Application of soluble carbon black in promoting conversion of agaricus bisporus fungus laccase into sulfonamide antibiotics

Abstract

The invention provides a method for preparing soluble carbon black from biomass and application of the soluble carbon black in promoting agaricus bisporus fungus laccase to convert sulfonamide antibiotics. The method for preparing the soluble carbon black from the biomass comprises the following steps: carrying out lower limit oxygen treatment on bamboo and wood chip biomass at the pyrolysis temperature of 300 DEG C for 3 hours to obtain bamboo and wood chip biochar, mixing the biochar with deionized water, carrying out ultrasonic treatment for 15 minutes, heating in a water bath, stirring for 45 minutes, repeating for 5 times, and carrying out suction filtration on the obtained liquid through a 0.45 mu m water-phase filter membrane to obtain the soluble carbon black. The obtained soluble carbon black can promote agaricus bisporus fungus laccase to degrade the sulfonamide antibiotics so as to effectively degrade the sulfonamide antibiotics in soil. Meanwhile, the soluble carbon black is wide in source, the preparation process is simple, and popularization and application in actual soil and water body remediation are facilitated.

Description

Technical Field

[0001] The invention relates to application of soluble carbon black in promoting Agaricus bisporus laccase to convert sulfonamide antibiotics. Background Art

[0002] Sulfonamide antibiotics are among the most widely used veterinary antibiotics in the world. They enter the soil through grazing livestock or the application of manure, and a large portion is retained in the soil. Sulfonamide antibiotics mainly include sulfamethoxazole, sulfamethoxazole, sulfadiazine, and sulfisoxazole. Due to the strong polarity and low volatility of antibiotics, they easily enter the soil environment. Sulfonamide antibiotics may pose a potential threat to human health through the food chain. Sulfonamide antibiotics have been reported to cause hypothyroidism and allergic hypersensitivity reactions. Therefore, effective and economical methods are needed to remove sulfonamide antibiotics to avoid environmental degradation and public health problems.

[0003] Laccases are abundant extracellular multi-copper oxidoreductases found in fungi and have been extensively studied. Laccases typically contain four copper catalytic centers: type I Cu2+ (T1 Cu) and type II Cu2+ (T2 Cu), both of which contain a single copper atom as a single-electron acceptor and exhibit paramagnetic properties. Type III Cu24+ (T3 Cu) contains two copper atoms, which act as diamagnetic two-electron acceptors. The substrate is oxidized by the T1 copper. Electrons are transferred to the T2 / T3 copper via a strongly conserved His-Cys-His tripeptide motif, where molecular oxygen is reduced. Laccases catalyze the one-electron oxidation of four substrate molecules while simultaneously reducing molecular oxygen to water. Several studies have demonstrated that extracellular laccase-driven autopolymerization can effectively eliminate pollutants from soils through free radical-activated C-C or CO covalent binding mechanisms.

[0004] Carbon black is a carbonaceous residue formed by the incomplete combustion of biomass or fossil fuels. It can release a water-soluble fraction, commonly referred to as dissolved carbon black. Dissolved carbon black is rich in aromatic domains and oxygen-containing functional groups, making it a good adsorbent for organic compounds and metals. Therefore, dissolved carbon black can act as a carrier for pollutants, influencing their fate and transport in the environment. Studies have shown that dissolved organic matter in biochar can retain hydrophobic pollutants, and the risk, transport, and bioavailability of such pollutants in the environment are significantly influenced by the quality of dissolved organic matter. Compared to dissolved organic matter in the natural environment, dissolved carbon black has a higher degree of fused rings but also contains a variety of chemical components with different properties and structures. Dissolved carbon black, produced from carbon black, has a strong ability to transport pollutants in the environment. Dissolved carbon black can be rapidly transported to soils receiving surface and groundwater through surface runoff, leaching, and infiltration, thereby affecting the fate and transport of certain nutrients and pollutants in the soil and altering the surrounding aquatic and soil environments. Similar to dissolved organic matter, dissolved carbon black is adsorbed onto suspended solids and particulate organic matter. Soluble carbon black can influence the adsorption behavior of organic pollutants and heavy metals through its own adsorption. The binding mechanism between DBC and organic pollutants is due to hydrophobic interactions between the aliphatic carbon chains, aromatic rings, and quinone or ester groups contained in the soluble carbon black and phenanthrene, as well as π-π electron donor-acceptor (EDA) interactions. DBC is rich in active functional groups such as aromatic, carboxyl, and hydroxyl groups, acting as a natural ligand and carrier, influencing the migration of heavy metals and organic pollutants in the environment.

[0005] In this invention, it is found that the presence of soluble carbon black significantly promotes the conversion of sulfonamide antibiotics by Agaricus bisporus fungus laccase, and can effectively improve the removal of sulfonamide antibiotics in soil. Summary of the Invention

[0006] The purpose of the present invention is to prepare soluble carbon black from biomass, so as to promote the degradation and removal of sulfonamide antibiotics by Agaricus bisporus fungus laccase.

[0007] The technical solution of the present invention:

[0008] A method for preparing soluble carbon black from biomass, comprising the following steps:

[0009] Step 1: The bamboo and sawdust biomass were air-dried, impurities were removed, and the biomass was crushed with a grinder, passed through an 80-mesh sieve, and heated at a pyrolysis temperature of 300°C with nitrogen for 3 hours to produce biochar;

[0010] Step 2: Weigh 25g of the prepared 300°C bamboo and 300°C sawdust biochar into a beaker, add 500mL of deionized water, and ultrasonicate for 15 minutes in an ultrasonic cleaner. Then, heat in a water bath at 40°C and stir for 45 minutes. Repeat this process five times. Pour the liquid obtained in the above steps into a filter, filter it with suction, and filter it through a 0.45μm filter membrane. Refrigerate the filtered liquid. The filtrate is the dissolved carbon black.

[0011] Step 3: Use a total organic carbon analyzer (TOC) to detect the total organic carbon content in the dissolved carbon black, thereby determining the organic carbon concentration of the extracted dissolved carbon black and diluting it to the required concentration range (about 50 mg / L).

[0012] The soluble carbon black prepared above is applied to the reaction of Agaricus bisporus fungus laccase degrading sulfonamide antibiotics, and the steps are as follows:

[0013] Step 1: Prepare 50 mg / L sulfamethoxazole and sulfamethoxazole pollutant solutions using 0 mg / L (organic carbon concentration), 10 mg / L, 25 mg / L, and 50 mg / L dissolved carbon black from bamboo and sawdust as biomass as background liquids, ultrasonicate for 15 minutes, and then transfer to an oscillator at 120 rpm in the dark and oscillate until completely dissolved.

[0014] Step 2: After shaking, perform oxygenation. After oxygenation, take 38 mL of the dissolved sample and 2 mL of 400 mg / L laccase solution into a 40 mL brown glass bottle and leave it uncovered.

[0015] Step 3: Sampling: Take 12 samples at 10 min, 20 min, 30 min, 45 min, 60 min, 80 min, 100 min, 120 min, 150 min, 180 min, 300 min and 480 min, each time sampling 1 mL, and filter through a 0.45 μm aqueous phase filter membrane and place into a 2 mL brown liquid vial.

[0016] Step 4: Termination of the reaction: Methanol was used to terminate the enzymatic reaction, and the concentrations of sulfamethoxazole and sulfamethoxazole were determined by high performance liquid chromatography (HPLC).

[0017] The present invention provides a method for producing soluble carbon black from biomass, comprising the following steps: subjecting bamboo and sawdust biomass to oxygen restriction at 300°C for 3 hours to produce bamboo and sawdust biochar; mixing the biochar with deionized water, ultrasonicating it for 15 minutes, heating it in a water bath with stirring for 45 minutes, and repeating this process five times; and filtering the resulting liquid through a 0.45 μm filter membrane to produce soluble carbon black. The soluble carbon black obtained by the present invention has humic and fulvic acid-like structures, which can promote the degradation of sulfonamide antibiotics by Agaricus bisporus laccase, thereby effectively degrading sulfonamide antibiotics in soil. Furthermore, the soluble carbon black is widely available and has a simple preparation process, facilitating its widespread application in practical soil remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the total amount of organic carbon black in preparing soluble carbon black with four different solid-liquid ratios.

[0019] Figure 2 is the total organic carbon black content of the dissolved carbon black extracted five times.

[0020] Figure 3(a) is the UV-visible absorption spectrum of dissolved carbon black in bamboo biochar.

[0021] Figure 3(b) shows the UV-visible absorption spectrum of dissolved carbon black in sawdust biochar. Figure 4(a) shows the conversion of sulfamethoxazole by Agaricus bisporus fungus laccase mediated by different concentrations of bamboo dissolved carbon black.

[0022] Figure 4(b) shows the conversion of sulfamethoxazole by Agaricus bisporus fungus laccase under different concentrations of bamboo-soluble carbon black. DETAILED DESCRIPTION

[0023] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0024] Example 1: Method for preparing soluble carbon black from biomass.

[0025] (1) The bamboo and sawdust biomass were dried naturally, impurities were removed, crushed with a grinder, passed through an 80-mesh sieve, and heated at a pyrolysis temperature of 300°C with nitrogen for 3 h to produce biochar;

[0026] (2) Weigh 5g, 10g, 20g, and 25g of the prepared 300℃ bamboo and 300℃ sawdust biochars into a beaker, add 500mL of deionized water, and place them in an ultrasonic cleaner for 15 minutes. Then heat them in a water bath at 40℃ and stir them for 45 minutes. Repeat the above operation 5 times. Pour the liquid obtained in the above steps into a filter device for suction filtration and filter it through a 0.45μm filter membrane. The liquid obtained after filtration is stored in a refrigerator. The filtrate obtained is the soluble carbon black.

[0027] (3) Use a total organic carbon analyzer (TOC) to detect the total organic carbon content in the dissolved carbon black, so as to determine the organic carbon concentration of the extracted dissolved carbon black, such as Figure 1 The total organic carbon black content of dissolved carbon black prepared with four different solid-to-liquid ratios is shown.

[0028] (4) Select a suitable solid-liquid ratio of 1:20 to prepare soluble carbon black. Weigh 25 g of the prepared 300 °C bamboo and 300 °C sawdust biochar into a beaker, add 500 mL of deionized water, and place it in an ultrasonic cleaner for 15 minutes. Then heat it in a water bath at 40 °C and stir it for 45 minutes. Repeat the above operation 5 times. Filter the liquids obtained in the 5 steps through a 0.45 μm filter membrane respectively. Use a total organic carbon analyzer (TOC) to detect the total organic carbon content in the soluble carbon black, so as to determine the organic carbon concentration of the soluble carbon black extracted each time, such as Figure 2 Total organic carbon black content of dissolved carbon black for the five extractions shown.

[0029] Example 2: UV-visible absorption spectra of dissolved carbon black in bamboo and sawdust biochars.

[0030] (1) Dissolved carbon black was diluted to 50 mg / L with deionized water. 3.5 mL of dissolved carbon black was added to a quartz cuvette (10 × 10 × 39 mm). The UV spectrum was measured using a UV spectrophotometer (UV-2600, Shimadzu, Japan). Ultrapure water was used as a blank background, and the reference blank was subtracted from the measured absorbance. The measurement range was set to 200–800 nm, the sampling interval was 1 nm, and the scanning speed was medium.

[0031] (2) Figure 3(a) shows the UV spectra of dissolved carbon black in bamboo biochar at 300°C, and Figure 3(b) shows the UV spectra of dissolved carbon black in sawdust biochar at 300°C. It can be seen that the absorption peak intensities of the two types of dissolved carbon black in the wavelength range of 190-800 nm are ranked as follows: dissolved carbon black from bamboo biomass (2.12) > dissolved carbon black from sawdust biomass (1.48). From the absorption values, it can be seen that the maximum UV absorption peak of dissolved carbon black from bamboo occurs near 195 nm. At the same time, it was found that the overall peak shape of the UV absorption peaks of dissolved carbon black from two different sources is relatively broad, with one maximum absorption peak. As the wavelength increases, the absorption values of the UV-visible absorption spectra of the two types of dissolved carbon black all decrease, which is very similar to the widely studied humic substances. The UV-visible spectrum can effectively reflect the relevant molecular structure of dissolved carbon black, the absorption value per unit concentration, or the ratio between the absorption values at specific wavelengths.

[0032] Example 3: Application of soluble carbon black prepared from biomass to the degradation of sulfonamide antibiotics by Agaricus bisporus fungus laccase.

[0033] (1) 50 mg / L sulfamethoxazole and sulfamethoxazole pollutant solutions were prepared using 0 mg / L (organic carbon concentration), 10 mg / L, 25 mg / L, and 50 mg / L soluble carbon black from bamboo and sawdust biomass as background solutions. After ultrasonication for 15 min, the solution was transferred to an oscillator at 120 rpm in the dark and oscillated until completely dissolved.

[0034] (2) After shaking, perform oxygenation treatment. After oxygenation treatment, take 38 mL of the dissolved sample and 2 mL of 400 mg / L laccase solution into a 40 mL brown glass bottle and leave it open.

[0035] (3) Sampling: Twelve samples were taken at 10 min, 20 min, 30 min, 45 min, 60 min, 80 min, 100 min, 120 min, 150 min, 180 min, 300 min, and 480 min, respectively. Each sample was 1 mL. The samples were filtered through a 0.45 μm aqueous filter membrane and placed in a 2 mL vial.

[0036] (4) Termination of reaction: Methanol was used to terminate the enzymatic reaction, and the concentrations of sulfamethoxazole and sulfamethoxazole were determined by high performance liquid chromatography (HPLC).

[0037] The above experiments measured that after the addition of soluble carbon black, as shown in Figure 4 (a) the conversion of sulfamethoxazole by Agaricus bisporus fungus laccase mediated by bamboo soluble carbon black at different concentrations, and Figure 4 (b) the conversion of sulfamethoxazole by Agaricus bisporus fungus laccase mediated by bamboo soluble carbon black at different concentrations, the efficiency of converting sulfamethoxazole by Agaricus bisporus fungus laccase was enhanced by 14.96%; the efficiency of converting sulfamethoxazole was enhanced by 13.39%, and the promotion effect increased with the concentration of soluble carbon black.

Claims

1. A method for preparing soluble carbon black from biomass, characterized in that: The following steps are involved: Step 1: The bamboo and sawdust biomass were air-dried, impurities were removed, and the biomass was crushed with a grinder, passed through an 80-mesh sieve, and heated at a pyrolysis temperature of 300°C with nitrogen for 3 hours to produce biochar; Step 2: Weigh 25g of the prepared 300°C bamboo and 300°C sawdust biochar into a beaker, add 500mL of deionized water, and ultrasonicate for 15 minutes in an ultrasonic cleaner. Then, heat in a water bath at 40°C and stir for 45 minutes. Repeat this process five times. Pour the liquid obtained in the above steps into a filter, filter it with suction, and filter it through a 0.45μm filter membrane. Refrigerate the filtered liquid. The filtrate is the dissolved carbon black. Step 3: Use a total organic carbon analyzer (TOC) to detect the total organic carbon content in the dissolved carbon black, thereby determining the organic carbon concentration of the extracted dissolved carbon black and diluting it to the required concentration range (about 50 mg / L).

2. Soluble carbon black prepared from biomass is used to degrade sulfonamide antibiotics by laccase from Agaricus bisporus fungi, characterized in that: Here are the steps: Step 1: Prepare 50 mg / L sulfamethoxazole and sulfamethoxazole pollutant solutions using 0 mg / L (organic carbon concentration), 10 mg / L, 25 mg / L, and 50 mg / L dissolved carbon black from bamboo and sawdust as biomass as background liquids, ultrasonicate for 15 minutes, and then transfer to an oscillator at 120 rpm in the dark and oscillate until completely dissolved. Step 2: After shaking, perform oxygenation. After oxygenation, take 38 mL of the dissolved sample and 2 mL of 400 mg / L laccase solution into a 40 mL brown glass bottle and leave it uncovered. Step 3: Sampling: Take 12 samples at 10 min, 20 min, 30 min, 45 min, 60 min, 80 min, 100 min, 120 min, 150 min, 180 min, 300 min and 480 min, each time sampling 1 mL, and filter through a 0.45 μm hydrophilic PTFE filter membrane and place into a 2 mL brown liquid vial. Step 4: Termination of the reaction: Methanol was used to terminate the enzymatic reaction, and the concentrations of sulfamethoxazole and sulfamethoxazole were determined by high performance liquid chromatography (HPLC).

Images