Method for removing antibiotics from antibiotic fermentation residue and application of the method

Abstract

This invention provides a method and application for removing antibiotics and synergistically recovering plant bioactive organic matter from antibiotic fermentation residue. The method employs a simple hydrothermal process combined with pyrolusite catalytic oxidation technology to achieve complete degradation of residual antibiotics and resistance genes in the antibiotic fermentation residue, achieving deep detoxification. Simultaneously, the high temperature and pressure conditions created by the hydrothermal technology disrupt the ultracolloidal structure of mycelial cell membranes, releasing soluble organic matter. This leads to the hydrolysis of macromolecular organic matter such as crude protein, crude fat, and sugars, intensifying caramelization and Maillard reactions to gradually generate humic acid-based plant bioactive organic matter. The resulting hydrothermal residue supernatant can be further processed into liquid ecological fertilizer. The liquid ecological fertilizer provided by this invention significantly promotes the biomass and stem length of *Phyllostachys edulis*.

Description

Technical Field

[0001] This invention belongs to the field of antibiotic fermentation residue technology, specifically relating to a method and application for removing antibiotics and synergistically recovering plant active organic matter from antibiotic fermentation residue. Background Technology

[0002] The microbial fermentation production of antibiotics generates a large amount of pharmaceutical waste—antibiotic fermentation residue, mainly composed of mycelium, fermentation medium, residual antibiotics, and other inorganic particles. Due to its rich organic matter content (>60%), antibiotic fermentation residue has strong resource utilization potential and is now widely used as soil organic fertilizer or amendment. However, residual antibiotics in the residue drive the spread and increase in abundance of drug-resistant bacteria and antibiotic resistance genes through the proliferation and horizontal gene transfer of host bacteria, posing a serious threat to ecosystems and human health. This patent aims to utilize a thermochemical treatment technology to simultaneously degrade residual antibiotics in bio-fermentation residue and recover plant bioactive organic matter.

[0003] In recent years, researchers have developed various technologies for the harmless treatment of antibiotic fermentation residues, including deep oxidation, microwave irradiation, and electrolysis. Chinese patent CN202210648383.8, "A Method and System for Treating Antibiotic Fermentation Residues," uses electron beam irradiation to generate hydroxyl radicals to degrade cephalosporin C, achieving a removal rate of up to 100%. Chinese patent CN202211184351.3, "A Method for Treating Antibiotic Fermentation Residues with Hydrothermal Synergistic nZVI Activation of Persulfate," uses a hydrothermal synergistic nZVI activation of persulfate system to achieve complete degradation of vancomycin. However, these methods primarily focus on antibiotic removal, neglecting the resource properties of the fermentation residues themselves. Furthermore, the intense oxidation of the residues through the consumption of large amounts of energy and chemicals leads to over-mineralization, resulting in low volume reduction and resource utilization. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a method and application for removing antibiotics and synergistically recovering plant bioactive organic matter from antibiotic fermentation residue.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first objective of this invention is to provide a method for removing antibiotics and synergistically recovering plant active organic matter from antibiotic fermentation residue. The method involves mixing the antibiotic fermentation residue with water, then adding pyrolusite, and conducting a first hydrothermal reaction at 160°C to 200°C. The pyrolusite is obtained by a second hydrothermal reaction using potassium permanganate solid, toluene, and deionized water.

[0007] Furthermore, the moisture content of the antibiotic fermentation residue is 60%~65%.

[0008] Furthermore, the mass ratio of the antibiotic fermentation residue to the pyrolusite is (50~60):1.

[0009] Furthermore, the mass ratio of the potassium permanganate solid, toluene, and water is (1~2):(2.4~3.0):(38~40).

[0010] Furthermore, the temperature of the second hydrothermal reaction is 175℃~185℃, and the reaction time is 24h~28h.

[0011] Furthermore, the antibiotics in the antibiotic fermentation residue include lincomycin antibiotics or macrolide antibiotics.

[0012] The second objective of this invention is to provide a liquid ecological fertilizer, which is obtained by centrifuging the reactants obtained by the above method and taking the supernatant.

[0013] Furthermore, the liquid ecological fertilizer contains fulvic acid and humic acid-like substances.

[0014] The third objective of this invention is to provide a method for cultivating Chinese cabbage, in which the above-mentioned liquid ecological fertilizer is used to hydroponically cultivate Chinese cabbage seedlings.

[0015] Furthermore, the aforementioned method for cultivating bok choy promotes root growth in bok choy seedlings.

[0016] In this invention, the term "antibiotic fermentation residue" refers to the solid waste generated by microbial fermentation during the production of antibiotics by pharmaceutical companies. It mainly consists of mycelium, fermentation culture medium, residual antibiotics, and other inorganic particles.

[0017] In this invention, the term "plant-active organic matter" refers to compounds with plant activity, which are one of the main sources of plant nutrition and can promote plant growth and development. These compounds mainly include dissolved organic carbon and microbial biomass.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] (1) This invention provides a method for removing antibiotics and synergistically recovering plant active organic matter from antibiotic fermentation residue. This method uses a simple hydrothermal method combined with pyrolusite catalytic oxidation technology to achieve complete degradation of residual antibiotics and resistance genes in antibiotic fermentation residue, thus achieving deep detoxification. At the same time, the high temperature and high pressure conditions created by the hydrothermal technology destroy the ultracolloidal structure of mycelial cell membranes, release soluble organic matter, hydrolyze macromolecular organic matter such as crude protein, crude fat, and sugars, and intensify caramelization and Maillard reactions to gradually generate humic acid-based plant active organic matter. The supernatant of the obtained hydrothermal fermentation residue can be further processed into liquid ecological fertilizer.

[0020] (2) This invention prepares a manganese oxide catalyst (pyrolusite) with excellent thermal stability via a hydrothermal method. Adding it to the hydrothermal system accelerates the dissolution and rupture of fermentation residue, releasing crude protein, crude fat, sugars, and antibiotics from its three-dimensional structural sites. Simultaneously, it generates highly oxidizing hydroxyl radicals, providing more redox sites and reducing reaction energy consumption. The combined effect of these two aspects enhances the hydrolysis and transformation of organic matter in the fermentation residue, while also enabling efficient removal of antibiotics. This invention employs a hydrothermal combined with pyrolusite treatment of fermentation residue to study the transformation behavior of organic matter and antibiotics in a water-manganese oxide system, providing a scientific basis for evaluating antibiotic degradation behavior and efficiently recovering active organic matter.

[0021] (3) The liquid ecological fertilizer provided by the present invention has a significant promoting effect on the biomass and stem length of the white carp. Attached Figure Description

[0022] Figure 1 XRD pattern of pyrolusite prepared in this invention;

[0023] Figure 2a The graph shows the change in lincomycin content in the filter residue after processing lincomycin fermentation residue using the method of the present invention.

[0024] Figure 2b A graph showing the change in lincomycin content in the filtrate after treating lincomycin fermentation residue using the method of the present invention;

[0025] Figure 2c The graph shows the change in tylosin content in the filter residue after processing tylosin fermentation residue using the method of the present invention.

[0026] Figure 2d The graph shows the change in tylosin content in the filtrate after treating tylosin fermentation residue using the method of the present invention.

[0027] Figure 3a A graph showing the change in the concentration of dissolved organic matter in the filtrate after treating lincomycin fermentation residue using the method of the present invention;

[0028] Figure 3b The graph shows the change in free amino acid concentration in the filtrate after treating lincomycin fermentation residue using the method of the present invention.

[0029] Figure 4 3D-EEM image of the filtrate after processing lincomycin fermentation residue using the method of the present invention;

[0030] Figure 5 The image shows the growth of Chinese cabbage (root length, stem length, and fresh weight) after the filtrate from the lincomycin fermentation residue was treated using the method of this invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0032] The lincomycin fermentation residue came from a pharmaceutical company in Ningxia and had a moisture content of 65%.

[0033] The tylosin fermentation residue came from a pharmaceutical company in Ningxia and had a moisture content of 60%.

[0034] Determination of antibiotics in solid-liquid phase:

[0035] Pretreatment of hydrothermal bacterial residue: 250 mL of water sample was taken, filtered through a filter membrane, and 0.2 g Na2EDTA was added to reduce the chelation between antibiotics and metal ions. An appropriate amount of concentrated hydrochloric acid was added to adjust the pH of the water sample to 3.0-4.0. Extraction and enrichment were performed using an Oasis HLB (200 mg / 6 cc) solid-phase extraction column. First, the HLB column was activated sequentially with 10 mL of methanol, 10 mL of pure water, and 10 mL of pure water (pH=4.0) at a rate of 5 mL / min; the sample was then loaded; after loading, the column was washed with 10 mL of pure water, dried under vacuum, and then dried again under nitrogen protection. It was eluted twice with 6 mL of methanol, purged with nitrogen until nearly dry, and concentrated to 1 mL for analysis.

[0036] Pretreatment of bacterial residue: Weigh 0.2 g of sample into a 50 mL centrifuge tube, add 20 mL of methanol / EDTA-Mcllvaine buffer solution (v / v = 1:1, pH=4), vortex for 15 min, sonicate for 15 min, centrifuge at 6000 r / min for 10 min, collect the supernatant, repeat the extraction three times and combine the extracts, add 0.2 g of Na2EDTA, add ultrapure water to 250 mL, adjust the pH to between 3.0 and 4.0 with hydrochloric acid, and pass the sample through an Oasis HLB (200 mg / 6 cc) solid-phase extraction column at a speed of 5 mL / min. The column is activated sequentially with 10 mL of methanol, 10 mL of pure water and 10 mL of pure water at pH 4.0, then loaded with the sample. The column is then washed with 10 mL of pure water, dried under nitrogen protection, and then slowly eluted twice with 6 mL of chromatographic grade methanol. The sample is then purged with nitrogen until nearly dry and concentrated to 1 mL for analysis.

[0037] Liquid chromatography-mass spectrometry (LC-MS) instrumentation conditions: Antibiotic concentrations were determined using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) (Agilent 1290 UPLC / 6550 Q-TOF, USA) equipped with a Waters BEH C18 column (2.1 × 100 mm, 1.7 µm). Column temperature was 40 °C, and flow rate was 0.20 mL / min. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and methanol:acetonitrile solution (v / v, 1:1) (B). The injection volume was 10 μL, and gradient elution was used: 0–1 min, 90% B; 1–18 min, 60% B; 18–24 min, 10% B; 24–28 min, 90% B. Multiple reaction monitoring (MRM) mode was used for mass spectrometry detection.

[0038] Example 1

[0039] This embodiment provides a method for removing antibiotics and synergistically recovering plant bioactive organic matter from lincomycin fermentation residue.

[0040] (1) Preparation of pyrolusite

[0041] 1.82 g of potassium permanganate solid was weighed and added to 5 mL of toluene and 70 mL of deionized water to prepare solution A. Solution A was added to a 100 mL hydrothermal reactor lined with polytetrafluoroethylene and reacted at 180 °C for 24 h. The treated sample was centrifuged at 8000 rpm to obtain a black solid, which was washed three times with deionized water and dried at 105 °C.

[0042] (2) Hydrothermal treatment

[0043] 70 g of lincomycin fermentation residue and 280 mL of water were added to a 500 mL hydrothermal reactor, followed by the addition of 1.4 g of pyrolusite, which was mixed thoroughly. The hydrothermal reactor was heated to the target temperature (25℃, 80℃, 120℃, 160℃, and 200℃) and kept at that temperature for a certain period of time (0h, 1h, 3h, 6h, and 12h) to obtain the reaction solution.

[0044] After cooling to room temperature, the reaction solution was centrifuged at 8000 rpm for 10 minutes. The solid was then air-dried and stored, and the supernatant was collected and filtered through a 0.45 μm filter membrane. The air-dried bacterial residue and hydrothermal supernatant were subjected to solid-phase extraction, and the concentration of hydrothermally degraded antibiotics was determined by LC-MS / MS.

[0045] The pyrolusite prepared in this embodiment is a brown-black fine particle, and its technical parameters are shown in Table 1.

[0046] Table 1. Technical parameters of pyrolusite.

[0047]

[0048] refer to Figure 1 The image shown is the XRD pattern of pyrolusite prepared in this embodiment. The diffraction peaks are located at 2θ=28°, 2θ=37°, and 2θ=56°, which are basically consistent with the characteristic diffraction peaks of manganese dioxide (PDF#24-0735). It can be determined that the sample prepared by the hydrothermal method in this patent is pyrolusite mineral.

[0049] refer to Figure 2a and Figure 2b Compared to hydrothermal systems alone, the hydrothermal-pyrolusite system significantly promotes the degradation of the antibiotic lincomycin. Furthermore, the treatment group incorporating pyrolusite exhibits a higher removal rate from the bacterial residue. At 200℃, the hydrothermal-pyrolusite system achieved degradation rates of 99.46% and 99.41% for lincomycin in bacterial residue and hydrothermal solution, respectively, while the hydrothermal system achieved degradation rates of 97.94% and 99.41%, respectively. Lincomycin has abundant hydroxyl substituents on its pyran ring, which are electron-donating activating groups, increasing the electron cloud density on the pyran ring and facilitating electron transfer, resulting in stronger reducing power. In addition, due to the two-dimensional layered structure of pyrolusite, other ions and molecules can easily be added or inserted between its layers. Upon contact with pyrolusite, the antibiotic is adsorbed onto its surface, undergoing a redox reaction and thus being oxidized and degraded, generating low-toxicity or non-toxic small molecule compounds. Further reactions ultimately produce CO2 and H2O.

[0050] Referring to Figure 3, the concentrations of protein, polysaccharide, humic acid, and free amino acids in lincomycin inoculum residue under different hydrothermal temperatures change. As the temperature increases from 25°C to 200°C, the concentrations of soluble biopolymers (especially proteins and humic acid) increase significantly, reaching a maximum at 200°C. The maximum humic acid content in the lincomycin inoculum residue is 17.29 g / L. However, when the temperature exceeds 120°C, a slight decrease in protein concentration is observed, indicating that soluble proteins undergo a higher degree of solubilization and transformation, leading to the formation of free amino acids in the lincomycin inoculum residue. The amino acids most enriched in the lincomycin inoculum residue produced at 120°C are hydrophobic components (isoleucine [Ile], leucine [Leu], and phenylalanine [Phe]) and basic components (lysine [Lys] and histidine [His]). This is because the addition of pyrolusite accelerates the dissolution and breakdown of the fermentation residue, releasing crude proteins, crude fats, sugars, and other organic molecules from its three-dimensional structural sites. Simultaneously, it generates highly oxidizing hydroxyl radicals, providing more redox sites, reducing reaction energy consumption, and significantly intensifying the decomposition of oxidative organic macromolecules (proteins, polysaccharides, etc.), thereby reducing hydrogen bonds and increasing the dissolution of hydrophobic proteins. Furthermore, the decrease in free amino acid concentration from 120℃ to 200℃ may be due to the secondary synthesis of these small-molecule amino acids through oxidative coupling reactions caused by pyrolusite, followed by complex polymerization and carboxylation to form humic acid-like structures, which also explains the increase in humic acid concentration.

[0051] To evaluate the changes in key organic matter in lincomycin bacterial residue, reference was made. Figure 4 The 3D-EEM image shows the bacterial residue solution at 25℃ hydrothermally. Figure 4 a) There are two main peaks, belonging to aromatic protein II (E) x / E m =200~250 nm / 320~380 nm) and soluble microbial metabolites (E x / E m =250~450 nm / 260~380 nm). With increasing hydrothermal temperature, the relative fluorescence intensity of these peaks increases significantly, and new peaks gradually appear in regions III and V. Figure 4 c). These results indicate that the hydrothermal-pyrolusite system decomposes soluble proteins and microbial metabolites, while simultaneously generating fulvic acid and humic acids. The enhancement of humic acids is attributed to the caramelization and Maillard reactions of polysaccharides and proteins in the lincomycin residue matrix at high temperatures.

[0052] Example 2

[0053] This embodiment provides a method for removing antibiotics and synergistically recovering plant bioactive organic matter from tylosin fermentation residue.

[0054] The process is basically the same as in Example 1, except that it uses tylosin fermentation residue.

[0055] refer to Figure 2c and Figure 2d The hydrothermal-pyrolusite system significantly promoted the degradation of the antibiotic tylosin compared to the hydrothermal system alone. Furthermore, the treatment group with added pyrolusite showed a higher removal rate of tylosin from the bacterial residue. At 200℃, the hydrothermal-pyrolusite system achieved degradation rates of 99.04% and 98.47% for tylosin in the bacterial residue and hydrothermal solution, respectively, while the hydrothermal system achieved degradation rates of 94.40% and 98.09% for tylosin in the bacterial residue and hydrothermal solution, respectively.

[0056] Example 3

[0057] This embodiment provides a method for removing antibiotics and synergistically recovering plant bioactive organic matter from lincomycin fermentation residue.

[0058] The process is basically the same as in Example 1, except that the mass ratio of lincomycin fermentation residue to pyrolusite is 55:1, and the mass ratio of potassium permanganate solid, toluene and water in the preparation of pyrolusite is 1.5:2.6:40.

[0059] Example 4

[0060] This embodiment provides a method for removing antibiotics and synergistically recovering plant bioactive organic matter from lincomycin fermentation residue.

[0061] The process is basically the same as in Example 1, except that the mass ratio of lincomycin fermentation residue to pyrolusite is 55:1, the mass ratio of potassium permanganate solid, toluene and water in the preparation of pyrolusite is 1.5:2.6:40, the temperature of the second hydrothermal reaction is 175°C, and the reaction time is 26 hours.

[0062] Example 5

[0063] This embodiment provides a method for removing antibiotics and synergistically recovering plant bioactive organic matter from lincomycin fermentation residue.

[0064] The process is basically the same as in Example 1, except that the mass ratio of lincomycin fermentation residue to pyrolusite is 50:1, the mass ratio of potassium permanganate solid, toluene and water in the preparation of pyrolusite is 1:3.0:40, the temperature of the second hydrothermal reaction is 175°C, and the reaction time is 28 hours.

[0065] At 200°C, the hydrothermal-pyrolusite system provided in Examples 3-5 achieved a degradation rate of 99% for lincomycin in bacterial residue and hydrothermal fluid.

[0066] Example 6

[0067] This embodiment provides a liquid ecological fertilizer.

[0068] After centrifuging the reaction solution obtained in Example 1 or Example 2, the supernatant is taken to obtain liquid ecological fertilizer.

[0069] Example 7

[0070] This embodiment provides a method for cultivating Chinese cabbage.

[0071] After centrifuging the reaction solution obtained in Example 1, the supernatant was removed to obtain liquid ecological fertilizer.

[0072] Chinese cabbage seeds were selected as the germination test material. First, the seeds were washed with 3% H2O2, and then selected plump seeds without obvious defects. Fifteen sterilized seeds were placed on filter paper and then in a 70 mm petri dish. 6 ml of pre-diluted liquid organic fertilizer to 20 ppm was added. The petri dishes were incubated in a dark chamber at 25±1℃ for 72 h. Germination rate, root biomass, stem biomass, and other plant phenotypic parameters were measured. Deionized water was used as a blank control, and each experiment was repeated three times. The results are shown in Table 2. Figure 5 As shown.

[0073] Table 2. Statistics on root length, stem length and fresh weight of Chinese cabbage.

[0074]

[0075] refer to Figure 5 Compared to the control group, all hydrothermal bacterial residue solutions significantly promoted the biomass and stem length of pak choi. With increasing hydrothermal temperature, the inhibitory effect on pak choi root growth gradually improved, which is closely related to the gradual degradation of lincomycin. The supernatant from the digested sludge after hydrothermal treatment at 200℃ showed the greatest promoting effect on rice seedling growth. Due to the complete degradation of lincomycin and the production of a large amount of plant-active organic matter (e.g., amino acids, humic acid), the 200℃ hydrothermal bacterial residue solution exhibits great fertilization potential and is expected to become a substitute for organic liquid fertilizers.

[0076] Comparative Example 1

[0077] This embodiment provides a treatment solution for cultivating Chinese cabbage using only pyrolusite.

[0078] Add 70 g of lincomycin fermentation residue and 280 mL of water to a 500 mL hydrothermal reactor, then add 1.4 g of pyrolusite and mix well. Stir at 25°C for 24 h. Centrifuge the resulting reaction solution at 8000 rpm for 10 minutes and take the supernatant to obtain the treatment solution.

[0079] The method for cultivating Chinese cabbage is the same as in Example 7, except that "6 ml of pre-diluted treatment solution to 20 ppm is added".

[0080] The applicant discovered that, compared to the control group, the treatment solution in this embodiment significantly inhibited the growth of pakchoi. The reason for this was that the pyrolusite did not completely oxidize the heterogeneous lincomycin-containing bacterial residue, resulting in a low content of plant-active organic matter released into the bacterial residue solution, insufficient to significantly promote plant growth and metabolism. Furthermore, the incomplete degradation of lincomycin by the pyrolusite also resulted in residual antibiotics inhibiting the anabolic and energy metabolism processes of pakchoi. Therefore, the "hydrothermal + pyrolusite" treatment process, due to its efficient removal of antibiotics while recovering plant-active organic matter, can become an important approach for the resource utilization of fermentation bacterial residue.

[0081] For any points not covered above, existing technologies shall apply.

[0082] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for removing antibiotics and synergistically recovering plant bioactive organic matter from antibiotic fermentation residue, characterized in that, After mixing antibiotic fermentation residue with water, pyrolusite is added, and a first hydrothermal reaction is carried out at 160℃~200℃. The mass ratio of antibiotic fermentation residue to pyrolusite is (50~60):

1. The pyrolusite is obtained by a second hydrothermal reaction of potassium permanganate solid, toluene, and deionized water. The mass ratio of potassium permanganate solid, toluene, and water is (1~2):(2.4~3.0):(38~40). The temperature of the second hydrothermal reaction is 175℃~185℃, and the reaction time is 24h~28h. The antibiotics in the antibiotic fermentation residue include lincomycin antibiotics or macrolide antibiotics.

2. The method as described in claim 1, characterized in that, The moisture content of the antibiotic fermentation residue is 60%~65%.

3. A liquid ecological fertilizer, characterized in that, After centrifuging the reactants obtained by the method as described in any one of claims 1-2, the supernatant is taken to obtain liquid ecological fertilizer.

4. The liquid ecological fertilizer as described in claim 3, characterized in that, The liquid ecological fertilizer contains fulvic acid and humic acid substances.

5. A method for cultivating bok choy, characterized in that, The liquid ecological fertilizer according to any one of claims 3-4 is used to hydroponically cultivate Chinese cabbage seedlings.

6. The method for cultivating bok choy as described in claim 5, characterized in that, Promotes root growth in bok choy seedlings.

Images