A polylysine-modified biochar and its preparation method and application

Abstract

A polylysine modified biochar and its preparation and application belong to the field of urban surface water treatment technology. The present invention is to solve the microbial contamination and potential public health risks caused by the presence of multiple pathogenic pathogens in existing surface water bodies. The method of the present invention: Step 1, disperse the carbon-rich solid in a Tris-HCl buffer solution, add ε-polylysine and dopamine hydrochloride, use NaOH solution to adjust the pH value of the solution to alkaline, stir the reaction at room temperature and ensure that there is sufficient dissolved oxygen in the solution; Step 2, after the reaction of step 1 is completed, vacuum filter, wash the collected solid with ultrapure water 5 to 8 times, dry at 50°C to 70°C and then sieve. The modified biochar prepared by the present invention can achieve efficient adsorption and removal of bacteria and viruses in water bodies in a short time, shows good removal effect and stability, is non-toxic and harmless to aquatic organisms and the ecological environment, and provides a basis for the design of environmentally friendly pathogen adsorption materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of urban surface water treatment, and specifically relates to a method for preparing modified biochar and using the same to adsorb and remove pathogens such as bacteria and viruses in water. Background Art

[0002] Waterborne and foodborne pathogen contamination of drinking and irrigation water poses a threat to human health. Pathogens (including bacteria, viruses, rickettsiae, fungi, and parasites) are significant surface water contaminants, and their presence can be inferred by elevated fecal indicator bacteria (FIB) concentrations. Populations most sensitive to pathogen contaminants include the elderly, children, and immunocompromised individuals. These individuals are not only at increased risk of infection but also experience more severe symptoms. Natural particles in water can create a protective shield for microorganisms through adsorption or coating, shielding them from disinfectants. This provides additional protection for microorganisms and facilitates viral latency. Viruses are generally believed to survive longer in aquatic environments than bacteria, yet many current technologies for bacterial inactivation in water often perform poorly against viral contaminants. Given the disinfection challenges posed by microbial contamination, the development of methods for the simultaneous removal of bacterial and viral pathogens from aquatic environments is essential. Summary of the Invention

[0003] The present invention aims to solve the problems of existing water disinfection technology, such as strong background organic matter interference, low virus removal rate, and high bacterial regeneration rate, and provides an environmentally friendly modified biochar prepared in one step and a preparation method, and a method for using the same to adsorb and remove pathogens in water.

[0004] The present invention discloses a polylysine-modified biochar, which is modified by a one-step method and consists of a positively charged coating layer and a carbon-rich solid. The positively charged coating layer comprises polydopamine and ε-polylysine, and the carbon-rich solid is a solid product generated by high-temperature thermal cracking of biomass (cellulose) in an oxygen-deficient or anaerobic environment. The particle size of the modified biochar is 10 to 200 meshes.

[0005] Principle: Biochar not only has high porosity, cation exchange capacity, huge surface area and nutrients, but also has a large amount of surface negative charge and high charge density. These surface properties make biochar have good adsorption properties. Existing studies have shown that biochar has a strong adsorption effect on heavy metals and ammonia nitrogen cations in solution. The enhanced pathogen removal ability of the modified biochar of the present invention is attributed to the fact that the positively charged polydopamine and ε-polylysine coated on the surface of the biochar reduce the negative Zeta potential of the biochar, improve its adsorption capacity for bacteria and viruses that usually have negative surface charges in surface water (electrostatic effects, hydrophobicity and spatial interactions), and because the rougher and irregularly shaped surface of the biochar is more conducive to the attachment of bacteria and viruses, and the chemical changes in the solution caused by the addition of biochar lead to changes in the Zeta potential of bacteria and viruses to a certain extent. At the same time, the antibacterial activity of ε-polylysine based on the destruction of the cell membrane structure of microorganisms leads to the death of Escherichia coli, which helps to improve the microbial removal rate of ε-polylysine modified biochar.

[0006] In order to achieve the above-mentioned object, the technical solution of the present invention is as follows: a polylysine-modified biochar is composed of a positively charged coating layer and a carbon-rich solid, wherein the materials of the positively charged coating layer are polydopamine and ε-polylysine, and the specific preparation method comprises the following steps: step 1, dispersing the carbon-rich solid in a Tris-HCl buffer solution, adding ε-polylysine hydrochloride and dopamine hydrochloride, adjusting the pH value of the solution to alkaline using a NaOH solution, and stirring the reaction at room temperature, wherein the dissolved oxygen in the solution is sufficient during the entire reaction process;

[0007] After the reaction in step 2 and step 1 is completed, vacuum filtration is performed, and the collected solid is washed with ultrapure water 5 to 8 times, dried at 50° C. to 70° C., and then sieved to obtain polylysine-modified biochar.

[0008] It is further defined that the carbon-rich solid is generated by high-temperature thermal cracking of biomass in an oxygen-deficient or anaerobic environment.

[0009] It is further defined that the carbon-rich solid is prepared by the following steps: 50 g of biomass is heated from room temperature to 700° C. at a rate of 5° C. / min in a tube furnace in an anaerobic or anaerobic environment, maintained for 6 hours, and then taken out and sieved after thorough cooling.

[0010] As a material with a wide range of sources, low cost and high adsorption capacity, biochar has been widely used in agriculture, environmental remediation and climate regulation. Biochar is a heterogeneous solid product obtained by high-temperature pyrolysis of plant biomass (such as straw, rice husks, sawdust, fruit peels, beetroots, and sugarcane bagasse) in an anoxic or anaerobic environment. The properties and specific composition of biochar depend on the raw materials and pyrolysis conditions. Its pyrolysis products contain a large amount of oil, tar, salt, metals, ash and aromatic compounds. Biochar is an inexpensive, easily available and environmentally friendly material. Its preparation process is a reprocessing of waste. Whether it is the biofuel produced by the preparation process or the application of biochar in agriculture, water treatment and greenhouse gas emission reduction, there is huge potential for development, application and research.

[0011] It is further defined that the biomass is cellulose, such as straw, rice husk, wood chips, fruit peel, beetroot, and sugarcane bagasse.

[0012] It is further defined that in step 1, 1 g of the carbon-rich solid is dispersed in a 10 mM Tris-HCl buffer solution, and 0.5 g of ε-polylysine hydrochloride and 0.3 g of dopamine hydrochloride are added.

[0013] It is further defined that in step 1, the pH value is adjusted to 8.5.

[0014] It is further defined that in step 2, drying is performed at 50°C to 70°C.

[0015] It is further defined that in step 2, the product is passed through a 200-mesh sieve.

[0016] The polylysine-modified biochar or the polylysine-modified biochar prepared by any of the above methods is used to capture bacteria and viruses in water.

[0017] The modified biochar has positive charge and can adsorb various bacteria and viruses that are usually negatively charged. Since the polylysine used has strong positively charged side chains and high isoelectric points, as well as excellent properties such as high safety, good antibacterial effect, broad antibacterial spectrum and high temperature resistance, the biochar modified by it will also have the ability to bind to bacteria and viruses and exhibit antibacterial efficacy.

[0018] The modified biochar prepared in the present invention has high efficiency in adsorbing and removing bacteria and viruses, and the removal rate of bacteria and bacteriophages in the suspended supernatant is higher than 90%, which is beneficial to reducing microbial health risks.

[0019] The present invention changes the negative charge of the original biochar surface by polylysine modification in a one-step process to reduce the energy barrier for biochar to adsorb bacteria and viruses. The preparation method has low cost, good stability, environmental friendliness, and is simple to prepare and operate.

[0020] The modified substance used in the present invention is a polylysine antibacterial substance that is harmless to the human body. Even if a trace amount of the modifier on the surface of the biochar is dissolved, it will not cause secondary pollution to the environment and can be absorbed and utilized by the human body. It has the advantages of high safety, good antibacterial effect and high temperature resistance.

[0021] The present invention utilizes biomass waste to prepare biochar, which can not only reduce waste emissions and alleviate environmental pollution caused by waste combustion, but also promote the sustainable development of various industries and improve the additional economic benefits of biomass.

[0022] The preparation method of the present invention has the advantages of simple process, convenient operation, low requirements on equipment and reaction parameters, etc., is suitable for large-scale preparation, and is conducive to industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a scanning electron microscope (SEM) image of the 1-0.5-0.3 biochar prepared in Example 1;

[0024] Figure 2 The following are the Fourier transform infrared spectroscopy (FTIR) test results of the biochar prepared in Example 1 before and after modification;

[0025] Figure 3 The MS2 phage removal kinetics results of the biochar prepared in Example 1 before and after modification;

[0026] Figure 4 The kinetic results of E. coli removal by the biochar prepared in Example 1 before and after modification are shown;

[0027] Figure 5 This is the removal effect of 1-0.5-0.3 biochar prepared in Example 1 on different concentrations of MS2 phage and E. coli for 6 h;

[0028] Figure 6 is the MS2 phage removal effect of the 1-0.5-0.3 biochar prepared in Example 1 in simulated surface water;

[0029] Figure 7 This is the E. coli removal effect of the 1-0.5-0.3 biochar prepared in Example 1 in simulated surface water. DETAILED DESCRIPTION

[0030] Specific embodiment 1: This embodiment is a polylysine-modified biochar. The biochar modified in one step consists of a positively charged coating layer and a carbon-rich solid. The positively charged coating layer involves polydopamine and ε-polylysine. The carbon-rich solid is a solid product generated by high-temperature thermal cracking of biomass (cellulose) in an oxygen-deficient or anaerobic environment. The particle size of the modified biochar is 10 mesh to 200 mesh. Specifically, 50g of cellulose (65μm) is heated at 700℃ for 6h in a tube furnace at a heating rate of 5℃ / min under oxygen-restricted conditions and passed through a 200-mesh sieve to obtain the original biochar. 1g of biochar is dispersed in a 10mM Tris-HCl buffer solution, 0.5g of ε-polylysine hydrochloride and 0.3g of dopamine hydrochloride are added, and the pH of the solution is adjusted to 8.5 with NaOH. The reaction is stirred at room temperature for 24h. The solution is exposed to oxygen throughout the reaction to ensure sufficient dissolved oxygen. After the reaction was complete, the composite was collected by vacuum filtration and washed 5-8 times with ultrapure water. After drying at 50-70°C, it was passed through a 200-mesh sieve to obtain a polydopamine-polylysine synergistically modified biochar, designated 1-0.5-0.3. Correspondingly, biochars containing either ε-polylysine or dopamine hydrochloride alone were prepared, designated 1-0.5 and 1-0.3, respectively. The pathogen removal efficacy of modified biochars containing different ε-polylysine hydrochloride dosages (0.25, 0.5, 1.0, and 1.5 g) was also investigated.

[0031] Table 1 Comparative effect of pathogen removal on biochar modified with different ε-polylysine ratios

[0032]

[0033] The modified biochar described in this embodiment is produced through a one-step modification process. Dopamine undergoes a self-polymerization reaction on the particle surface in a weakly alkaline, dissolved oxygen-containing aqueous solution to form a polydopamine coating. Dopamine can act as an intermediate layer to react with the primary amines on polylysine through Michael addition or Schiff base reaction to graft polylysine functional molecules. The above modification process changes the negative charge and hydrophobicity of the original biochar surface to reduce the energy barriers (electrostatic effects, hydrophobicity, and spatial interactions) for biochar adsorption of bacteria and viruses. The rougher and irregularly shaped surface of the biochar and the macroporous spaces introduced by the coating layer are conducive to the attachment of bacteria and viruses. The chemical changes in the solution caused by the addition of biochar lead to changes in the Zeta potential of bacteria and viruses to a certain extent, which helps to improve the microbial removal rate of ε-polylysine-modified biochar.

[0034] The modified biochar agent described in this embodiment uses the environmentally friendly positively charged antibacterial substance polylysine and the main biomass "cellulose" in waste crop residues. It can not only guide the resource utilization of agricultural waste and reduce the environmental pollution caused by waste combustion, but also promote the sustainable development of various industries and increase the additional economic benefits of biomass.

[0035] Specific embodiment 2: A method for capturing bacteria and viruses in water using the modified biochar prepared in embodiment 1 is specifically completed by the following steps:

[0036] 1) MS2 phage and E. coli culture: MS2 stock culture and E. coli cells were added to Luria Broth medium at a ratio of 1:1 and cultured at 37°C and 150 rpm for 24 hours. E. coli cells and cell debris were removed by centrifugation at 10,000 × g for 20 minutes, and the supernatant was filtered through a 0.22 μm filter to obtain MS2 phage stock solution, which was stored in a refrigerator at ~4°C until use. E. coli was cultured in sterile Luria Broth medium at 37°C and 200 rpm for 16 hours, then transferred to a sterile centrifuge tube and centrifuged at 3000 rpm for 15 minutes. The supernatant was discarded, and physiological saline was added to shake and disperse the precipitate, and then centrifuged twice. Finally, the precipitate was resuspended in physiological saline and stored in a refrigerator at ~4°C until use. 2) Biochar adsorption of phage / E. coli: 0.5 g of original biochar / modified biochar was mixed with 300 mL of sterile pure water containing MS2 phage (concentration of ~1.0×10 6 PFU / mL) were mixed in a 500 mL wide-mouth bottle and stirred at 380 rpm for 6 h. 1.0 mL of sample solution was taken at a series of time intervals (0 / 15 / 30 / 60 / 120 / 240 / 360 min) and filtered through a 0.22 μm nylon membrane filter to retain biochar particles and impurities. The filtrate was diluted according to a certain concentration gradient and counted on MS2 double-layer agar plates. 4 , 1.0×10 5 , 1.0×10 6 , 1.0×10 7 , 1.0×10 8 The phage suspension with PFU / mL was mixed with the modified biochar and stirred for 6 h, and then the same sampling, dilution and counting operation as above was performed. 0.5 g of original biochar / modified biochar was added to a 500 mL wide-mouth bottle, and 300 mL of Escherichia coli suspension (concentration of 1.0×10 7CFU / mL). Subsequently, the sealed jar was placed in a constant temperature shaker at 25°C and 200 rpm for 6 hours. 1.0 mL of the suspension was taken at a series of time intervals (0 / 15 / 30 / 60 / 120 / 240 / 360 min) and filtered through a 10 μm nylon membrane filter to retain biochar particles and impurities. The bacterial concentration in the filtrate was calculated by plate count. The bacterial adsorption capacity was obtained based on the initial bacterial concentration and the bacterial concentration in the suspension phase after adding biochar for a certain period of time. 4 , 1.0×10 5 , 1.0×10 6 , 1.0×10 7 , 1.0×10 8 ±10% CFU / mL E. coli suspension was mixed with modified biochar and shaken for 6 h before sampling, dilution and counting as above. 3) Application of modified biochar in simulated surface water: The original biochar or modified biochar was dispersed in 300 mL of sterile pure water, and then 0.3 mL of phage or E. coli stock solution was added to the suspension to ensure that the final concentration of MS2 phage or E. coli was 1.0 × 10 6 PFU / mL, ~1.0×10 7 By introducing three types of natural organic matter to simulate actual surface water, the effects of competitive adsorption of natural organic matter (humic acid, bovine serum albumin, and sodium alginate, with concentration gradients of 1, 2, 4, and 6 mg / L, calculated as TOC) coexisting in actual surface water on the capture of bacteriophages and Escherichia coli were studied.

[0037] The modified biochars with different ε-polylysine ratios prepared in the first embodiment were investigated for their ability to inhibit MS2 phage (~10 6 PFU / mL) and Escherichia coli (~10 7 The results showed that the 6-hour removal effect of biochar was 1-0.5-0.3, which showed the best removal effect for MS2 bacteriophage and Escherichia coli at higher concentrations. The excess ε-polylysine at high dosage directly coupled with polydopamine in the liquid phase instead of being loaded on the biochar, which led to the deterioration of its anti-pathogen effect. The original biochar, 1-0.5-0.3, 1-0.5 and 1-0.3 biochars were further selected for Fourier transform infrared spectroscopy testing ( Figure 1 ) to detect the functional group distribution of each biochar. FTIR spectroscopy showed that there were abundant functional groups (such as C=C, C=O, -COOH, CO, and CH) on the surface of the original biochar. However, the degree of dehydration of the biochar was high at a pyrolysis temperature of 700°C, specifically at 730-885 cm -1 The peak at (CH group) is weak and 1700~1725cm -1The peak at (-COOH group) disappears [4] , and aromatic C=O(1631cm -1 )、C=C(1520~1600cm -1 ) peak is more obvious. The main infrared absorption peak of polydopamine is between 1200 and 1600 cm -1 The thickness of the polydopamine layer coated on the substrate surface is only 40-50 nm, and its content in the whole sample is relatively low. Therefore, the infrared spectrum of the biochar coated with polydopamine only (1-0.3) is still dominated by the infrared absorption of biochar. The characteristic peaks in the spectrum of the biochar synergistically coated with ε-polylysine and polydopamine (1-0.5-0.3) are enhanced, and three obvious characteristic peaks appear at 3429 cm -1 (overlapping peaks of -NH2, -NH- and -OH group stretching vibrations), 950cm -1 (NH out-of-plane deformation vibration peak) and 1631 cm -1 (C=O stretching vibration), indicating that both ε-polylysine and polydopamine were successfully coated on the biochar surface.

[0038] In the second embodiment, the biochar adsorption of bacteriophages / killing of E. coli was tested in sterilized water and simulated surface water. Figure 2 It can be seen that the original biochar has an adsorption removal rate of only 1.74LRV for MS2 phage, and desorption occurs over time, indicating that the original biochar has only a weak binding force with MS2 phage. The biochar (1-0.3) coated only with polydopamine has a much improved hydrophilicity, but its adsorption effect on hydrophobic phages is worsened. The polylysine-modified biochar (1-0.5) has a good adsorption effect on phages due to the increase in positive charge. The polydopamine-polylysine synergistically modified biochar (1-0.5-0.3) showed 100% adsorption removal, that is, a 6.52LRV removal rate. Dopamine undergoes self-polymerization reaction on the surface of the particles in a weakly alkaline, dissolved oxygen-containing aqueous solution to form a polydopamine coating, and the rich amine groups on the ε-polylysine chain are covalently bound to the dopamine coating surface through the Schiff base reaction, thereby promoting the positive charge of the biochar surface and the formation of a stable complex. By Figure 3 It can be seen that for high concentration E. coli suspension (~1.0×10 7 CFU / mL), the adsorption and bactericidal effect of 1-0.5-0.3 biochar increased with time, and the removal rate was 1.61LRV at 6 h, which showed good antibacterial potential. Figure 4 The adsorption and removal effects of 1-0.5-0.3 biochar on different concentrations of MS2 phage / E. coli are shown. It can be seen that the adsorption and removal effects of 1-0.5-0.3 biochar on ~1.0×10 6The MS2 phages below the PFU / mL concentration were all 100% removed by adsorption, while for the MS2 phages at the concentration of ~1.0×10 7 PFU / mL of MS2 can still achieve a 6.13LRV removal rate; 1-0.5-0.3 biochar for ~1.0×10 4 The antibacterial effect on Escherichia coli below the concentration of PFU / mL is excellent, and the antibacterial effect on Escherichia coli is excellent. 5 coli with a CFU / mL still has a 3.73LRV removal rate, proving that it has high application potential for harmful bacteria in the common concentration range of surface water.

[0039] The 1-0.5-0.3 biochar was further used to remove pathogens in simulated surface water in the presence of organic pollutants. Figure 5 As shown in the figure, even with the interference of bovine serum albumin and sodium alginate, the removal rates of at least 4.90LRV and 4.56LRV can still be achieved, which proves that these two types of organic matter have weak competitive adsorption on the active sites on the modified biochar. However, negatively charged humic acid has a strong binding force with the modified biochar, and high concentrations of humic acid inhibit the adsorption and removal effect of MS2 bacteriophage. The results for Escherichia coli are shown in the figure. Figure 6 As shown, the presence of NOM improves the adsorption efficiency of modified biochar for E. coli to a certain extent. This is attributed to the fact that the natural organic matter adheres to the modified biochar surface, further expanding the attachment sites of E. coli to the surface NOM. Unlike humic acid and bovine serum albumin, sodium alginate has a hydrophilic surface, which slightly reduces the adhesion of E. coli.

[0040] The modified biochar prepared in this embodiment has a high pathogen adsorption efficiency, with an adsorption rate of 100% for MS2 bacteriophage and an antibacterial rate of over 90% for E. coli, ensuring the biosafety of water bodies. This embodiment uses a polydopamine interlayer to reduce the hydrophobicity and micropore exposure of the biochar, achieving better dispersion in water bodies and expanding the mesoporous channels for pathogen adsorption. The polylysine loading enhances the biochar's positive charge and antibacterial properties, improves the electrostatic adsorption efficiency of bacteriophages, and enhances the sterilization efficiency by attacking the outer membrane of E. coli.

[0041] The biochar prepared in this embodiment is less susceptible to interference from common natural organic matter in surface water bodies, and its MS2 phage adsorption and Escherichia coli antibacterial effects can still reach a relatively good level. It also shows stable efficacy against pathogens in different concentration ranges and has wide applicability to surface water bodies with different biological risks.

Claims

1. A polylysine modified biochar, characterized in that The modified biochar is composed of a positively charged coating layer and a carbon-rich solid. The positively charged coating layer is made of polydopamine and ε-polylysine. The carbon-rich solid is a solid product generated by high-temperature thermal cracking of biomass in an oxygen-deficient or anaerobic environment. The carbon-rich solid is prepared according to the following steps: 50 g of biomass is heated from room temperature to 700° C. at a rate of 5° C. / min in an oxygen-deficient or anaerobic environment in a tube furnace, maintained for 6 hours, and then removed and sieved after thorough cooling. The modified biochar is prepared by the following steps: Step 1: Disperse the carbon-rich solid in a Tris-HCl buffer solution, add ε-polylysine hydrochloride and dopamine hydrochloride, adjust the pH value of the solution to alkaline using a NaOH solution, and react by stirring at room temperature. During the entire reaction process, there is sufficient dissolved oxygen in the solution; After the reaction in step 2 and step 1 is completed, vacuum filtration is performed, and the collected solid is washed with ultrapure water 5 to 8 times, dried at 50° C. to 70° C., and then sieved to obtain polylysine-modified biochar.

2. The modified biochar according to claim 1, characterized in that The biomass is cellulose.

3. The method for preparing modified biochar according to any one of claims 1 or 2, characterized in that The preparation method is carried out according to the following steps: Step 1: Disperse the carbon-rich solid in a Tris-HCl buffer solution, add ε-polylysine hydrochloride and dopamine hydrochloride, adjust the pH value of the solution to alkaline using a NaOH solution, and react by stirring at room temperature. During the entire reaction process, there is sufficient dissolved oxygen in the solution; After the reaction in step 2 and step 1 is completed, vacuum filtration is performed, and the collected solid is washed with ultrapure water 5 to 8 times, dried at 50° C. to 70° C., and then sieved to obtain polylysine-modified biochar.

4. The preparation method according to claim 3, characterized in that In step 1, 1 g of carbon-rich solid was dispersed in 10 mM Tris-HCl buffer solution, and 0.5 g of ε-polylysine hydrochloride and 0.3 g of dopamine hydrochloride were added.

5. The preparation method according to claim 3, characterized in that In step 1, the pH is adjusted to 8.

5.

6. The preparation method according to claim 3, characterized in that In step 2, pass through a 200-mesh sieve.

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