Modified biochar matrix loaded with electroactive bacteria as well as preparation method and application of modified biochar matrix

Abstract

The invention discloses a modified biochar matrix loaded with electroactive bacteria and a preparation method and application thereof, and relates to the technical field of environmental protection, and the preparation method comprises the following steps: (1) preparing a biochar carrier; 2) modifying the surface of the ferro-manganese metal oxide; and (3) carrying out attachment culture on the electroactive bacteria on the surface of the substrate. Biochar is prepared by a hydrothermal method to serve as a carrier, modification of metal oxides such as iron and manganese is carried out on the surface of the biochar, modified hydrothermal biochar is obtained, the material is used as the biochar carrier, on one hand, directional enrichment of electroactive bacteria is achieved through directional culture, on the other hand, the extracellular electron transfer process is enhanced, and the electroactive bacteria can be effectively enriched. Therefore, antibiotic degradation is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of environmental protection, and particularly relates to a modified biochar matrix loaded with electroactive bacteria and a preparation method and application thereof. BACKGROUND

[0002] With the development of industry and agriculture, new pollutants are emerging in wastewater, among which antibiotics have become a type of pollutant that is focused on. The widespread use of antibiotics in medical and livestock breeding fields has led to an increase in their concentration in the water environment, posing a potential threat to the ecological environment and human health. Antibiotics and their metabolites can disrupt the structure and function of aquatic microbial communities and interfere with the balance of aquatic ecosystems. At the same time, long-term low-concentration exposure can promote the accumulation and spread of antibiotic resistance genes, leading to new challenges to public health. However, the existing wastewater treatment processes have poor removal efficiency of antibiotics and antibiotic resistance genes, and the removal efficiency needs to be further improved.

[0003] Current treatment technologies for antibiotic degradation and removal include physical adsorption, chemical oxidation, and biological treatment. Physical adsorption mainly separates antibiotics from contaminated media through adsorbents with porous and large specific surface area. Although adsorbents such as activated carbon and resin can quickly enrich antibiotics, they need to be frequently replaced or regenerated, which is costly and cannot achieve complete degradation of pollutants. Chemical oxidation methods generally include photochemical oxidation, catalytic wet oxidation, ozone oxidation, electrochemical oxidation, and Fenton oxidation. Although chemical oxidation has high degradation efficiency, it can produce toxic by-products (such as halogenated organic compounds), and has high energy consumption and complex operation. Biological treatment is a method that uses microorganisms to produce enzymes and other substances in a specific environment, which directly or indirectly changes the structure of antibiotics, making them inactive. Traditional biological treatment methods such as activated sludge have low removal efficiency for some antibiotics (such as sulfonamides), and microorganisms are easily inhibited by antibiotics, resulting in poor system stability. However, biological treatment has advantages in environmental compatibility and long-term cost, and by adding high-efficiency degrading bacteria or electron mediators, the system performance is improved, which has become a research hotspot in recent years.

[0004] In the present invention, researchers have isolated and characterized a variety of high-efficiency antibiotic-degrading bacteria. Larcher et al. found that Rhodococcus maris (ATCC 13557) removed 29% of sulfamethoxazole (SMX) in a basic salt medium (MSM) with SMX and glucose concentrations of 6 mg / L and 0.5 g / L, respectively. Guo Xia-li et al. found that Phanerochaete chrysosporium removed 53% of sulfamethoxazole (SMX) in 50 mL of a well-defined medium with SMX concentration of 10 mg / L after 24 hours, and 71% after three days. However, due to environmental and antibiotic characteristics, it is difficult for high-efficiency bacteria to be stably enriched in the reaction system for a long time, affecting the treatment efficiency of antibiotics. In addition, electrically active bacteria have good effect on antibiotic degradation through extracellular electron transfer. Chen et al. studied the ability of Shewanella oneidensis MR-1 to degrade sulfamethoxazole (SMX) and reduce Fe3+ with anthraquinone-2,6-disulfonate (AQDS) and riboflavin. The results showed that when the initial SMX concentration was 0.04 mM, the SMX degradation rate was 38.5%. However, this method also has the above problems. Improving the degradation efficiency of strains to antibiotics can be achieved through enrichment culture and synergistic enhancement. Enrichment culture can enrich strains with stronger tolerance and higher degradation efficiency by applying selective pressure. However, due to the relatively low concentration of pollutants in the environment, this method is not very effective.

[0005] In summary, the existing antibiotic treatment technology generally has the problems of low efficiency, high cost, and high risk of secondary pollution. Although biological treatment has the advantages of low cost and environmental friendliness, it is difficult to achieve directional enrichment of high-efficiency bacteria and electrically active bacteria through selective culture due to the low concentration of antibiotics in the natural environment and the presence of other easily degradable organic matter. In addition, the extracellular electron transfer of electrically active bacteria in the natural environment is limited by the interface energy barrier and insufficient metabolic activity, making it difficult to achieve efficient removal of antibiotics.

[0006] Therefore, those skilled in the art are committed to providing a new antibiotic degradation method to achieve efficient removal of antibiotics while reducing cost and secondary pollution risk. SUMMARY

[0007] The present application provides a modified biochar matrix loaded with electrically active bacteria, as well as a preparation method and application thereof, aiming to solve the problems of low efficiency, high cost, and high risk of secondary pollution in the existing antibiotic treatment technology.

[0008] To achieve the above technical purposes, the present application mainly adopts the following technical solutions:

[0009] The application discloses a preparation method of a modified biochar matrix loaded with electroactive bacteria.

[0010] (1) preparing a biochar carrier: raw material rice husk is cleaned, dried, crushed, and sieved to obtain a biomass raw material, which is then mixed with ultrapure water and placed into a hydrothermal synthesis reaction kettle to perform a reaction in an electric heating constant-temperature air drying oven to obtain original biochar HBC;

[0011] (2) surface modification of iron-manganese metal oxides: the original biochar in step (1) is ground and sieved, iron-manganese mixed metal salt solution is added thereto, stirring is performed until the mixture is uniform, soaking is performed for a set time, then biochar powder and metal salt solution are separated by filtration, and the biochar powder is washed until the washing liquid becomes colorless, the biochar powder is collected, dried and ground, the obtained biochar powder is calcined under a nitrogen atmosphere to remove impurities and promote the combination of metal ions and biochar, and the obtained metal ion modified biochar is denoted as HBC-Fe / Mn;

[0012] (3) attachment culture of electroactive bacteria on the surface of the matrix: HBC-Fe / Mn is weighed in a transparent anaerobic bottle, LB culture medium is added, then activated and diluted electroactive bacteria Shewanella oneidensis MR-1 bacterial liquid is inoculated on the LB culture medium, the transparent anaerobic bottle is placed in a constant-temperature shaker for shaking culture under an anaerobic condition, and the biochar material is combined with the bacteria.

[0013] In the preferable embodiment of the application, in step (1), the mass ratio of the biomass raw material to the ultrapure water is 1:50, the reaction temperature is 300 DEG C, and the reaction time is 3h.

[0014] In the preferable embodiment of the application, in step (2), the iron-manganese mixed metal salt solution is a ferric chloride solution and a manganese chloride solution, and the concentration of the ferric chloride solution and the manganese chloride solution is 0.5 mol / L,

[0015] Further, the mass-volume ratio of the original biochar to the iron-manganese mixed metal salt solution is 1:20 (g / mL), the soaking time is 3h, and the calcination temperature is 300 DEG C and the calcination time is 3h.

[0016] In the preferred embodiment of the present application, in step (3), the OD600 of the diluted electrically active bacteria Shewanella oneidensis MR-1 is 0.1, the volume ratio of the diluted electrically active bacteria Shewanella oneidensis MR-1 to the LB culture medium is 1:100, and the mass-volume ratio of the HBC-Fe / Mn to the diluted electrically active bacteria Shewanella oneidensis MR-1 is 1:10 (g / mL).

[0017] In the preferred embodiment of the present application, in step (3), the shock culture condition is: shock culture at 150 rpm and 30℃ for 24 h.

[0018] The present application also discloses a modified biochar matrix loaded with electrically active bacteria prepared by the above method.

[0019] The present application also discloses an application of the modified biochar matrix loaded with electrically active bacteria in degrading antibiotics.

[0020] In the preferred embodiment of the present application, the antibiotic is sulfamethoxazole.

[0021] The present application also discloses a method for degrading antibiotics, which comprises adding the modified biochar matrix loaded with electrically active bacteria into a sample containing antibiotics and shock culture.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The present application uses hydrothermal method to prepare biochar as a carrier, and modifies the surface of the biochar with metal oxides such as iron and manganese to obtain a modified hydrothermal biochar. Using this material as a biochar carrier, on the one hand, the directional enrichment of electrically active bacteria is realized through directional culture, and on the other hand, the extracellular electron transfer process is strengthened, thereby promoting the degradation of antibiotics. The main performances are as follows:

[0024] 1. The biochar prepared by the hydrothermal method can retain more redox groups compared with the pyrolysis method, and the electron transfer capacity of the material is improved;

[0025] 2. The surface is modified with iron and manganese oxides to improve the electron storage capacity, larger specific surface area and developed pore structure, which is a medium for extracellular electron transfer of electrically active bacteria, can promote the adhesion and growth of electrically active bacteria, increase the biomass and activity of electrically active bacteria on the filler surface, more efficiently promote the electron transfer between electrically active bacteria and the external environment, accelerate the metabolic process of microorganisms, and thereby improve the degradation efficiency of antibiotics;

[0026] 3. Directly fixing electroactive bacteria Shewanella on the surface of biochar carrier, the degradation effect of electroactive bacteria on refractory new pollutants such as antibiotics is obviously better than that of conventional activated sludge mixed bacteria, and the addition of conductive filler further enhances the degradation effect of electroactive bacteria on refractory pollutants by enhancing the activity of the electron transfer system of electroactive bacteria, the content of cytochrome c and the content of NADH enzyme, and using the characteristics of electroactive bacteria that can directly transfer electrons without adding medium or electron shuttle, a functional material with high electrical conductivity and high capacitance is constructed as a biological carrier, and the degradation effect of antibiotics is strengthened by increasing the electron transfer efficiency of electroactive bacteria in a microbial electrochemical system.

[0027] The concept, specific structure and generated technical effects of the present application will be further described below in combination with the drawings, so as to fully understand the purpose, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 SEM images of HBC, HBC-Fe / Mn and BC provided by the present application;

[0029] Figure 2 XRD spectra of HBC and HBC-Fe / Mn provided by the present application;

[0030] Figure 3 Electrical conductivity of biochar under different pressures provided by the present application;

[0031] Figure 4 Adsorption kinetics curve of HBC-Fe / Mn and BC provided by the present application;

[0032] Figure 5 SMX degradation fold line graph provided by the present application. DETAILED DESCRIPTION

[0033] The following reference description of the drawings introduces a plurality of preferred embodiments of the present application, so that the technical content thereof is more clear and convenient to understand. The present application can be embodied in many different forms of embodiments, and the protection scope of the present application is not limited to the embodiments mentioned herein.

[0034] The List of Key-Controlled New Pollutants (2023 Edition) implemented on March 1, 2023, and the New Pollutant Control Action Plan issued by the General Office of the State Council in May 2022 both explicitly require "strengthening end-of-pipe treatment." Among the 14 types of new pollutants that are the focus of control, antibiotics are prominently listed. With the issuance of the two policies, there is a huge demand in related fields for technologies that can effectively treat antibiotic pollution. Antibiotic pollution has a wide range of sources, including medical wastewater, livestock and poultry wastewater, and pharmaceutical industry wastewater, all of which contain large amounts of antibiotics. Traditional wastewater treatment technologies have limited effectiveness in removing these pollutants. Biodegradation technology has significant technical and economic advantages in treating antibiotic pollution. Microorganisms can break down and convert antibiotics into harmless or less harmful substances through their metabolic activities. From an economic cost perspective, biodegradation technology does not require high equipment procurement and maintenance costs. It only needs to properly regulate the growth environment of microorganisms to achieve stable degradation results, with long-term operating costs much lower than physical and chemical treatment methods. With further research on antibiotics, increasing the electron transfer efficiency of electroactive microorganisms in microbial electrochemical systems to enhance the degradation of antibiotics has gradually become a research hotspot.

[0035] Biochar and its metal-modified materials are simple to prepare and do not require complex equipment or high investment. Biochar is obtained by pyrolysis or gasification of biomass or organic materials, and metal-modified biochar is obtained by mixing metal precursors or metal compounds with raw materials, impregnating or coating the surface of the materials, and then treating them (such as heat treatment) to combine the metal with the material surface, thereby improving the performance or imparting specific functions. After modification, biochar-based materials often have a larger specific surface area and rich pore structure, allowing them to adsorb more pollutants and provide a good electron transfer channel for electroactive bacteria. Compared with ordinary carbon materials, it can provide a suitable growth environment and attachment surface for electroactive bacteria, promote the attachment and growth of electroactive bacteria, increase the biomass and activity of electroactive bacteria on the filler surface, and is conducive to the formation of stable biofilms. It can also more efficiently promote electron transfer between electroactive bacteria and the external environment, accelerate the metabolic process of microorganisms, and thus improve the degradation efficiency of pollutants.

[0036] Because the raw materials for biochar are very diverse and readily available, almost all biomass resources, including various organic or industrial waste, agricultural and forestry waste (such as straw, rice husk, corn cob, tree bark, wood chips, etc.), can be used. Therefore, compared with some traditional functional materials, the raw material cost of biochar can be ignored, which provides favorable conditions for its large-scale production and application.

[0037] The following is illustrated by specific examples.

[0038] Example 1 Preparation of modified biochar substrate loaded with electroactive bacteria

[0039] Firstly, the present application prepares the biochar carrier by hydrothermal method: the raw material rice husk is washed, dried, crushed and passed through a 100 mesh screen to obtain the biomass raw material. The biomass raw material and ultrapure water are placed in a hydrothermal synthesis reaction kettle at a mass ratio of 1:50, and the reaction is carried out in an electric heating constant temperature air drying oven, and the reaction temperature is set to 300℃, and the reaction time is 3h. The biochar with high conductivity value and mild reaction conditions is obtained, which is denoted as HBC.

[0040] Secondly, surface modification of iron and manganese metal oxides: the corresponding hydrothermal biochar is ground and passed through a 100 mesh screen. A mixed metal salt solution with a concentration of 0.5mol / L of each of the two metal ions is prepared using ferric chloride and manganese chloride salt. The hydrothermal biochar is mixed with the solution at a ratio of 5g:100mL and stirred uniformly with a magnetic stirrer. After soaking for 3h, the biochar powder and metal salt solution are separated by filtration with an organic filter paper, and the biochar powder is washed with ultrapure water and anhydrous ethanol alternately until the washing liquid becomes colorless. The biochar powder on the filter paper is collected, dried and ground. The obtained biochar powder is calcined in a tube furnace at a temperature of 300℃ for 3h under a nitrogen atmosphere to remove impurities and promote the combination of metal ions and biochar, and the obtained metal ion modified biochar is denoted as HBC-Fe / Mn.

[0041] Thirdly, attachment and culture of electroactive bacteria on the surface of the substrate: first, the electroactive bacteria are prepared, and Shewanella oneidensis MR-1 is used in the experiment and stored in a-80℃ refrigerator. Before the experiment, Shewanella oneidensis MR-1 is transferred from the-80℃ refrigerator to the-20℃ refrigerator for storage and activation. According to the actual use instructions, LB culture medium is prepared, 100mL of LB culture medium is taken into a 250mL conical flask, 1ul of thawed bacterial solution is inoculated into the culture medium in a biological safety cabinet, the opening is sealed with tin paper and placed in a constant temperature shaker, and the culture is carried out at 150rpm and 30℃ for 24h. The OD600 value of the bacterial solution is measured before use, and the OD600 value is diluted to 0.1. 0.1g of HBC-Fe / Mn is weighed into a 250mL transparent anaerobic bottle, 100mL of LB culture medium is added, 1mL of activated and diluted Shewanella oneidensis MR-1 bacterial solution with an OD600 value of 0.1 is inoculated into the LB culture medium in a biological safety cabinet, the air in the bottle is pumped out with a vacuum pump, and nitrogen is introduced. The opening of the bottle is sealed with a matching butyl rubber plug to ensure an anaerobic state. The transparent anaerobic bottle is placed in a constant temperature shaker and cultured at 150rpm and 30℃, and the biochar material is combined with the bacteria after 24h of culture.

[0042] Test Example 1

[0043] The original biochar HBC, modified biochar HBC-Fe / Mn and pyrolytic carbon as a reference prepared by the present application were subjected to performance characterization. Among them, the pyrolytic carbon was made by pyrolysis firing with sludge as raw material, denoted as BC. The microstructure of the surface of the biochar sample was observed by scanning electron microscopy-energy dispersive spectrometer (SEM-EDS), as shown in Figure 1 The mass percentages of carbon (C), hydrogen (H), nitrogen (N), sulfur (S) and oxygen (O) in the biochar were determined by an elemental analyzer, as shown in Table 1; the crystal structure information of the biochar was analyzed by an X-ray diffractometer, as shown in Figure 2 The specific surface area and pore structure parameters of the material were determined by a specific surface area analyzer, as shown in Table 2; the biochar powder conductivity was determined by a four-probe method, as shown in Figure 3

[0044] Table 1 Elemental analysis of three kinds of biochar

[0045]

[0046] Table 2 BET fitting parameters of three kinds of biochar

[0047]

[0048] It can be seen from Figure 1 that the modified biochar HBC-Fe / Mn has more abundant surface pores than BC and more smooth and uniform holes than HBC. The elemental analysis of the three kinds of biochar in Table 1 shows that the pyrolytic biochar has a relatively high carbon content and a good carbonization rate, while the hydrothermal biochar has a high oxygen content. After modification, the carbon content of the biochar decreases and the oxygen content increases. Figure 2 In the XRD spectrum, the modified biochar HBC-Fe / Mn increases the crystal diffraction peaks of ferroferric oxide and manganese dioxide. The BET analysis of the three kinds of biochar in Table 2 shows that the specific surface area values of HBC and HBC-Fe / Mn are much larger than that of BC, which reflects the pore advantage of hydrothermal carbon over pyrolytic carbon. Figure 3 The results show that the HBC-Fe / Mn and HBC have similar values as a whole, while the conductivity of BC is significantly higher than that of the two hydrothermal biochars.

[0049] Test Example 2 investigated the adsorption capacity of HBC-Fe / Mn and BC for antibiotics

[0050] ​Take 0.1 g of HBC-Fe / Mn and BC respectively in a 250 mL transparent anaerobic bottle, add 100 mL of LB medium, and add SMX solution of a specific concentration to make the SMX concentration in the medium 10 mg / L. Use a vacuum pump to remove air from the bottle and introduce nitrogen. Use a matching butyl rubber plug to seal the bottle opening to ensure an anaerobic state. Place the transparent anaerobic bottle in a constant temperature shaker and shake at 150 rpm and 30°C for 6 days. Take 2 mL of the medium every 24 hours. Use a needle filter to take the sample and pass it through a 0.22 um water filter membrane. Use solid phase extraction to purify the obtained culture solution to remove the interference of LB medium and other substances in the medium. Use high performance liquid chromatography to measure the content of antibiotics in the medium.

[0051] The results are as follows Figure 4 The adsorption kinetics test shows that the capture effect of HBC-Fe / Mn and BC on SMX, and the adsorption rate of HBC-Fe / Mn in the initial stage (0-60 min) and the final equilibrium adsorption capacity are significantly higher than those of BC.

[0052] Test Example 3 investigates the adsorption capacity of HBC-Fe / Mn and BC loaded with electrically active bacteria for antibiotics

[0053] Take 0.1 g of HBC-Fe / Mn and BC respectively in a 250 mL transparent anaerobic bottle, add 100 mL of LB medium, and add SMX solution of a specific concentration to make the SMX concentration in the medium 10 mg / L. Use a vacuum pump to remove air from the bottle and introduce nitrogen. Use a matching butyl rubber plug to seal the bottle opening to ensure an anaerobic state. Place the transparent anaerobic bottle in a constant temperature shaker and shake at 150 rpm and 30°C for 6 days. Take 2 mL of the medium every 24 hours. Use a needle filter to take the sample and pass it through a 0.22 um water filter membrane. Use solid phase extraction to purify the obtained culture solution to remove the interference of LB medium and other substances in the medium. Use high performance liquid chromatography to measure the content of antibiotics in the medium.

[0054] The results are as follows Figure 5 It can be seen that the degradation ability of electrically active bacteria for SMX is significantly stronger than that of activated sludge, and the addition of HBC-Fe / Mn and BC has a strengthening effect on the degradation of SMX by electrically active bacteria, and the strengthening effect of BC on the degradation of SMX is slightly weaker than that of HBC-Fe / Mn. Figure 5

[0055] ​The preferred embodiments of the present application have been described above in detail. It should be understood that modifications and variations to the preferred embodiments could be made by those skilled in the art without departing from the spirit and scope of the application. Accordingly, it is intended that there be included within the scope of the application, all such modifications and variations as would be apparent to those skilled in the art upon reading this disclosure. It is intended to obtain for the inventors such patent rights as are available for any patent granted on the present application.

Claims

1. A method of preparing a modified biochar substrate loaded with electroactive bacteria, characterized in that, Comprising the following steps: (1) Preparation of biochar carrier: the raw material rice husk is washed, dried, crushed, and sieved to obtain a biomass raw material, which is then mixed with ultrapure water and placed in a hydrothermal synthesis reaction kettle, and the reaction is carried out in an electric heating constant temperature air drying oven to obtain the original biochar HBC; (2) Surface modification of iron-manganese metal oxide: the original biochar of step (1) is ground and sieved, and iron-manganese mixed metal salt solution is added thereto, stirred and mixed uniformly, soaked for a set time, then the biochar powder and metal salt solution are separated by filtration, and the biochar powder is washed until the washing liquid becomes colorless, the biochar powder is collected, dried and ground, and the obtained biochar powder is calcined under a nitrogen atmosphere to remove impurities and promote the combination of metal ions and biochar, to obtain a metal ion modified biochar, denoted as HBC-Fe / Mn; (3) Attachment culture of electroactive bacteria on the surface of the substrate: HBC-Fe / Mn is weighed into a transparent anaerobic bottle, LB culture medium is added, then activated and diluted electroactive bacteria Shewanella oneidensis MR-1 bacterial solution is inoculated into the LB culture medium, and the transparent anaerobic bottle is placed in a constant temperature shaker for culture under anaerobic conditions, so that the biochar material is combined with the bacteria. In step (1), the mass ratio of the biomass raw material to the ultrapure water is 1:50, the reaction temperature is 300°C, and the reaction time is 3h.

2. The production method according to claim 1, characterized by, In step (2), the iron-manganese mixed metal salt solution is a ferric chloride solution and a manganese chloride solution, and the concentration of the ferric chloride solution and the manganese chloride solution is 0.5mol / L.

3. The preparation method according to claim 1, characterized in that, The mass-volume ratio of the original biochar to the iron-manganese mixed metal salt solution is 1:20 (g / mL); the soaking time is 3h; and the calcination temperature is 300°C for 3h.

4. The production method according to claim 3, characterized by, In step (3), the OD600 of the diluted electroactive bacteria Shewanella oneidensis MR-1 is 0.1, the volume ratio of the diluted electroactive bacteria Shewanella oneidensis MR-1 bacterial solution to the LB culture medium is 1:100, and the mass-volume ratio of the HBC-Fe / Mn to the diluted electroactive bacteria Shewanella oneidensis MR-1 bacterial solution is 1:10 (g / mL).

5. The preparation method according to claim 1, characterized in that, In step (3), the shaking culture conditions are as follows: shaking culture at 150rpm and 30°C for 24h.

7. The modified biochar substrate loaded with electroactive bacteria prepared by the method of any one of claims 1-6.

6. The method of claim 1, wherein, 8. The use of the modified biochar substrate loaded with electroactive bacteria of claim 7 in degrading antibiotics. The antibiotic is sulfamethoxazole. The modified biochar substrate loaded with electroactive bacteria is added to a sample containing antibiotics and subjected to shaking culture.

9. Use according to claim 6, characterized in that, ​ 10. A method of degrading an antibiotic, characterized by, ​

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