niger cells. Figure 4 In the expansion column, the intermediate phenoxyacetic acid is catalytically degraded. Finally, the secondary degradation solution is pumped back to the fixed bed, where KDSPL-02 immobilized cells catalyze complete mineralization degradation. The system is operated in a cyclic manner until antibiotic residues and intermediates are undetectable by HPLC. The treated water is collected and reused for washing mycelia. The system's run time depends on the initial substrate concentration; complete mineralization degradation can be achieved within 36 hours when the penicillin V concentration is 100-200 mg / L.

[0043] Example 14: Harmless treatment of penicillin V fermentation waste mycelium by coupling membrane system with FBR-FBB reactor system Combination 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 (i.e., the Ceramic Membrane Filtration shown in the figure). The antibiotic-containing wastewater was collected as the feed liquid for the fixed-bed reactor. The waste wet mycelium was collected as harmless solids. 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. The results showed that penicillin V in water was completely mineralized and degraded after 16 hours.

[0044] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort 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 a combined cyclic cascade of Paracoccus and Aspergillus niger to deeply degrade penicillin V; The method includes the following steps: First, using whole cells of Paracoccus to degrade penicillin V once to obtain intermediate products 6-aminopenicillanic acid and phenoxyacetic acid; then using whole cells of Aspergillus niger to degrade the intermediate product phenoxyacetic acid a second time to obtain catechol; finally, returning to whole cells of Paracoccus to degrade the intermediate catechol and 6-aminopenicillanic acid a third time, ultimately achieving complete mineralization and degradation of penicillin V.

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

3. The method for deep degradation of penicillin V according to claim 1, characterized in that, The Aspergillus niger is a fungus with accession number CGMCC No. 3.3928; the Aspergillus niger in the method includes the immobilized whole cells of this fungus.

4. The method for deep degradation of penicillin V according to claim 2, characterized in that, Immobilized whole cells of Paracoccus were prepared using chitosan, sodium alginate, and biochar as carriers, employing an embedding immobilization technique.

5. The method for deep degradation of penicillin V according to claim 3, characterized in that, Immobilized whole cells of Aspergillus niger were prepared using sodium alginate, polyvinyl alcohol, and biochar as carriers, employing an embedding immobilization technique.

6. The method for deep degradation of penicillin V according to claim 1, characterized in that, The apparatus 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.

7. The application of the method for deep degradation of penicillin V according to any one of claims 1-6, characterized in that, Coupled with membrane technology, the treatment of solid hazardous waste is transformed into wastewater treatment; the application includes the following steps: taking solid hazardous waste and washing it with water in a ceramic membrane system, collecting the antibiotic-containing wastewater as the feed liquid of a fixed-bed reactor, and then treating the feed liquid according to the method of any one of claims 1-6.

8. The application according to claim 7, characterized in that, Coupled with membrane technology, it can be used for the treatment of mycelial solid waste generated in penicillin V production.

9. The application according to claim 7, characterized in that, Coupled with membrane technology, it can be used for the harmless treatment of mycelial solid waste generated in the production of β-lactam antibiotics.

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