Method for degrading new pollutants by co-culturing microalgae with graphene oxide

Abstract

The present application belongs to the field of environmental governance and biology, and provides a method for degrading new pollutants by co-culturing microalgae and graphene oxide, comprising the following steps: S1, preparing graphene oxide by Hummers method and performing ultrasonic dispersion pretreatment; S2, expanding freshwater algae Synedra ulna to the logarithmic growth phase in SE culture medium, collecting the algal cells and adjusting to the target density; S3, mixing the graphene oxide prepared in S1 with the algal liquid in S2, and co-culturing under the conditions of light and shaking table; S4, adding wastewater containing new pollutants to the co-culturing system, and removing the pollutants by the adsorption of graphene oxide and the metabolic degradation of microalgae; S5, regularly sampling and analyzing the concentration change of the pollutants, and evaluating the degradation efficiency; by co-culturing graphene oxide and Synedra ulna, the present application completely mineralizes the pollutants by the synergistic effect of GO adsorption and algal degradation, significantly improves the removal efficiency of refractory pollutants, and widens the application range.

Description

Technical Field

[0001] This invention belongs to the fields of environmental remediation and biology, specifically a method for co-culturing microalgae and graphene oxide to degrade new pollutants. Background Technology

[0002] With accelerated industrialization, new pollutants (such as pharmaceuticals, perfluorinated compounds, and microplastics) have accumulated in the environment, becoming a serious problem. These pollutants have unique chemical properties, making them difficult to remove effectively using traditional physical, chemical, and physicochemical methods. While biological methods (such as microbial degradation) have shown some effectiveness, they suffer from long treatment cycles, high energy consumption, and high costs. In recent years, research on wastewater treatment using plants and algae has attracted attention, particularly the potential of *Raphidocelis subcapitata* in water purification. This freshwater algae can adsorb and degrade organic pollutants, but its effectiveness is limited when used alone, and it has a weak ability to treat recalcitrant new pollutants. Graphene is mainly used for the physical adsorption of pollutants, but its adsorption rate is slow, and its removal effect on some poorly soluble organic pollutants is limited. *Raphidocelis subcapitata* has a weak degradation capacity and a slow degradation rate when treating certain types of pollutants (especially recalcitrant new pollutants).

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

[0004] To address the problems raised in the background art, those skilled in the art have proposed a method for co-culturing microalgae with graphene oxide to degrade novel pollutants. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for co-culturing microalgae and graphene oxide to degrade novel pollutants, thereby solving the problems of long processing cycles, high energy consumption, and high costs in existing methods for treating novel pollutants.

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

[0007] S1. Graphene oxide was prepared by the Hummers method and subjected to ultrasonic dispersion pretreatment;

[0008] S2. The freshwater algae *Neptune cephalopoda* was cultured in SE medium to the logarithmic growth phase, and the algal cells were collected and adjusted to the target density.

[0009] S3. Mix the graphene oxide prepared in S1 with the algal solution in S2 and co-cultivate them under light and shaking conditions.

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

[0011] S5. Regularly sample and analyze changes in pollutant concentrations to assess degradation efficiency.

[0012] Preferably, the preparation steps of graphene oxide in S1 are as follows: graphite powder is mixed with concentrated sulfuric acid, potassium permanganate is added at low temperature to carry out oxidation reaction; the temperature is raised to 35-45℃ to continue oxidation, and hydrogen peroxide is added to terminate the reaction after the reaction is completed; after washing with deionized water to pH≈5, graphene oxide powder is obtained 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 before dispersion, with a power of 200-400 W.

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

[0015] Preferably, the conditions for co-culturing in S3 include: graphene oxide concentration of 0.1-0.5 g / L, light intensity of 20,000-40,000 lux, shaking speed of 120-200 rpm, and culture temperature of 20-25℃.

[0016] Preferably, the new pollutant in S4 includes at least one of drug residues, perfluorinated compounds, and microplastics, with an initial concentration of 0.1-100 mg / L.

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

[0018] Preferably, the verification method for the synergistic effect of graphene oxide and microalgae includes: setting up 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, 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, the method also includes a graphene oxide recovery step: after co-cultivation, graphene oxide and algae are separated by centrifugation or filtration; the 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] This invention utilizes the co-cultivation of graphene oxide and *Alternaria septemlobus* to thoroughly remove pollutants through the synergistic effect of GO adsorption and algal degradation, significantly improving the removal efficiency of recalcitrant pollutants and broadening the application scope; it also shortens degradation time and reduces energy consumption; GO is recyclable, and algae can be utilized as resources, reducing operating costs; the operation is simple and easy to scale up; no chemical reagents are added, and the biodegradation products are CO2 and H2O, with no secondary pollution. Attached Figure Description

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

[0023] Figure 2 This is a schematic diagram of the co-culture complex of graphene oxide and *Neptunus cephalopoda*. Detailed Implementation

[0024] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0025] As attached Figure 1 To be continued Figure 2 As shown: This invention provides a method for co-culturing microalgae and graphene oxide to degrade novel pollutants, comprising the following steps:

[0026] S1. Preparation of 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. Before dispersion, it is ultrasonically treated for 20-40 min with a power of 200-400 W.

[0027] Preparation steps of graphene oxide:

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

[0029] (b) Heat to 35-45℃ and continue oxidation. After the reaction is complete, add hydrogen peroxide to terminate the reaction.

[0030] (c) After washing with deionized water to pH≈5, graphene oxide powder was obtained after drying.

[0031] S2. Microalgae culture: The freshwater algae Raphidocelis subcapitata was cultured in SE medium to the logarithmic growth phase, and the algal cells were concentrated, collected, and adjusted to the target density.

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

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

[0034] Light-dark cycle: 14-18 hours of light / 6-10 hours of darkness; algal solution density adjusted to 3.0 × 10⁻⁶. 6 -4.0×10 6 cells / mL.

[0035] S3. Construction of co-culture system: The graphene oxide prepared in S1 was mixed with the algal solution in S2 and co-cultured under light and shaking conditions.

[0036] The co-cultivation conditions include: graphene oxide concentration of 0.1-0.5 g / L; light intensity of 20,000-40,000 lux; shaking speed of 120-200 rpm; and cultivation temperature of 20-25℃.

[0037] S4. Pollutant Degradation Treatment: Wastewater containing new pollutants is added to the co-culture system. The pollutants are removed through the synergistic effect of graphene oxide adsorption and microalgae metabolic degradation. The new pollutants include at least one of drug residues, perfluorinated compounds, and microplastics, with an initial concentration of 0.1-100 mg / L.

[0038] S5. Dynamic monitoring, regular sampling and analysis of pollutant concentration changes, and assessment of removal efficiency;

[0039] Monitoring steps for pollutant removal efficiency:

[0040] (a) Centrifugation to separate algal cells from graphene oxide;

[0041] (b) The supernatant was filtered through a 0.22 μm filter membrane and the residual concentration of contaminants was analyzed by high performance liquid chromatography (LC).

[0042] The synergistic effect of graphene oxide and microalgae was verified as follows: 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 were set up; the pollutant removal rates of each group were compared, and the synergistic effect was 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 a graphene oxide recovery step: after co-cultivation, graphene oxide and algae are separated by centrifugation or filtration; the 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 1.0g of natural graphite powder (particle size <20μm, purity ≥99.9%) and place it in a dry beaker; measure 50mL of concentrated sulfuric acid (H2SO4, 98%) and cool it to 10℃ in an ice bath.

[0049] ② Low-temperature oxidation: Under continuous stirring (magnetic stirrer, speed 500 rpm), 3.0 g of potassium permanganate (KMnO4) was slowly added to the graphite-sulfuric acid mixture in 4 portions, and the reaction temperature was controlled at ≤20℃ for 2 h.

[0050] ③Medium-temperature oxidation: Remove the ice bath, raise the temperature of the reaction system to 40±2℃, and continue stirring for 4 hours until the mixture becomes dark brown and viscous.

[0051] ④ Termination of reaction: Slowly add 100 mL of pre-cooled deionized water (4℃), stir for 15 min, then add 5 mL of hydrogen peroxide (H2O2, 30%) dropwise. The solution turns bright yellow and is allowed to stand to precipitate.

[0052] ⑤ Purification and drying: The product was collected using a vacuum filtration device (filter membrane pore size 0.45μm), and repeatedly washed with deionized water until the pH of the filtrate was 5.0±0.2; it was then dried in a vacuum drying oven at 60℃ for 24h to obtain graphene oxide powder.

[0053] GO Dispersion and Characterization:

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

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

[0056] 2. Cultivation and Pretreatment of *Nepaliformis*

[0057] Culture medium preparation: Prepare SE culture medium according to the standard formula (components: NaNO3: 250mg / L, CaCl2·2H2O: 25mg / L, MgSO4·7H2O: 75mg / L, K2HPO4·3H2O: 75mg / L, KH2PO4: 175mg / L, NaCl: 25mg / L, FeCl3·6H2O: 5mg / L, Fe-EDTA: 1mL / L, trace element solution 1mL / L), adjust the pH to 7.0±0.2, and autoclave at 121℃ for 20min.

[0058] Algal culture expansion: In a clean bench, the preserved algal culture is inoculated onto sterile SE medium;

[0059] Placed in a light incubator (model S-LI350B), with air filtered through a 0.22μm filter membrane (flow rate 0.5L / min), incubation conditions:

[0060] Temperature: 22±2℃;

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

[0062] Light-dark cycle: 16 hours of light / 8 hours of darkness;

[0063] Logarithmic phase collection: After 5-7 days of cultivation, the algal density reaches 3.5 × 10⁻⁶. 6 Cells / mL (detected by hemocytometer), centrifuged at 8000 rpm for 10 min (centrifuge model Sorvall STEAM 8R), supernatant discarded, and resuspended in sterile SE medium to the target density.

[0064] 3. Construction of co-cultivation system and pollutant treatment

[0065] GO-Algae Mixed System:

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

[0067] Placed in a light-controlled shaking incubator (model ZQLY-300G), co-culture conditions:

[0068] Illumination intensity: 30,000 lux (LED light source, spectral range 400-700nm);

[0069] Temperature: 22±2℃;

[0070] Shaking speed: 160 rpm;

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

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

[0073] 4. Removal efficiency monitoring

[0074] Dynamic sampling:

[0075] Time points: 0, 24, 48, 72, 96 h; take 5 mL of the mixture each time and immediately store it at 4℃ away from light.

[0076] Sample processing:

[0077] ① Centrifugation: Centrifuge at 8000 rpm, 4℃ for 10 min, and filter the supernatant through a 0.22 μm filter membrane;

[0078] ②LC analysis:

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

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

[0081] 5. Validation of synergistic effects

[0082] Control group setup:

[0083] GO group only: 0.5g / L GO + SMX wastewater (algae-free);

[0084] Algal section only: 3.5 × 10 6 cells / mL algal solution + SMX wastewater (no GO);

[0085] Blank group: SMX wastewater (no GO, no algae).

[0086] In this method, GO can efficiently adsorb pollutants through its high specific surface area and abundant oxygen-containing functional groups. The near-cephalopoda algae completely degrade the adsorbed pollutants into harmless substances (such as CO2 and H2O) through metabolism, thus avoiding secondary pollution. In the co-culture system, GO continuously releases the adsorbed pollutants for algal degradation, which extends the service life of GO and reduces regeneration costs.

[0087] GO adsorption reduces the toxicity of pollutants to algal cells and enriches pollutants on the surface of algal cells, which can significantly improve degradation efficiency. For example, it breaks through the limitations of traditional biological methods for drug residues (such as sulfamethoxazole).

[0088] The synergistic effect of GO and algae can significantly shorten the degradation time of pollutants. For example, sulfamethoxazole (SMX) can achieve significant removal in a short time, while traditional methods are usually time-consuming; it only requires light and shaking conditions, without the need for additional energy input (such as high temperature and high pressure), thus reducing operating costs.

[0089] Furthermore, GO can be recycled and reused (e.g., through magnetic separation and acid washing regeneration); algal biomass can be converted into biomethane through anaerobic fermentation or used as biofertilizer, thus realizing resource utilization.

[0090] No chemical reagents are needed. Both GO and algae are environmentally friendly materials, and their degradation products are CO2 and H2O, with no secondary pollution. Flow cytometry analysis showed that the algal cell survival rate in the co-culture system was >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 industrial wastewater treatment plants, surface water remediation and other scenarios. Only basic parameters such as light, temperature and shaking speed need to be controlled, without the need for complex equipment or professional technical support.

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

[0093] This method involves adding graphene oxide to the culture medium of *Plasmodium septum*, utilizing the high specific surface area and abundant oxidizing groups of graphene oxide to adsorb pollutants in the water. At the same time, *Plasmodium septum* accelerates the degradation of pollutants through photosynthesis and metabolic degradation.

[0094] In this scheme, the preparation method of graphene oxide (Hummers method) is relatively mature, and graphene oxide itself is relatively economical and has a strong pollutant adsorption capacity. Through the synergistic effect of graphene oxide and *Neptunia spicata*, 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, which can reduce the negative impact on the environment. It is suitable for removing various types of water pollutants, including new pollutants and recalcitrant organic pollutants.

[0095] The embodiments of the present invention are given for the purposes of illustration and description. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled 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 this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. "A plurality of" means two or more, unless otherwise explicitly specified.

[0097] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0098] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0099] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions 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 suitable manner in one or more embodiments or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0100] The accompanying drawings of the embodiments disclosed in this invention only involve structures related to the embodiments disclosed in this invention. Other structures can refer to general designs. In the absence of conflict, the same embodiment and different embodiments of this 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 make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for degrading emerging pollutants by co-culturing microalgae with graphene oxide, characterized in that, The method comprises the following steps: S1, preparing graphene oxide by Hummers method and performing ultrasonic dispersion pretreatment; the preparation step of the graphene oxide is: mixing graphite powder with concentrated sulfuric acid, adding potassium permanganate at low temperature to perform oxidation reaction, continuing oxidation after heating to 35-45 DEG C, adding hydrogen peroxide to terminate the reaction after the reaction is completed, washing with deionized water until pH is about 5, and drying to obtain graphene oxide powder; the concentration of the graphene oxide is 0.1-1.0 g / L, and the graphene oxide is treated by ultrasonic wave with a power of 200-400 W for 20-40 min before dispersion; S2, the fresh water algae, Pinguiacola capensis, was expanded in SE medium to the logarithmic growth phase, and the culture conditions of the microalgae were as follows: temperature 20-25 °C, light intensity 6000-10000 lux, light and dark cycle 14-18 h light / 6-10 h dark, the algal cells were collected and adjusted to a target density of 3.0x10 6 -4.0x10 6 cells / mL; S3, mixing the graphene oxide prepared in S1 with the algal liquid in S2, and performing co-culture under light and oscillation conditions; S4, adding wastewater containing new pollutants to the co-culture system, and removing the pollutants by the adsorption of the graphene oxide and the metabolic degradation of the microalgae; S5, periodically sampling and analyzing the concentration change of the pollutants, and evaluating the degradation efficiency.

2. The method for co-culturing microalgae and graphene oxide to degrade new pollutants as described in claim 1, characterized in that: The co-culture conditions in S3 include: the graphene oxide is added at a concentration of 0.1-0.5 g / L; the light intensity is 20000-40000 lux; the shaking speed is 120-200 rpm; and the culture temperature is 20-25 DEG C.

3. The method according to claim 2, wherein the microalgae and graphene oxide are co-cultured in a medium containing the new pollutant. The new pollutants 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.

4. The method for co-culturing microalgae and graphene oxide to degrade new pollutants as described in claim 1 or 3, characterized in that: The monitoring step of the degradation efficiency of the pollutants in S5 is: centrifuging the algal cells and the graphene oxide; filtering the supernatant through a 0.22 mu m filter membrane, and analyzing the residual concentration of the pollutants by high performance liquid chromatography.

5. The method of claim 1, wherein the microalgae and graphene oxide co-culture degrades the new pollutant. The method further comprises a graphene oxide recovery step: after the co-culture is completed, the graphene oxide and the algal body are separated by centrifugation or filtration; and the graphene oxide is repeatedly used after acid washing or high temperature treatment.

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