Method for slowing down biological pollution of graphene oxide material based on regulation and control of energy-taxis motion capability
By regulating the redox degree of graphene oxide materials and adsorbing metal ions, and using electrode protection, the biological pollution problem of graphene oxide materials caused by bacterial energy movement is solved, and the effect of slowing down biological pollution and improving the sustainability of water treatment is achieved.
Patent Information
- Application Number
- CN202510060913.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-04-25
AI Technical Summary
Graphene oxide materials are susceptible to bacterial energy-bearing movement during water treatment, resulting in biological contamination. The prior art focuses on the modification of membrane materials but fails to effectively solve the problem of biological activity itself.
By changing the redox degree of graphene oxide materials, it absorbs stable negative-oriented metal ions, and uses electrodes as insoluble electron acceptors to protect bacterial energy-bearing and movement behavior, thereby alleviating biological contamination.
Effectively alleviate the biological pollution of graphene oxide materials caused by bacterial energy-bearing movement, delay the development of biocontamination on the surface of membrane materials, and improve the sustainability of the water treatment process.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of nano material application, and relates to a method for mitigating biological contamination of graphene oxide materials based on regulating kinetic movement ability. Background Art
[0002] Membrane technology based on water treatment plays an important role in drinking water purification and wastewater reuse. Graphene oxide (GO) has a large number of hydrophilic functional groups and is often used as a water treatment membrane material. Graphene oxide can play a molecular sieve role, allowing particles smaller than the channel size of the nanosheet to penetrate, while blocking other larger substances, and allowing water to flow quickly due to the presence of hydrophobic channels. However, microorganisms in water are easily deposited on the surface of membrane materials, and then multiply to form biofilms and produce extracellular secretions, resulting in biological fouling of membrane materials. Membrane biofouling is a major limiting factor in various membrane separation processes, which will lead to operational problems such as reduced flux, increased operating pressure, increased energy consumption, limited sustainable operation of water treatment processes, increased cleaning frequency, and shortened membrane life in the water treatment process. Therefore, seeking methods to resist or reduce membrane biofouling is crucial for water treatment processes such as wastewater reuse and seawater desalination. At present, the methods to reduce membrane biofouling are mainly based on the strategy of membrane material modification, while less attention is paid to the fundamental factor that causes membrane biofouling-biological activity itself.
[0003] In a paper published in Chemical Engineering Journal, 281, pp. 53-59, 2015, Sun XF et al. reported a method for preparing a graphene oxide-silver nanoparticle (GO-AgNPs) composite antibacterial fouling membrane on a cellulose acetate membrane. During application, the flux of the GO-AgNPs composite membrane was higher than that of the graphene oxide membrane and the Ag membrane alone, and it showed strong antibacterial activity, which could cause 86% of Escherichia coli to be inactivated within 2 hours after contact with the membrane, effectively preventing biological fouling on the membrane surface. In a paper published in Desalination, 451, pp. 45-58, 2019, Armendáriz-Ontiveros MM et al. evaluated the anti-biological fouling performance of commercial polyamide reverse osmosis membranes coated with iron nanoparticles (FeNPs) and graphene oxide (GO), and the bacteria used in the experiment were halotolerant Bacillus. The results show that the reverse osmosis membrane containing GO-FeNP coating has a greater flux after being biofouled, and is most efficient in reducing membrane biofouling, which helps to alleviate the biofouling problem of reverse osmosis membranes. Cheng W et al., in a paper published in Environmental Science & Technology, Vol. 54, No. 1, pp. 517-526 in 2019, functionalized the surface of graphene oxide materials, exposed graphene oxide nanosheets on the membrane surface by coating, and enhanced the antibacterial activity and biofouling resistance of graphene oxide membranes.
[0004] Shewanella is a typical dissimilatory metal-reducing bacterium that has attracted much attention. It is widely distributed in almost all mesophilic environments on Earth. As a facultative anaerobe, Shewanella mainly uses oxygen as the final electron acceptor under aerobic conditions, and can use a variety of soluble and insoluble substances as electron acceptors under anaerobic conditions, including iron / manganese minerals, various metal ions and non-metal ions, and organic pollutants such as azo dyes and nitroaromatic compounds. In the actual environment, Shewanella can effectively sense and find pollutants through tropism, which is accompanied by the metabolism of cellular chemotactic effectors. It is called tropism, that is, chemotactic effectors disturb the redox state and proton motive force of the electron transport chain during bacterial metabolism. This signal is sensed by chemotactic receptors and initiates bacterial tropism response behavior. This tropism behavior greatly affects the migration and distribution of bacteria in the environment.
[0005] Salas EC et al. reported in a paper published in ACS Nano, Vol. 4, No. 8, pp. 4852-4856 in 2010 that Shewanella can bioreduced graphene oxide to reduced graphene oxide, and this process is completed through extracellular electron transfer (EET). In addition to the Shewanella strains reported by Salas et al. that showed different degrees of graphene oxide reduction ability under strict exclusion of oxygen, Wang G et al. reported in a paper published in Nano Research, Vol. 4, pp. 563-570 in 2011 that Shewanella also showed similar or even stronger graphene oxide reduction ability under aerobic and anaerobic culture conditions. Zhang C et al. demonstrated in a paper published in Nano Letters, Vol. 20, No. 2, pp. 1455-1460 in 2020 that graphene oxide can be used as a chemical attractant to attract bacteria to the surface of nanoparticles by releasing signal molecules, promoting bacterial aggregation and migration to graphene oxide materials. Therefore, the biological contamination of graphene oxide materials in practical applications is likely caused by the kinetic behavior of bacteria towards graphene oxide. If the kinetic behavior of bacteria towards graphene oxide materials can be weakened, the biological contamination of graphene oxide membrane materials in practical applications can be slowed down.
[0006] Based on the above analysis, a method for mitigating the biofouling of graphene oxide materials by regulating the kinetic motility of bacteria is proposed. This method is simple to operate, low in cost and highly practical, and can effectively mitigate the biofouling of graphene oxide membrane materials in practical applications. Summary of the invention
[0007] The invention belongs to the technical field of nanomaterial application, and provides a method for slowing down the biological contamination of graphene oxide materials based on regulating the tropism movement ability of bacteria, which can be used to slow down the biological contamination of materials such as graphene oxide membranes in water treatment processes.
[0008] The technical solution of the present invention is as follows:
[0009] The method for slowing down the biological contamination of graphene oxide materials based on regulating the kinetic movement ability comprises the following steps:
[0010] Step 1: Cultivation of microorganisms
[0011] Shewanella oneidensis MR-1 was selected as the object of bacterial movement research. MR-1 was inoculated into 25 mL of soybean nutrient medium, and cultured in a constant temperature shaker at 32°C and 160 rpm for 10 hours, then expanded and taken out for use after 15 hours.
[0012] Preparation of soybean nutrient medium: weigh 0.8 g of soybean liquid medium and dissolve it in 25 mL of ultrapure water. After high temperature and high pressure sterilization, obtain the soybean nutrient medium, which is placed on an ultraclean bench for use.
[0013] Step 2: Preparation of graphene oxide
[0014] Graphene oxide was synthesized by a modified Hummer method. Graphite powder, concentrated nitric acid (98%), and concentrated sulfuric acid (98%) were mixed and stirred in a ratio of 1g:(9-11)mL:(44-48)mL under ice bath conditions, and 7-8g KMnO4 was slowly added. Stirring was continued under ice bath conditions for 110-120min, and then the reaction solution was moved to a 33-35℃ water bath and stirred overnight. 44-48mL ultrapure water was slowly added to the reaction system, and the system temperature was gradually increased. The oil bath was kept at 96-98℃ for 150-160min. After the reaction solution was cooled, 200-210mL ultrapure water was slowly added to the system, and 23-25mL 30% H2O2 solution was added dropwise to remove excess KMnO4. The obtained graphite oxide precipitate was collected by centrifugation (6000g, 30min), washed twice with 10% dilute hydrochloric acid, and then washed several times with ultrapure water until the supernatant was neutral. The collected graphite oxide is redispersed in high-purity water and subjected to ultrasonic exfoliation for several hours to obtain a single layer or several layers of graphene oxide.
[0015] Step 3: Preparation of graphene oxide with different redox degrees
[0016] Reduced graphene oxide (rGO) was prepared by reducing graphene oxide with ascorbic acid. The concentration ratio of ascorbic acid to graphene oxide was 3:1. The ascorbic acid solution and graphene oxide suspension were mixed evenly in an anaerobic chamber, and the reduction time was 30 min, 120 min, and 1440 min, respectively. After being taken out, it was washed three times with deionized water to remove excess ascorbic acid, and three r-graphene oxides with different redox degrees were obtained.
[0017] Step 4: Preparation of graphene oxide adsorbed with Cu(II)
[0018] The prepared graphene oxide powder was dispersed into deionized water under continuous stirring and ultrasonic treatment to form a uniform dispersion of 1 mg / mL. An appropriate amount of CuSO4·5H2O was weighed and dissolved in deionized water to prepare a series of CuSO4 solutions with different concentrations. The graphene oxide suspension was mixed evenly with the CuSO4 solution and placed in a 150 rpm shaker for adsorption for 2 hours. After being taken out, it was washed three times with deionized water to remove the unadsorbed Cu(Ⅱ) to obtain graphene oxide adsorbed with Cu(Ⅱ).
[0019] Step 5: Preparation of graphene oxide film material
[0020] The prepared graphene oxide powder was dispersed into deionized water under continuous stirring and ultrasonic treatment to form a uniform dispersion of 1 mg / mL. In order to obtain a uniform and stable graphene oxide film, Fe(Ⅲ) (ferric chloride, analytical reagent) was used as a crosslinker, and the diluted graphene oxide suspension was mixed with the same volume of 0.5-0.7 mmol / L Fe(Ⅲ) for corresponding crosslinking. Subsequently, the mixed dispersion was vacuum filtered through a mixed cellulose ester support membrane with a pore size of 0.45 μm, the vacuum pump suction was -1 bar, and the graphene oxide loading was 7-9 g / m 2 Finally, the synthesized graphene oxide membrane was washed by deionized water for several hours to remove unbound Fe(III) and impurities.
[0021] Step 6: Preparation of modified LM medium
[0022] A modified Lactate-Medium (LM) culture medium was prepared. The culture medium components included 12 mM HEPES, 12 mM NaHCO3, 0.3% sodium lactate by mass, 0.04% yeast extract powder by mass and 0.02% peptone by mass. The pH of the LM culture medium was adjusted to 6.8-7.2, nitrogen was aerated for 30 minutes, and the culture medium was sterilized at 121°C under high pressure for 20 minutes before use.
[0023] Step 7: Using Electrodes as Electron Acceptor Protection
[0024] A three-electrode static water treatment system was constructed and a constant potential steady-state test was performed. The graphene oxide membrane was installed at the bottom of the electrolytic cell, and about 30 mL of modified LM medium was added to the electrolytic cell, and 10 8 -10 9 cells / mL of bacterial cells. A carbon felt sheet with a specification of 1cm×1cm was used as the working electrode, a platinum sheet was used as the counter electrode, a constant potential of 0.5V was applied, and the reaction time was 6h.
[0025] Step 8: Construction of static water treatment system
[0026] The graphene oxide membrane was installed in a water treatment frame with an exposed area of 10-13 cm 2 The modified LM medium was added to the surface of the graphene oxide membrane and inoculated with 10 8 -10 9 cells / mL of bacterial cells. After 6 h, the bacterial suspension was discarded and the graphene oxide membrane was gently washed with a buffer solution to remove unattached cells. The washed graphene oxide membrane was transferred to 20 mL of buffer solution, soaked and placed in a 150 rpm shaker for 10 min to separate the deposited bacterial cells. The cell suspension was then diluted to 10 4 -105 cells / mL, and the plate colony counting method was used to count the concentration of bacteria attached to the surface of graphene oxide membrane to characterize the degree of biological contamination of graphene oxide membrane.
[0027] Preparation of buffer solution: Weigh the required amount of hydroxyethylpiperazineethanesulfonic acid and dissolve it in ultrapure water to a concentration of 20 mmol / L, adjust the pH to 6.8-7.2, and sterilize at high temperature and high pressure to obtain a buffer solution.
[0028] The beneficial results of the present invention are:
[0029] The present invention regulates the kinetic behavior of bacteria by three methods: changing the redox degree of graphene oxide materials, allowing graphene oxide to adsorb stable negative kinetic metal ions, and using electrodes as insoluble electron acceptor protection, thereby reducing the biological contamination of graphene oxide materials caused by bacteria through kinetic movement, and can slow down the biological contamination of graphene oxide membrane materials in actual water treatment processes. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 is the cell concentration of bacteria attached to the graphene oxide / reduced graphene oxide film under the condition of changing the redox degree of graphene oxide.
[0031] Figure 2 It is the cell concentration of bacteria attached to graphene oxide / Cu(II)-doped graphene oxide film when the ratio of Cu(II) to graphene oxide concentration is changed.
[0032] FIG3( a ) is a photograph of the plate colony counting method of bacteria attached to the graphene oxide membrane in the absence of an electrode.
[0033] FIG3( b ) is a photograph of the plate colony counting method of bacteria attached to the graphene oxide film using the electrode as the electron acceptor.
[0034] Figure 4 is the cell concentration of bacteria attached to the graphene oxide membrane without electrode and with electrode as electron acceptor. DETAILED DESCRIPTION
[0035] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.
[0036] Example 1
[0037] In this example, membrane materials were prepared using graphene oxide and reduced graphene oxide with different redox degrees, and the effect of changing the redox degree of graphene oxide on mitigating biological contamination of graphene oxide membrane materials was explored.
[0038] (1) Inoculate MR-1 into 25 mL of soybean nutrient medium, place in a constant temperature shaker at 32°C and 160 rpm for 10 h, then expand the culture and remove after 15 h. Pipette 20 mL of soybean nutrient medium with MR-1 bacteria and centrifuge at low speed (5500 rpm, 4 min) to avoid damaging the bacterial flagella and affecting its swimming ability. Collect the MR-1 bacteria and wash them three times with buffer solution (20 mmol / L, pH 7.0), mix and resuspend to 200 μL with an oscillating mixer as MR-1 bacterial suspension for later use.
[0039] (2) Preparation of graphene oxide: 1.0 g of graphite powder was mixed with 9 mL of concentrated nitric acid (98%) and 48 mL of concentrated sulfuric acid (98%) under ice bath conditions, and 8 g of KMnO4 was slowly added. The mixture was stirred for 120 min under ice bath conditions, and then the reaction solution was moved to a 35°C water bath and stirred overnight. 44 mL of ultrapure water was slowly added to the reaction system, and the system temperature was gradually increased. The reaction solution was placed in an oil bath at 96°C for 160 min. After the reaction solution was cooled, 210 mL of ultrapure water was slowly added to the system, and 25 mL of 30% H2O2 solution was added dropwise to remove excess KMnO4. The obtained graphite oxide precipitate was collected by centrifugation (6000 g, 30 min), and washed twice with 10% dilute hydrochloric acid, and then washed several times with ultrapure water until the supernatant was neutral. The collected graphite oxide was redispersed in high-purity water, and ultrasonically exfoliated for several hours to obtain a single layer or several layers of graphene oxide.
[0040] (3) Preparation of graphene oxide with different redox degrees: Reduced graphene oxide was prepared by reducing graphene oxide with ascorbic acid. Weigh 0.72g of ascorbic acid and dissolve it in 40mL of deionized water, mix it evenly, and obtain an ascorbic acid solution with a concentration of 18mg / mL. Weigh 0.24g of graphene oxide and disperse it in 100mL of deionized water, mix it evenly, and obtain a graphene oxide suspension with a concentration of 2.4mg / mL. In an anaerobic chamber, mix 10mL of ascorbic acid solution and 25mL of graphene oxide suspension evenly, so that the ratio of ascorbic acid to graphene oxide is 3:1, and the reduction time is 30min, 120min, and 1440min respectively. After taking it out, wash it three times with deionized water to remove excess ascorbic acid, and obtain three reduced graphene oxides with different redox degrees (respectively denoted as rGO-1, rGO-2, and rGO-3).
[0041] (4) Preparation of graphene oxide or reduced graphene oxide membrane: 1 g of graphene oxide powder prepared in step (2) or reduced graphene oxide powder prepared in step (3) is dispersed in 1 L of deionized water under continuous stirring and ultrasonic treatment to form a uniform dispersion of 1 mg / mL. The diluted graphene oxide or reduced graphene oxide suspension is mixed with the same volume of 0.5 mmol / LFe (III) for corresponding crosslinking. Subsequently, the mixed dispersion is vacuum filtered through a mixed cellulose ester support membrane with a pore size of 0.45 μm, the vacuum pump suction is -1 bar, and the graphene oxide or reduced graphene oxide loading is 7 g / m 2 Finally, the synthesized graphene oxide or reduced graphene oxide film was washed with deionized water for 2 h to remove unbound Fe(III) and impurities.
[0042] (5) Prepare a modified LM medium containing 12 mM HEPES, 12 mM NaHCO3, 0.3% sodium lactate, 0.04% yeast extract powder and 0.02% peptone, adjust the pH to 6.8-7.2, aerate with nitrogen for 30 min, sterilize at 121°C for 20 min and set aside. Add 100 μL of the MR-1 bacterial suspension described in step (1) to the modified LM medium and mix well to make the bacterial concentration about 10 8 -10 9 cells / mL.
[0043] (6) Install the graphene oxide or reduced graphene oxide membrane in a water treatment frame with an exposed area of 10.3 cm 2 . Add 30 mL of the modified LM medium inoculated with MR-1 bacteria described in step (5) into the static water treatment system in an anaerobic chamber so that it is in full contact with the surface of the graphene oxide or reduced graphene oxide membrane. After 6 hours, discard the bacterial suspension and gently rinse the graphene oxide or reduced graphene oxide membrane with a buffer solution to remove unattached MR-1 bacterial cells. Transfer the washed graphene oxide or reduced graphene oxide membrane to 20 mL of buffer solution, soak and place in a 150 rpm shaker for 10 minutes to separate the deposited MR-1 bacterial cells to obtain a bacterial suspension.
[0044] (7) Dilute the MR-1 bacterial suspension in step (6) to 10 4 -10 5 cells / mL, and the plate colony counting method was used to count the concentration of MR-1 bacteria attached to the surface of graphene oxide or reduced graphene oxide membrane. The effective counting range was 30-300 cfu (colony forming units) to characterize the degree of biological contamination of graphene oxide or reduced graphene oxide membrane.
[0045] Figure 1is the cell concentration of MR-1 bacteria attached to the graphene oxide / reduced graphene oxide film when the redox degree of graphene oxide is changed. When graphene oxide is not reduced, the cell concentration of bacteria attached to the graphene oxide film material is (9.2±0.3)×10 5 cells / cm 2 The cell concentrations of bacteria attached to the rGO-1, rGO-2, and rGO-3 membranes obtained after the graphene oxide was reduced for 30 min, 120 min, and 1440 min were (8.1±0.1)×10 5 cells / cm 2 、(5.2±0.2)×10 5 cells / cm 2 and (3.0±0.5)×10 5 cells / cm 2 Compared with the unreduced graphene oxide membrane material, the concentration of bacterial cells attached to the membrane decreased by 12.0%, 43.5% and 67.4%, respectively. The cell concentration of bacteria attached to the reduced graphene oxide membrane was significantly reduced, indicating that the higher the degree of reduction of graphene oxide, the weaker the MR-1 bacteria's ability to move towards graphene oxide materials, which helps to slow down the formation of biological contamination of graphene oxide membrane materials.
[0046] Example 2
[0047] In this example, a static water treatment system was constructed using graphene oxide and graphene oxide membrane materials doped with Cu(II), and the effect of graphene oxide adsorbing negatively oriented metal ions on mitigating biological contamination of graphene oxide membrane materials was investigated.
[0048] (1) Collect 1 L of water sample from a natural water body and store it in a refrigerator at 4°C.
[0049] (2) Preparation of graphene oxide: 1.0 g of graphite powder was mixed with 10 mL of concentrated nitric acid (98%) and 46 mL of concentrated sulfuric acid (98%) under ice bath conditions, and 7 g of KMnO4 was slowly added. The mixture was stirred for 110 min under ice bath conditions, and then the reaction solution was moved to a 34°C water bath and stirred overnight. 46 mL of ultrapure water was slowly added to the reaction system, and the system temperature was gradually increased, and the mixture was placed in an oil bath at 98°C for 150 min. After the reaction solution was cooled, 200 mL of ultrapure water was slowly added to the system, and 24 mL of 30% H2O2 solution was added dropwise to remove excess KMnO4. The obtained graphite oxide precipitate was collected by centrifugation (6000 g, 30 min), and washed twice with 10% dilute hydrochloric acid, and then washed several times with ultrapure water until the supernatant was neutral. The collected graphite oxide was redispersed in high-purity water, and ultrasonically exfoliated for several hours to obtain a single layer or several layers of graphene oxide.
[0050] (3) Preparation of graphene oxide adsorbing Cu(Ⅱ): The prepared graphene oxide powder was dispersed in deionized water under continuous stirring and ultrasonic treatment to form a uniform dispersion of 1 mg / mL. An appropriate amount of CuSO4·5H2O was weighed and dissolved in deionized water to prepare a series of CuSO4 solutions with different concentrations: 10 mg / L, 13 mg / L, 20 mg / L and 40 mg / L. The graphene oxide suspension and the CuSO4 solution were mixed in equal volumes to make the ratio of Cu(Ⅱ) to graphene oxide concentration 0, 1:100, 1:75, 1:50 and 1:25, respectively, and placed in a 150 rpm shaker for adsorption for 2 hours. After taking out, it was washed three times with deionized water to remove the unadsorbed Cu(Ⅱ) to obtain graphene oxide adsorbing Cu(Ⅱ).
[0051] (4) Preparation of graphene oxide film doped with Cu(II): The graphene oxide adsorbed with Cu(II) described in step (3) was dispersed in deionized water to form a uniform dispersion of 1 mg / mL. Subsequently, the suspension was vacuum filtered through a mixed cellulose ester support membrane with a pore size of 0.45 μm, the vacuum pump suction was -1 bar, and the graphene oxide adsorbed with Cu(II) was loaded with 8 g / m 2 Finally, the synthesized membrane material was washed by deionized water for 2 h to remove unbound Cu(II) and impurities.
[0052] (5) The Cu(II)-doped graphene oxide membrane was installed in a water treatment frame with an exposed area of 11.8 cm 2 . Add 30 mL of the water sample described in step (1) into the static water treatment system in an anaerobic chamber so that it is in full contact with the surface of the Cu(Ⅱ)-doped graphene oxide membrane. After 6 hours, discard the water sample and gently rinse the Cu(Ⅱ)-doped graphene oxide membrane with a buffer solution to remove unattached bacterial cells. Transfer the washed Cu(Ⅱ)-doped graphene oxide membrane to 20 mL of buffer solution, soak and place it in a 150 rpm shaker for 10 minutes to separate the deposited bacterial cells and obtain a bacterial suspension.
[0053] (6) Dilute the bacterial suspension in step (5) to 10 4 -10 5 cells / mL, and the plate colony counting method was used to count the concentration of bacteria attached to the surface of graphene oxide membrane. The effective counting range was 30-300 cfu to characterize the degree of biological contamination of graphene oxide membrane.
[0054] Figure 2The cell concentration of bacteria attached to graphene oxide / Cu(Ⅱ)-doped graphene oxide membranes was shown when the ratio of Cu(Ⅱ) to graphene oxide concentration was changed. When there was no Cu(Ⅱ) adsorption on the graphene oxide membrane, the cell concentration of bacteria attached to the membrane was (9.5±0.1)×10 5 cells / cm 2 When the concentration ratio of Cu(Ⅱ) to graphene oxide is 1:100, 1:75, 1:50 and 1:25, the cell concentration of bacteria attached to the Cu(Ⅱ)-doped graphene oxide film is (8.5±0.4)×10 5 cells / cm 2 、(7.0±0.2)×10 5 cells / cm 2 、(4.9±0.5)×10 5 cells / cm 2 and (2.3±0.1)×10 5 cells / cm 2 Compared with the graphene oxide membrane without Cu(Ⅱ) adsorption, the concentration of bacterial cells attached to the membrane decreased by 10.5%, 26.3%, 48.4% and 75.8%, respectively. The decrease in the concentration of bacterial cells attached to the membrane indicates that as the amount of Cu(Ⅱ) adsorbed by the graphene oxide membrane increases, the ability of bacteria to move toward the Cu(Ⅱ)-doped graphene oxide membrane material gradually weakens, which is beneficial to slow down the biological contamination of the graphene oxide membrane material caused by bacteria.
[0055] Example 3
[0056] In this example, electrodes were used as insoluble electron acceptor protection to investigate the effect of electrodes on mitigating biofouling of graphene oxide membrane materials.
[0057] (1) Inoculate MR-1 into 25 mL of soybean nutrient medium, place in a constant temperature shaker at 32°C and 160 rpm for 10 h, then expand the culture and take it out after 15 h. Pipette 20 mL of soybean nutrient medium with MR-1 bacteria, centrifuge at low speed (5500 rpm, 4 min) to avoid damaging the bacterial flagella and affecting its swimming ability, collect the MR-1 bacteria and wash them three times with buffer solution (20 mmol / L, pH 7.0), mix and resuspend to 200 μL with an oscillating mixer as MR-1 bacterial suspension for later use.
[0058] (2) Preparation of graphene oxide: 1.0 g of graphite powder was mixed with 11 mL of concentrated nitric acid (98%) and 44 mL of concentrated sulfuric acid (98%) under ice bath conditions, and 8 g of KMnO4 was slowly added. The mixture was stirred for 120 min under ice bath conditions, and then the reaction solution was moved to a 33°C water bath and stirred overnight. 48 mL of ultrapure water was slowly added to the reaction system, and the system temperature was gradually increased. The reaction solution was placed in an oil bath at 97°C for 155 min. After the reaction solution was cooled, 200 mL of ultrapure water was slowly added to the system, and 23 mL of 30% H2O2 solution was added dropwise to remove excess KMnO4. The obtained graphite oxide precipitate was collected by centrifugation (6000 g, 30 min), washed twice with 10% dilute hydrochloric acid, and then washed several times with ultrapure water until the supernatant was neutral. The collected graphite oxide was redispersed in high-purity water, and ultrasonically exfoliated for several hours to obtain a single layer or several layers of graphene oxide.
[0059] (3) Preparation of graphene oxide membrane: 1 g of graphene oxide powder prepared by the improved Hummer method was dispersed into 1 L of deionized water under continuous stirring and ultrasonic treatment to form a 1 mg / mL uniform dispersion. The diluted graphene oxide suspension was mixed with the same volume of 0.7 mmol / L Fe(III) for corresponding crosslinking. Subsequently, the mixed dispersion was vacuum filtered through a mixed cellulose ester support membrane with a pore size of 0.45 μm, the vacuum pump suction was -1 bar, and the graphene oxide loading was 9 g / m 2 Finally, the synthesized graphene oxide membrane was washed with deionized water for 2 h to remove unbound Fe(III) and impurities.
[0060] (4) Prepare a modified LM medium containing 12 mM HEPES, 12 mM NaHCO3, 0.3% sodium lactate, 0.04% yeast extract powder and 0.02% peptone, adjust the pH to 6.8-7.2, aerate with nitrogen for 30 min, sterilize at 121°C for 20 min and set aside. Add 100 μL of the MR-1 bacterial suspension described in step (1) to the modified LM medium and mix well to make the bacterial concentration about 10 8 -10 9 cells / mL.
[0061] (5) Construct a three-electrode static water treatment system and conduct a steady-state test using a constant potential method. Install the graphene oxide membrane at the bottom of the electrolytic cell, and add 30 mL of the modified LM medium inoculated with MR-1 bacteria described in step (3) to the electrolytic cells of the experimental group (using the electrode as an insoluble electron acceptor for protection) and the control group (without the electrode). Use a carbon felt sheet with a specification of 1 cm × 1 cm as the working electrode and a platinum sheet as the counter electrode, connect to the electrochemical workstation, and apply a constant potential of 0.5 V. After 6 hours, discard the bacterial suspension and gently rinse the graphene oxide membrane with a buffer solution to remove the unattached MR-1 bacterial cells. Transfer the washed graphene oxide membrane to 20 mL of buffer solution, soak and place it in a 150 rpm shaker for 10 minutes to separate the deposited MR-1 bacterial cells to obtain a bacterial suspension.
[0062] (6) Dilute the MR-1 bacterial suspension in step (5) to 10 4 -10 5 cells / mL, and the plate colony counting method was used to count the concentration of MR-1 bacteria attached to the surface of graphene oxide membrane. The effective counting range was 30-300 cfu to characterize the degree of biological contamination of graphene oxide membrane.
[0063] FIG3(a) is a photo of the plate colony count method of bacteria attached to the graphene oxide membrane in the absence of an electrode, and FIG3(b) is a photo of the plate colony count method of bacteria attached to the graphene oxide membrane in the case of using an electrode as an electron acceptor. Figure 4 is the cell concentration of MR-1 bacteria attached to the graphene oxide membrane without electrode and using electrode as electron acceptor. Figure 4 As shown in the figure, the concentration of bacterial cells attached to the graphene oxide membrane in the control group without electrode was (9.3±0.1)×10 5 cells / cm 2 In the experimental group using the electrode as an insoluble electron acceptor, the concentration of bacterial cells attached to the graphene oxide membrane was (2.9±0.4)×10 5 cells / cm 2 , which was 68.8% lower than that of the control group, and significantly lower than that of the control group. The cell concentration of bacteria attached to the graphene oxide membrane was significantly reduced, indicating that under the experimental conditions of using the electrode as an insoluble electron acceptor for protection, the electrode has an attractive effect on MR-1 bacteria, which can slow down the kinetic movement and attachment of bacteria to the graphene oxide membrane material, thereby delaying the development of biological contamination on the surface of the graphene oxide membrane material.
Claims
1. A method for mitigating biological contamination of graphene oxide materials based on regulating kinetic motility, characterized in that: The steps include: Step 1: Cultivation of microorganisms Step 2: Preparation of graphene oxide Graphene oxide was synthesized using a modified Hummer method; Step 3: Preparation of graphene oxide with different redox degrees Reduced graphene oxide (rGO) was prepared by reducing graphene oxide with ascorbic acid; the concentration ratio of ascorbic acid to graphene oxide was 3:1, and the ascorbic acid solution and the graphene oxide suspension were mixed evenly in an anaerobic chamber, and the reduction time was 30 min, 120 min, and 1440 min, respectively; after being taken out, the mixture was washed three times with deionized water to remove excess ascorbic acid, and three r-graphene oxides with different redox degrees were obtained; Step 4: Preparation of graphene oxide adsorbed with Cu(II) The prepared graphene oxide powder was dispersed in deionized water under continuous stirring and ultrasonic treatment to form a uniform dispersion of 1 mg / mL; an appropriate amount of CuSO4·5H2O was weighed and dissolved in deionized water to prepare a series of CuSO4 solutions with different concentrations; the graphene oxide suspension was evenly mixed with the CuSO4 solution and placed in a 150 rpm shaker for adsorption for 2 hours; after being taken out, it was washed three times with deionized water to remove the unadsorbed Cu(II) to obtain graphene oxide adsorbing Cu(II); Step 5: Preparation of graphene oxide film material The prepared graphene oxide powder was dispersed into deionized water under continuous stirring and ultrasonic treatment to form a uniform dispersion of 1 mg / mL; in order to obtain a uniform and stable graphene oxide film, Fe(III) (ferric chloride, analytical reagent) was used as a crosslinking agent, and the diluted graphene oxide suspension was mixed with the same volume of 0.5-0.7 mmol / L Fe(III) for corresponding crosslinking; Subsequently, the mixed dispersion was vacuum filtered through a mixed cellulose ester support membrane with a pore size of 0.45 μm, a vacuum pump suction of -1 bar, and a graphene oxide loading of 7-9 g / m 2 ;Finally, the synthesized graphene oxide membrane was washed with deionized water for several hours to remove unbound Fe(III) and impurities; Step 6: Preparation of modified LM medium Prepare a modified Lactate-Medium (LM) culture medium, the culture medium components include 12mM HEPES, 12mM NaHCO3, 0.3% sodium lactate by mass, 0.04% yeast extract powder by mass and 0.02% peptone by mass, adjust the pH of the LM culture medium to 6.8-7.2, aerate with nitrogen for 30 minutes, sterilize at 121°C for 20 minutes, and then take out for use; Step 7: Using Electrodes as Electron Acceptor Protection A three-electrode static water treatment system was constructed to conduct a steady-state test using a constant potential method. The graphene oxide membrane was installed at the bottom of the electrolytic cell. About 30 mL of modified LM medium was added to the electrolytic cell, and 10 8 -10 9 cells / mL of bacterial cells; a carbon felt sheet with a specification of 1cm×1cm was used as the working electrode, a platinum sheet was used as the counter electrode, a constant potential of 0.5V was applied, and the reaction time was 6h; Step 8: Construction of static water treatment system The graphene oxide membrane was installed in a water treatment frame with an exposed area of 10-13 cm 2 ; Add modified LM medium to the surface of graphene oxide membrane and inoculate 10 8 -10 9 cells / mL of bacterial cells; after 6 h, the bacterial suspension was discarded and the graphene oxide membrane was gently washed with a buffer solution to remove unattached cells; the washed graphene oxide membrane was transferred to 20 mL of buffer solution, immersed and placed in a 150 rpm shaker for 10 min to separate the deposited bacterial cells; the cell suspension was then diluted to 10 4 -10 5 cells / mL, and the plate colony counting method was used to count the concentration of bacteria attached to the surface of graphene oxide membrane to characterize the degree of biological contamination of graphene oxide membrane.
2. The method for mitigating biological contamination of graphene oxide materials based on regulating kinetic movement ability as claimed in claim 1, characterized in that: The specific operation of step 1 is as follows: Shewanella oneidensis MR-1 is selected as the bacterial movement research object; MR-1 is inoculated into 25 mL of soybean nutrient medium, and the medium is placed in a constant temperature shaker at 32° C. and 160 rpm for 10 hours, and then expanded and cultured, and taken out for use after 15 hours; Preparation of soybean nutrient medium: weigh 0.8 g of soybean liquid medium and dissolve it in 25 mL of ultrapure water. After high temperature and high pressure sterilization, obtain the soybean nutrient medium, which is placed on an ultraclean bench for use.
3. The method for mitigating biological contamination of graphene oxide materials based on regulating kinetic movement ability as claimed in claim 1, characterized in that: The specific operations of step 2 are as follows: Graphite powder, concentrated nitric acid (98%), and concentrated sulfuric acid (98%) were mixed and stirred in a ratio of 1 g: (9-11) mL: (44-48) mL under ice bath conditions, 7-8 g KMnO4 was slowly added, and stirring was continued for 110-120 min under ice bath conditions, and then the reaction solution was moved to a 33-35 ° C water bath and stirred overnight; 44-48 mL ultrapure water was slowly added to the reaction system, and the system temperature was gradually increased, and an oil bath of 96-98 ° C was placed for 150-160 min; after the reaction solution was cooled, 200-210 mL ultrapure water was slowly added to the system, and 23-25 mL was added dropwise 30% H2O2 solution to remove excess KMnO4; collect the obtained graphite oxide precipitate by centrifugation (6000g, 30min), wash it twice with 10% dilute hydrochloric acid, and then wash it several times with ultrapure water until the supernatant is neutral; redisperse the collected graphite oxide in high-purity water, and ultrasonically peel it to obtain a single layer or several layers of graphene oxide.
4. The method for mitigating biological contamination of graphene oxide materials based on regulating kinetic movement ability as claimed in claim 1, characterized in that: In step 8, the buffer solution is prepared by weighing the required amount of hydroxyethylpiperazineethanesulfonic acid and dissolving it in ultrapure water to a concentration of 20 mmol / L, adjusting the pH to 6.8-7.2, and sterilizing with high temperature and high pressure to obtain a buffer solution.