Method for degrading new pollutants through co-culture of microalgae and graphene oxide

Abstract

The invention belongs to the field of environmental governance and biology, and provides a method for degrading new pollutants through co-culture of microalgae and graphene oxide, which comprises the following steps: S1, preparing graphene oxide through a Hummers method, and carrying out ultrasonic dispersion pretreatment; s2, carrying out expanding culture on freshwater algae, namely the clostridium clostridium, in an SE culture medium to a logarithmic phase, collecting algae cells, and adjusting the algae cells to target density; s3, mixing the graphene oxide prepared in the step S1 with the algae liquid in the step S2, and carrying out co-culture under the conditions of illumination and shaking table; s4, adding wastewater containing new pollutants into the co-culture system, and removing the pollutants through the synergistic effect of the adsorption effect of graphene oxide and the metabolic degradation effect of microalgae; s5, regularly sampling and analyzing the concentration change of the pollutants, and evaluating the degradation efficiency; according to the method disclosed by the invention, through co-culture of the graphene oxide and the acuminate clobetosa and the synergistic effect of GO adsorption and algae degradation, the pollutants are thoroughly mineralized, the removal efficiency of the refractory pollutants is remarkably improved, and the application range is widened.

Description

Technical Field

[0001] The present invention belongs to the fields of environmental governance and biology, and specifically relates to a method for co-culturing microalgae and graphene oxide to degrade new pollutants. Background Art

[0002] With the acceleration of industrialization, new pollutants (such as pharmaceuticals, perfluorinated compounds, plastic particles, etc.) have accumulated in the environment, becoming a serious problem. These pollutants have unique chemical properties and are difficult to effectively remove using traditional physical, chemical, and physicochemical methods. Although biological methods (such as microbial degradation) have achieved results, they have problems such as long treatment cycles, high energy consumption, and high costs; in recent years, research on using plants and algae to treat wastewater has received attention, especially the potential of Raphidocelis subcapitata in water purification. This freshwater alga can adsorb and degrade organic pollutants, but its effect is limited when used alone, and its ability to treat recalcitrant new pollutants is weak; graphene is mainly used for physically adsorbing pollutants, but its adsorption speed is slow, and its removal effect on some pollutants such as certain poorly soluble organic compounds is limited; Raphidocelis subcapitata has a weak degradation ability and a slow degradation speed when treating certain types of pollutants (especially recalcitrant new pollutants).

[0003] Existing methods for treating emerging pollutants have problems such as long treatment cycles, high energy consumption, and high costs.

[0004] Therefore, those skilled in the art have proposed a method for co-culturing microalgae and graphene oxide to degrade new pollutants to solve the problems raised in the background art. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a method for co-culturing microalgae and graphene oxide to degrade new pollutants, so as to solve the problems of long treatment cycles, high energy consumption, high costs, etc. in the existing methods for treating new pollutants.

[0006] A method for co-culturing microalgae and graphene oxide to degrade new pollutants includes the following steps:

[0007] S1. Prepare graphene oxide by the Hummers method and perform ultrasonic dispersion pretreatment;

[0008] S2. Expand the freshwater alga Raphidocelis subcapitata in SE medium to the logarithmic growth phase, collect the algal cells and adjust them to the target density;

[0009] S3. Mix the graphene oxide prepared in S1 with the algal solution in S2 and perform co-culturing under light and oscillation conditions;

[0010] S4. Add wastewater containing new pollutants to the co-culture system, and synergistically remove the pollutants through the adsorption of graphene oxide and the metabolic degradation of microalgae;

[0011] S5. Regularly sample and analyze the change of pollutant concentration to evaluate the degradation efficiency.

[0012] Preferably, the preparation steps of graphene oxide in S1: Mix graphite powder with concentrated sulfuric acid, add potassium permanganate for oxidation reaction at low temperature; raise the temperature to 35 - 45 °C for continuous oxidation, and add hydrogen peroxide to terminate the reaction after the reaction is completed; wash with deionized water until pH≈5, and obtain graphene oxide powder after drying.

[0013] Preferably, the concentration of graphene oxide in S1 is 0.1 - 1.0 g / L, and it is ultrasonically treated for 20 - 40 min with a power of 200 - 400 W before dispersion.

[0014] Preferably, the culture conditions of the microalgae in S2 are: temperature: 20 - 25 °C, light intensity: 6000 - 10000 lux, light - dark cycle: 14 - 18 h light / 6 - 10 h dark, and the density of the algal solution is adjusted to 3.0×10 6 -4.0×10 6 cells / mL.

[0015] Preferably, the co - culture conditions in S3 include: the added concentration of graphene oxide is 0.1 - 0.5 g / L, light intensity: 20000 - 40000 lux, shaker speed: 120 - 200 rpm, and culture temperature: 20 - 25 °C.

[0016] Preferably, the new pollutants in S4 include at least one of pharmaceutical residues, perfluorinated compounds, and microplastics, and the initial concentration of the pollutants is 0.1 - 100 mg / L.

[0017] Preferably, the monitoring steps of the pollutant degradation efficiency in S5: Centrifuge to separate algal cells and graphene oxide; after the supernatant is filtered through a 0.22 - μm filter membrane, analyze the residual concentration of the pollutants by high - performance liquid chromatography (LC).

[0018] Preferably, the verification method of the synergistic effect between graphene oxide and microalgae includes: setting an adsorption group containing only graphene oxide (GO), a degradation group containing only microalgae, a co - culture group of GO and microalgae, and a blank control group; comparing the pollutant removal rates of each group, and the synergistic effect is defined as the removal rate of the co - culture group being higher than the sum of the removal rates of the GO adsorption group and the microalgae degradation group.

[0019] Preferably, it also includes the recovery step of graphene oxide: After the co - culture is completed, separate graphene oxide and algal bodies by centrifugation or filtration; graphene oxide is reused after acid washing or high - temperature treatment.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] Through the co - culture of graphene oxide and Raphidocelis subcapitata, the present invention thoroughly removes pollutants through the synergistic effect of GO adsorption and algal degradation, significantly improves the removal efficiency of refractory pollutants, and broadens the application scope; shortens the degradation time and reduces energy consumption; GO can be recycled and algae can be utilized resourcefully, reducing the operating cost; the operation is simple and easy to scale up; no chemical reagents are added, and the biodegradation product is CO 2 and H 2 O, without secondary pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a schematic diagram of the overall structure of the present invention;

[0023] Figure 2 is a schematic diagram of the co - culture complex of graphene oxide and Raphidocelis subcapitata. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The following further describes the embodiments of the present invention in detail with reference to the drawings and examples. The following examples are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0025] As shown in the attached Figure 1 to the attached Figure 2 figures: The present invention provides a method for co - culturing microalgae and graphene oxide to degrade new pollutants, including the following steps:

[0026] S1. Prepare graphene oxide. Graphene oxide is prepared by the Hummers method and subjected to ultrasonic dispersion pretreatment. The concentration of graphene oxide is 0.1 - 1.0 g / L, and it is ultrasonically treated for 20 - 40 min before dispersion, with a power of 200 - 400 W;

[0027] Steps for preparing graphene oxide:

[0028] (a) Mix graphite powder with concentrated sulfuric acid and add potassium permanganate for oxidation reaction at low temperature;

[0029] (b) Raise the temperature to 35 - 45 °C and continue oxidation. After the reaction is completed, add hydrogen peroxide to terminate the reaction;

[0030] (c) Wash with deionized water until pH≈5, and dry to obtain graphene oxide powder.

[0031] S2. Microalgae culture. The freshwater alga Raphidocelis subcapitata is expanded in SE medium to the logarithmic growth phase, and the algal cells are concentrated, collected and adjusted to the target density;

[0032] The culture conditions of microalgae are as follows:

[0033] Temperature: 20 - 25°C; Light intensity: 6000 - 10000 lux;

[0034] Light - dark cycle: 14 - 18 h light / 6 - 10 h dark; The density of the algal solution is adjusted to 3.0×10 6 -4.0×10 6 cells / mL.

[0035] S3. Construction of the co - culture system: Mix the graphene oxide prepared in S1 with the algal solution in S2, and conduct co - culture under light and shaker conditions;

[0036] The conditions for co - culture include: The addition concentration of graphene oxide is 0.1 - 0.5 g / L; Light intensity: 20000 - 40000 lux; Shaker rotation speed: 120 - 200 rpm; Culture temperature: 20 - 25°C.

[0037] S4. Pollutant degradation treatment: Add wastewater containing new pollutants to the co - culture system, and remove pollutants through the synergistic effect of the adsorption of graphene oxide and the metabolic degradation of microalgae; The new pollutants include at least one of drug residues, perfluorinated compounds, and microplastics, and the initial concentration of pollutants is 0.1 - 100 mg / L

[0038] S5. Dynamic monitoring: Regularly take samples to analyze the change of pollutant concentration and evaluate the removal efficiency;

[0039] Monitoring steps for pollutant removal efficiency:

[0040] (a) Centrifuge to separate algal cells and graphene oxide;

[0041] (b) After the supernatant is filtered through a 0.22 - μm filter membrane, analyze the residual concentration of pollutants by high - performance liquid chromatography (LC).

[0042] The verification method of the synergistic effect between graphene oxide and microalgae is as follows: Set up an adsorption group containing only GO, a degradation group containing only microalgae, a co - culture group of GO and microalgae, and a blank control group; Compare the pollutant removal rates of each group, and the synergistic effect is defined as the removal rate of the co - culture group being higher than the sum of the removal rates of the GO adsorption group and the microalgae degradation group.

[0043] It also includes the recovery step of graphene oxide: After co - culture, separate graphene oxide and algal bodies by centrifugation or filtration; Graphene oxide is reused after acid washing or high - temperature treatment.

[0044] This method is applicable to the treatment of new pollutants in industrial wastewater, surface water or domestic sewage.

[0045] Example: Removal of sulfonamide antibiotics (sulfamethoxazole, SMX)

[0046] 1. Preparation and Characterization of Graphene Oxide

[0047] Synthesis of GO by Hummers method:

[0048] ① Raw material pretreatment: Weigh 1.0 g of natural graphite powder (particle size < 20 μm, purity ≥ 99.9%) and place it in a dry beaker; Measure 50 mL of concentrated sulfuric acid (H 2 SO 4 , 98%) and cool it to 10 °C in an ice bath.

[0049] ② Low-temperature oxidation: Under continuous stirring (magnetic stirrer, rotation speed 500 rpm), slowly add 3.0 g of potassium permanganate (KMnO 4 ) to the graphite-sulfuric acid mixture in 4 portions, control the reaction temperature ≤ 20 °C, and react for 2 h.

[0050] ③ Medium-temperature oxidation: Remove the ice bath, heat the reaction system to 40 ± 2 °C, and continue stirring and reacting for 4 h until the mixture becomes dark brown and viscous.

[0051] ④ Terminate the reaction: Slowly add 100 mL of pre-cooled deionized water (4 °C), stir for 15 min, and then dropwise add 5 mL of hydrogen peroxide (H 2 O 2 , 30%). The solution turns bright yellow, and let it stand for precipitation.

[0052] ⑤ Purification and drying: Collect the product using a vacuum filtration device (filter membrane pore size 0.45 μm), wash it repeatedly with deionized water until the pH of the filtrate is 5.0 ± 0.2; Dry it in a vacuum drying oven at 60 °C for 24 h to obtain graphene oxide powder.

[0053] Dispersion and Characterization of GO:

[0054] Add the dried GO powder to deionized water to prepare a 0.5 g / L suspension, and ultrasonically treat it for 30 min (ultrasonic cleaner, power 200 W, frequency 40 kHz);

[0055] Observe the GO sheet structure through transmission electron microscopy (TEM, model JEM-2100), showing that the monolayer rate > 80%; Raman spectroscopy (intensity ratio of D peak to G peak ID / IG = 1.12) confirms moderate oxidation degree.

[0056] 2. Cultivation and Pretreatment of

[0057] Configuration of culture medium: Prepare SE culture medium according to the standard formula (composition: NaNO 3 : 250 mg / L, CaCl 2 ·2H 2 O: 25 mg / L, MgSO4 ·7H 2 O: 75 mg / L, K 2 HPO 4 ·3H 2 O: 75 mg / L, KH 2 PO 4 : 175 mg / L, NaCl: 25 mg / L, FeCl3·6H 2 O: 5 mg / L, Fe-EDTA: 1 mL / L, trace element solution 1 mL / L), adjust the pH to 7.0 ± 0.2, autoclave at 121 °C for 20 min.

[0058] Algal strain expansion culture: In a laminar flow hood, inoculate the preserved algal strain into the sterilized SE medium;

[0059] Place it in a light incubator (model S-LI350B), introduce air filtered through a 0.22 μm filter membrane (flow rate 0.5 L / min), and the culture conditions are:

[0060] Temperature: 22 ± 2 °C;

[0061] Light intensity: 8000 lux (cool white fluorescent lamp, light quality ratio of red to blue light = 3:1);

[0062] Light-dark cycle: 16 h light / 8 h dark;

[0063] Collection in the logarithmic phase: After culturing for 5 - 7 days, when the algal density reaches 3.5×10 6 cells / mL (detected by a hemocytometer), centrifuge at 8000 rpm for 10 min (centrifuge model Sorvall STEAM 8R), discard the supernatant, and resuspend with sterile SE medium to the target density.

[0064] 3. Construction of the co-culture system and treatment of pollutants

[0065] GO-algae mixed system:

[0066] Mix the 0.5 g / L GO suspension with the algal solution (3.5×10 6 cells / mL) at a volume ratio of 1:10, transfer it to a 250 mL conical flask, wrap the mouth of the flask with tin foil, and seal it with a sealing film.

[0067] Place it in a light shaking incubator (model ZQLY-300G), and the co-culture conditions are:

[0068] Light intensity: 30000 lux (LED light source, spectral range 400 - 700 nm);

[0069] Temperature: 22 ± 2 °C;

[0070] Shaking table speed: 160 rpm;

[0071] Initial pH: 7.0 ± 0.2 (adjusted with 1 mol / L NaOH and HCl solutions).

[0072] Pollutant addition: Preparation of sulfamethoxazole (SMX) stock solution: Weigh 100 mg of SMX (purity ≥ 98%), dissolve it in 100 mL of ultrapure water, filter it through a 0.22 μm membrane filter, and prepare a 1000 mg / L simulated wastewater; add the SMX wastewater to the co-culture system with a final concentration of 10 mg / L.

[0073] 4. Monitoring of removal efficiency

[0074] Dynamic sampling:

[0075] Time points: 0, 24, 48, 72, 96 h; Take 5 mL of the mixed solution each time and immediately store it in the dark at 4 °C.

[0076] Sample treatment:

[0077] ① Centrifugal separation: 8000 rpm, 4 °C, centrifuge for 10 min, and filter the supernatant through a 0.22 μm membrane filter;

[0078] ② LC analysis:

[0079] Instrument: Waters 2695 high performance liquid chromatograph; Chromatographic column: ZORBAX Eclipse Plus C18 (4.6 × 250 mm, 5 μm);

[0080] Mobile phase: Phase A (0.1% 0.01 mol / L phosphoric acid water), Phase B (20% acetonitrile).

[0081] 5. Verification of synergistic effect

[0082] Setting of control groups:

[0083] Only GO group: 0.5 g / L GO + SMX wastewater (without algae);

[0084] Only algae group: 3.5×10 6 cells / mL algal solution + SMX wastewater (without GO);

[0085] Blank group: SMX wastewater (without GO and without algae).

[0086] In this method, GO can efficiently adsorb pollutants through its high specific surface area and abundant oxygen-containing functional groups, and the adsorbed pollutants can be completely degraded into harmless substances (such as CO 2 and H 2O), secondary pollution is avoided; GO continuously releases the adsorbed pollutants in the co-culture system for algae degradation, prolonging the service life of GO and reducing the regeneration cost.

[0087] The adsorption of GO reduces the toxicity of pollutants to algal cells, and at the same time enriches the pollutants on the surface of algal cells, significantly improving the degradation efficiency. For example, the drug residue (such as sulfamethoxazole) breaks through the limitations of traditional biological methods.

[0088] Through the synergistic effect of GO and algae, the degradation time of pollutants can be significantly shortened. For example, sulfamethoxazole (SMX) can achieve a significant removal effect in a short time, while traditional methods usually take a long time; only light and shaker conditions are required, without additional energy input (such as high temperature, high pressure), reducing the operating cost.

[0089] And GO can be recycled and reused (such as magnetic separation, pickling regeneration); algal biomass can be converted into biogas through anaerobic fermentation or used as bio-fertilizer to achieve resource utilization.

[0090] No chemical reagents need to be added. Both GO and algae are environmentally friendly materials, and the degradation products are CO 2 and H 2 O, without secondary pollution; through flow cytometry detection, the survival rate of algal cells in the co-culture system is > 90%, indicating that GO has no significant toxicity to algae.

[0091] The GO-algae co-culture system can be integrated into a photobioreactor (PBR) to achieve continuous flow treatment, which is suitable for scenarios such as industrial wastewater treatment plants and surface water restoration; only basic parameters such as light, temperature, and shaker speed need to be controlled, without complex equipment or professional technical support.

[0092] Through control group experiments (GO only group, algae only group, co-culture group) and characterization methods such as LC and SEM, the synergistic mechanism of GO adsorption and algae degradation was clarified; detailed methods for GO preparation, algae culture, co-culture conditions, and pollutant monitoring were provided, which are easy to promote and apply.

[0093] In this method, graphene oxide is added to the culture medium of Centroceras clavulatum, and the high specific surface area and abundant oxygen-containing groups of graphene oxide are used to adsorb pollutants in water. At the same time, Centroceras clavulatum accelerates the degradation of pollutants through photosynthesis and metabolic degradation.

[0094] In this solution, the preparation method of graphene oxide (Hummers method) is relatively mature, and graphene oxide itself is relatively economical and has strong pollutant adsorption capacity. Through the synergistic effect of graphene oxide and Raphidiopsis subcapitata, the degradation efficiency of pollutants in wastewater is significantly improved. This method does not require high energy consumption and a large amount of chemical reagents, and can reduce the negative impact on the environment. It is applicable to the removal of various types of water pollutants, including emerging pollutants and refractory organic pollutants.

[0095] The embodiments of the present invention are given for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

[0096] In the description of the present invention, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. The meaning of "a plurality" is two or more unless otherwise specifically defined.

[0097] In the present invention, unless otherwise clearly defined and limited, the terms such as "installed", "connected", "connected to", "fixed" and the like should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or integrated; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and it may be the internal communication of two components or the interaction relationship between two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0098] In the present invention, unless otherwise clearly defined and limited, the first feature being "on" or "under" the second feature may be that the first and second features are in direct contact, or the first and second features are indirectly in contact through an intermediate medium. Moreover, the first feature being "above", "over" and "on top of" the second feature may be that the first feature is directly above or obliquely above the second feature, or merely indicates that the first feature has a higher horizontal height than the second feature. The first feature being "under", "below" and "beneath" the second feature may be that the first feature is directly below or obliquely below the second feature, or merely indicates that the first feature has a lower horizontal height than the second feature.

[0099] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0100] In the drawings of the disclosed embodiments of the present invention, only the structures related to the disclosed embodiments are involved, and other structures can refer to the general design. Without conflict, the same embodiment and different embodiments of the present invention can be combined with each other.

[0101] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for degrading new pollutants by co-culturing microalgae and graphene oxide, characterized in that: The following steps are involved: S1. preparing graphene oxide by Hummers method and performing ultrasonic dispersion pretreatment; S2, expanding the freshwater algae Achyranthes subcapitata in SE medium to the logarithmic growth phase, collecting algal cells and adjusting to the target density; S3, mixing the graphene oxide prepared in S1 with the algae solution in S2, and co-culturing under illumination and shaking conditions; S4, adding wastewater containing new pollutants into the co-cultivation system, and removing pollutants through the synergistic effect of graphene oxide adsorption and microalgae metabolic degradation; S5. Take samples regularly to analyze changes in pollutant concentrations and evaluate degradation efficiency.

2. A method for degrading new pollutants by co-culturing microalgae and graphene oxide as claimed in claim 1, characterized in that: The preparation steps of graphene oxide in S1 are as follows: mixing graphite powder with concentrated sulfuric acid, adding potassium permanganate at low temperature for oxidation reaction; heating to 35-45°C to continue oxidation, adding hydrogen peroxide to terminate the reaction after the reaction is completed; washing with deionized water to pH≈5, and drying to obtain graphene oxide powder.

3. A method for degrading new pollutants by co-culturing microalgae and graphene oxide as claimed in claim 2, characterized in that: The concentration of graphene oxide in S1 is 0.1-1.0 g / L, and it is ultrasonically treated for 20-40 min at a power of 200-400 W before dispersion.

4. A method for degrading new pollutants by co-culturing microalgae and graphene oxide as claimed in claim 3, characterized in that: The culture conditions of the microalgae described in S2 are as follows: temperature: 20-25°C; light intensity: 6000-10000 lux; light-dark cycle: 14-18 h light / 6-10 h dark; the algae solution density was adjusted to 3.0×10 6 -4.0×10 6 cells / mL.

5. A method for degrading new pollutants by co-culturing microalgae and graphene oxide as claimed in claim 4, characterized in that: The co-culture conditions in S3 include: graphene oxide addition concentration of 0.1-0.5 g / L; light intensity: 20000-40000 lux; shaker speed: 120-200 rpm; culture temperature: 20-25°C.

6. A method for degrading new pollutants by co-culturing microalgae and graphene oxide as claimed in claim 5, characterized in that: The new pollutants described in S4 include at least one of drug residues, perfluorinated compounds, and microplastics, and the initial concentration of the pollutants is 0.1-100 mg / L.

7. A method for degrading new pollutants by co-culturing microalgae and graphene oxide as claimed in claim 1 or 6, characterized in that: Monitoring steps of pollutant degradation efficiency in S5: centrifugal separation of algae cells and graphene oxide; filtering the supernatant through a 0.22 μm filter membrane, and analyzing the residual concentration of pollutants by high performance liquid chromatography.

8. The method for degrading new pollutants by co-culturing microalgae and graphene oxide as claimed in claim 1, characterized in that: The process also includes a step of recovering graphene oxide: after the co-cultivation, the graphene oxide and the algae are separated by centrifugation or filtration; and the graphene oxide is reused after acid washing or high temperature treatment.

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