Preparation of a recyclable 3D microalgae gel and method for removing antibiotics from water

Abstract

The present invention discloses a method for preparing a recyclable 3D microalgae gel and removing antibiotics from water. Based on 3D gel encapsulation technology and constructing an isolating semipermeable membrane on the surface of the gel matrix, a recyclable microalgae solid-state purification system has been developed, which synergizes the mild environment provided by the gel matrix and the isolating effect of the semipermeable membrane. While protecting the activity of the microalgae and achieving efficient biodegradation, it prevents leakage of the microalgae and ensures the containment of potential microbial biohazards. At the same time, through 3D printing technology, recyclable, low-cost, and rapid large-scale industrial manufacturing can be achieved, overcoming the shortcomings of traditional microalgae water treatment and having broad application prospects.

Description

Technical Field

[0001] The present invention relates to the application of algae composite materials in the field of wastewater treatment, and in particular to a method for preparing a recyclable 3D microalgae gel and removing antibiotics from water. Background Art

[0002] The advent of antibiotics has greatly facilitated the treatment of bacterial infections in humans and animals. However, the release of large quantities of untreated antibiotics into natural waterways can disrupt ecosystems and exacerbate the crisis of antibiotic resistance, resulting in approximately 1.2 million deaths annually from antibiotic-resistant bacterial infections. Consequently, the World Health Organization (WHO) has identified antibiotic resistance as one of the most serious challenges facing human health. While various methods for removing antibiotics, including advanced oxidation, adsorption, and ultrafiltration, have demonstrated good efficiency, these methods are complex and costly to prepare and may produce harmful byproducts. In contrast, bioremediation, which utilizes microorganisms to degrade antibiotics, offers an environmentally friendly technology for addressing antibiotic pollution. In particular, photosynthetic algae are a candidate for low-carbon bioremediation, accumulating valuable biomass and producing oxygen while processing antibiotics, enabling both wastewater bioremediation and carbon sequestration. For example, the published invention patent CN 114561293 A discloses a method for degrading cefradine in water using microalgae. However, the degradation efficiency is low, and the activity of the microalgae is inhibited at high antibiotic concentrations. At the same time, typical microalgae-mediated remediation technologies require the construction of large treatment ponds or pools, which require significant floor space and water volumes. Furthermore, suspended microalgae in wastewater are often difficult to recycle, and the influx of these microorganisms (especially engineered species) into natural water bodies can affect the distribution of local microbiota (e.g., causing algal blooms).

[0003] Cell-living materials provide an isolated environment by embedding cells in a matrix to protect them from environmental damage while allowing material exchange to ensure cell growth. Currently, most hydrogel matrices (such as sodium alginate) encapsulate cells in the form of gel particles. These systems have weak mechanical properties, limited diffusion of nutrients, and easy leaching of microorganisms, which limits their application. Summary of the Invention

[0004] To address the difficulties of traditional microalgae processing and recycling, poor microalgae bioactivity at high concentrations, and low degradation efficiency, as well as the problems of traditional packaging materials with weak mechanical properties, easy leakage of microorganisms leading to algal blooms, and difficulty in industrial manufacturing, the present invention provides a method for preparing a recyclable 3D microalgae gel network and removing antibiotics from water, which mainly includes the following steps:

[0005] 1. Prepare microalgae bio-ink: Dissolve polyethylene glycol diacrylate in deionized water, then add the photoinitiator LAP. After complete dissolution, add sodium alginate. Finally, mix the concentrated algae solution with the system to obtain bio-ink.

[0006] 2. 3D Printing Microalgae Bio-Ink: The bio-ink is transferred to a printing cartridge and loaded into a 3D printer. The printer's software is used to construct the model structure, and the diameter of the monofilament is adjusted by switching the needle. Light-curing 3D printing is performed according to the set parameters. Once the model is printed, it is further cured by UV light and calcium chloride cross-linking.

[0007] 3. Constructing an isolation coating: Immerse the printed and solidified microalgae gel in a tannic acid solution to construct an isolation coating. The coating can be applied multiple times to ensure uniformity.

[0008] Furthermore, the addition ratio of the polyethylene glycol diacrylate in step 1 is 4 wt% to 8 wt%;

[0009] Furthermore, the addition ratio of the photoinitiator LAP in step 1 is 0.2 wt% to 0.6 wt%;

[0010] Furthermore, the sodium alginate added in step 1 is 5 wt% to 7 wt%;

[0011] Furthermore, the microalgae in step 1 is Chlorella, Chlamydomonas reinhardtii, Spirulina or diatom;

[0012] Furthermore, the microalgae cell density OD 680 0.5~2.0;

[0013] Furthermore, the amount of algae liquid added in step 1 is 50 to 300 mL.

[0014] Furthermore, the printing parameters described in step 2 are as follows: layer height: 0.25-0.4 mm; number of layers: 6-12 layers; random printing fill, print outline, and starting point; model: quadrilateral; speed: 10-20 mm / s; broken wire lift: 0.4-0.8 mm; air pressure: 0.2-0.4 MPa; early wire release: 200-400 ms; early wire closing: 0.1-0.4 mm; light curing power: 0.2%-3%; filling mode: linear filling; filling method: linear filling; filling offset: 0.7 mm; filling spacing: 2 mm; rotation angle: 90°; number of rotations: 2 times;

[0015] Furthermore, the UV curing time in step 2 is 2 to 10 minutes, and the ionic crosslinking time is 5 to 20 minutes; and the calcium chloride concentration in step 2 is 2 wt% to 6 wt%.

[0016] Furthermore, the concentration of tannic acid in step 3 is 5 to 15 mg / mL;

[0017] Furthermore, the coating time in step three is 1 to 10 minutes.

[0018] The present invention also provides a method for removing antibiotics from wastewater using a recyclable 3D microalgae gel membrane, which comprises the following steps:

[0019] The printed microalgae gel film is placed in the antibiotic wastewater to be treated. After 1 to 5 days of degradation, the antibiotic wastewater is purified. After treatment, the microalgae gel film can be directly taken out for recycling or enter the next wastewater treatment cycle.

[0020] Further, the antibiotic wastewater includes one or more of tetracyclines, macrolides, and sulfonamides;

[0021] Furthermore, the antibiotic wastewater concentration is 0 to 500 mg / L;

[0022] Furthermore, the degradation conditions include a temperature of 22-25°C, a humidity of 30-70%, and a light intensity of 45-55 μmolphotons m -2 s -1 .

[0023] The technical advantages of the present invention are:

[0024] 1. The present invention realizes recyclable antibiotic wastewater treatment by constructing a foldable microalgae gel membrane;

[0025] 2. Compared with the widely used suspended microalgae, the solid microalgae purification system has a much higher tolerance to antibiotics and a stronger degradation ability;

[0026] 3. The constructed semi-permeable membrane isolation layer can prevent the leakage of microalgae and avoid potential microbial biohazards;

[0027] 4. Microalgae gel is biodegradable and can be used as a biofertilizer without generating solid waste;

[0028] 5. Microalgae gel membrane can be mass-produced on a large scale using 3D printing technology and has the potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a rheological curve of the microalgae bio-ink of Example 1;

[0030] Figure 2 is the tensile stress-strain curve of the gel of Example 1;

[0031] Figure 3This is a graph of microalgae leaching before and after the microalgae gel (denoted as MHN) in Example 1 is coated with a tannic acid isolation layer;

[0032] Figure 4 This is a comparison of the effects of suspended microalgae (denoted as SM), MHN, and MHN@TA in treating different concentrations of tetracycline in Example 2, and a graph showing the removal efficiency of tetracycline by MHN@TA at different times; C e represents the antibiotic concentration in the solution after degradation, and C0 represents the initial antibiotic concentration in the solution;

[0033] Figure 5 This is a graph showing the degradation efficiency of antibiotics under different treatments;

[0034] Figure 6 This is a graph showing the efficiency of MHN@TA cyclic degradation of antibiotics in Example 3; Specific embodiments

[0035] Example 1:

[0036] A method for preparing a recyclable 3D microalgae gel network, the specific implementation method is as follows:

[0037] 1. Preparation of microalgae bio-ink: 0.4 g polyethylene glycol diacrylate was dissolved in 5 mL deionized water, and then 10 mg of photoinitiator LAP was added. After the solution was completely dissolved, 0.3 g of sodium alginate was added. Finally, 200 mL of OD 680 The Chlorella algae liquid with a pH of 1.0 was centrifuged at 5000 g for 4 minutes, and then the precipitate was collected and mixed evenly with the system to obtain bio-ink;

[0038] 2. 3D printing microalgae bio-ink: Transfer the bio-ink prepared in step 1 into the printing cartridge and load it into the 3D printer (Bio- SR, Regenovo, Hangzhou, China). The printer's software was used to construct the model structure, and the filament diameter was adjusted by switching the needle. Light-curing 3D printing was performed according to the set parameters. After printing, the model was further cured by UV irradiation and calcium chloride cross-linking. The specific printing parameters were: layer height: 0.3 mm; number of layers: 10; randomized print fill, print outline, and starting point; model: quadrilateral; speed: 15 mm / s; filament break lift: 0.5 mm; air pressure: 0.3 MPa; early filament exit: 300 ms; early filament shutoff: 0.3 mm; light curing power: 1%; infill mode: linear infill; infill method: linear infill; infill offset: 0.7 mm; infill spacing: 2 mm; rotation angle: 90°; number of rotations: 2. The printed microalgae gel was cured under UV light for 3 minutes and then further cross-linked in a 5 wt% calcium chloride solution for 10 minutes to obtain the final microalgae gel, designated as MHN.

[0039] 3. Constructing an isolation coating: The printed and solidified microalgae gel was immersed in a 10 mg / mL tannic acid solution for 2 minutes to construct an isolation coating. The coating process was repeated twice to ensure a uniform coating, which was recorded as MHN@TA.

[0040] Example 2:

[0041] The printed microalgae gel film with a size of 40 cm × 50 cm × 0.5 cm was placed in 1000 mL of wastewater containing tetracycline at 23 °C, 50% humidity, and a light intensity of 50 μmol photons m -2 s -1 , and the purification of antibiotic wastewater is completed after 3 days of degradation.

[0042] Example 3: The microalgae gel after purification in Example 2 was taken out and placed in new antibiotic wastewater to enter the next cycle of degradation.

[0043] Comparative Example 1: Preparation method of PEGDA single network hydrogel

[0044] 0.4 g of polyethylene glycol diacrylate was dissolved in 5 mL of deionized water, and then 10 mg of photoinitiator LAP was added. The solution was poured into a mold and cured under UV light for 3 minutes to obtain a formed hydrogel, which was recorded as PEGDA.

[0045] Comparative Example 2: Preparation Method of SA Single Network Hydrogel

[0046] Preparation of sodium alginate single network hydrogel: 0.3 g of sodium alginate was dissolved in 5 mL of deionized water, the solution was poured into a mold, and cross-linked with a 5 wt % calcium chloride solution for 10 min to obtain a formed hydrogel, which was recorded as SA.

[0047] Comparative Example 3: Preparation method of 3D double network hydrogel network

[0048] The preparation method was the same as in Example 1, except that no microalgae were added, denoted as DNH and DNH@TA.

[0049] Comparative Example 4: Suspended microalgae treatment method

[0050] The exponential growth phase of Chlorella was collected and centrifuged at 5000 g for 4 minutes. The sediment was then collected and the microalgae cells were resuspended in tetracycline wastewater and incubated at 23 °C, 50% humidity, and a light intensity of 50 μmol photons m - 2 s -1 After 3 days of degradation, the antibiotic wastewater was purified. It is recorded as SM.

[0051] Detection Example

[0052] Tetracycline Detection: 1 mL of the sample was filtered through a 0.22 μm filter membrane, and the residual antibiotic concentration was determined by high-performance liquid chromatography. The detection conditions were: a COSMOSIL C18 column (4.6 × 150 mm, 5 μm); the mobile phase consisted of ultrapure water and acetonitrile-trifluoroacetic acid (0.1%, v / v); the mobile phase gradient was 85% to 20% (v / v); the column temperature was 25°C; the flow rate was 2 mL / min; the injection volume was 20 μL; and the detection wavelength was 350 nm.

[0053] Figure 1 It can be seen that the bio-ink has shear-thinning properties, and the addition of microalgae does not affect its rheological properties, indicating that the bio-ink has good printability;

[0054] Figure 2 It can be seen that the tensile strength of the double-network hydrogel (denoted as DNH) is 134.3 kPa, which is higher than that of the single-network hydrogel (polyethylene glycol diacrylate denoted as PEGDA, sodium alginate denoted as SA). At the same time, due to the hydrogen bond interaction between PEGDA and TA, the mechanical properties of the DNH@TA hydrogel coated with tannic acid (denoted as TA) are further improved, indicating that the microalgae gel film has good tensile properties and can be folded and bent;

[0055] Figure 3 It can be seen that after 7 days of immersion, the microalgae leakage rate of MHN without TA coating was 12.9%, while the leakage rate of MHN@TA was only 1.2%, showing excellent sealing performance;

[0056] Depend on Figure 4 a It can be seen that compared with SM, MHN and MHN@TA have higher degradation efficiency, especially in the environment of high concentration of antibiotics; Figure 4 b It can be seen that after 72h of treatment, the degradation efficiency of MHN@TA for tetracycline at a concentration of 100mg / L is as high as 99.3%, indicating that MHN@TA has excellent degradation performance and its degradation efficiency is not affected before and after coating.

[0057] Figure 5Water(dark) and Water(light) indicate the natural degradation of tetracycline wastewater without any treatment under light and dark conditions; DNH@TA indicates the treatment of tetracycline wastewater by hydrogel without the addition of microalgae (the preparation method of DNH@TA is the same as that of MHN@TA, the difference is that DNH@TA does not add microalgae); it can be seen that under light or dark conditions, the natural degradation efficiency of tetracycline in water is negligible. The tetracycline removal efficiency of DNH@TA hydrogel without microalgae is about 7.7%, indicating that the gel has little adsorption effect on tetracycline, while the tetracycline removal efficiency of MHN@TA is as high as 96.3%, indicating that microalgae biodegradation is the main driving force for the removal of antibiotics.

[0058] The removal rate was calculated according to the formula: Removal rate (%) = (1-(Ce / C0)) x 100%, where Ce represents the concentration of antibiotics in the solution after degradation and C0 represents the initial antibiotic concentration in the solution;

[0059] Figure 6 It can be seen that the microalgae gel film can be conveniently subjected to multiple antibiotic degradation treatments without a significant decrease in degradation efficiency. After five cycles of degradation, the degradation efficiency is still greater than 90%.

Claims

1. A method for preparing a recyclable 3D microalgae gel, comprising the following steps: Step 1: Prepare microalgae bio-ink: Dissolve polyethylene glycol diacrylate in deionized water, then add photoinitiator LAP. After complete dissolution, add sodium alginate. Finally, mix the concentrated microalgae solution with the system to obtain bio-ink. Step 2: 3D printing of microalgae bio-ink: The bio-ink is transferred to a printing cartridge and loaded into a 3D printer. The model structure is constructed using the printer's software, and the diameter of the monofilament is adjusted by switching the needle. Light-curing 3D printing is performed according to the set parameters. After the model is printed, it is further cured by UV light irradiation and calcium chloride cross-linking. Step 3: Constructing an isolation coating: Immersing the printed and solidified microalgae gel into a tannic acid solution to construct an isolation coating.

2. The method for preparing a recyclable 3D microalgae gel according to claim 1, characterized in that: The addition ratio of the polyethylene glycol diacrylate in step 1 is 4 wt % to 8 wt %.

3. The method for preparing a recyclable 3D microalgae gel according to claim 1, characterized in that: The addition ratio of sodium alginate in step 1 is 5wt% to 7wt%.

4. The method for preparing a recyclable 3D microalgae gel according to claim 1, characterized in that: The microalgae in step 1 are Chlorella, Chlamydomonas reinhardtii, Spirulina or diatom.

5. The method for preparing a recyclable 3D microalgae gel according to claim 1, characterized in that: The microalgae cell density OD 680 The concentration of algae solution is 0.5-2.0, and the amount of algae solution added is 50-300mL.

6. The method for preparing a recyclable 3D microalgae gel according to claim 1, characterized in that: The printing parameters in step 2 are: layer height: 0.25-0.4mm; Number of layers: 6-12; Print fill, print outline, random starting point; Model: quadrilateral; Speed: 10-20mm / s; Broken wire lifting: 0.4-0.8mm; Air pressure: 0.2-0.4MPa; Early wire drawing: 200-400ms; Early wire closing: 0.1-0.4mm; Light curing power: 0.2%-3%; Filling mode: Linear filling; Filling method: Linear filling; Filling offset: 0.7mm; Filling spacing: 2mm; Rotation angle: 90°; Number of rotations: 2 times.

7. The method for preparing a recyclable 3D microalgae gel according to claim 1, characterized in that: In step 2, the UV curing time is 2 to 10 minutes, and the ionic crosslinking time is 5 to 20 minutes; and in step 2, the calcium chloride concentration is 2 wt% to 6 wt%.

8. The method for preparing a recyclable 3D microalgae gel according to claim 1, characterized in that: In step 3, the concentration of tannic acid is 5 to 15 mg / mL; and the coating time is 1 to 10 minutes.

9. The method for removing antibiotics from wastewater using a recyclable 3D microalgae gel according to any one of claims 1 to 8, characterized in that: The recyclable 3D microalgae gel prepared by any one of the methods according to claims 1-8 is placed in the antibiotic wastewater to be treated, and the antibiotic wastewater is purified after degradation for 1 to 5 days. After treatment, the microalgae gel membrane is directly taken out for recycling or enters the next wastewater treatment cycle.

10. The method for removing antibiotics from wastewater using a recyclable 3D microalgae gel according to claim 9, characterized in that: Antibiotic wastewater contains one or more of tetracyclines, macrolides, and sulfonamides; the antibiotic wastewater concentration is 0-500 mg / L; the degradation conditions include temperature of 22-25°C, humidity of 30-70%, and light intensity of 45-55 μmolphotons·m -2 s -1 .

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