Periphyton carrier for enriching and killing viruses in aquaculture tail water and preparation method of periphyton carrier

Abstract

The invention discloses a periphyton carrier for breeding tail water virus enrichment and killing and a preparation method thereof.The periphyton carrier is of a core-shell structure, the core is a signal layer with catalytic particles loaded on the surface, the shell is a supporting layer, the signal layer is gel with dispersed cyclic diguanylic acid, and the supporting layer is a gel layer with dispersed cyclic diguanylic acid. The catalytic particles are nano-carriers loaded with sodium percarbonate and potassium ferrocyanide, and the supporting layer is a polymer containing pores. By integrating signal molecule regulation and control and a free radical synergistic inactivation mechanism, the virus enrichment capacity of the biological membrane is optimized, and the in-situ killing efficiency is enhanced. According to the periphyton carrier, the pertinence and response speed of virus treatment are improved, the environmental risk and cost bottleneck of a traditional method are overcome, an innovative solution is provided for safe management of aquaculture water, and the virus inactivation rate can be larger than or equal to 99.9%.

Description

Technical Field

[0001] The present invention belongs to the technical field of water treatment, and specifically relates to a periphyton biological carrier for virus enrichment and disinfection of aquaculture tail water and a preparation method thereof, belonging to the cross technical field of environmental functional materials and biofilm water treatment. Background Art

[0002] Waterborne pathogens, including bacteria, viruses, fungi and parasites, are widely present in aquaculture water bodies, posing a serious threat to the health of aquatic animals and the stability of the ecosystem. Among them, viruses, due to their tiny volume, strong infectivity and ability to tolerate harsh environments, have become one of the most destructive pathogens. Viruses can not only cause large-scale infections at low concentrations, but also spread pathogens across regions and species through water exchange, wastewater discharge and infected fish bodies, causing long-term harm to aquaculture systems and natural ecosystems. In areas with weak infrastructure, the problem of virus pollution is particularly prominent, threatening not only the economic benefits of the aquaculture industry, but also human health.

[0003] Traditional virus treatment methods, such as chlorination disinfection, ozonation and ultraviolet irradiation, although they can effectively inactivate viruses, face problems such as by-product generation, high energy consumption and sensitivity to water quality conditions. Although emerging methods such as photocatalytic technology show potential, they are still limited by challenges such as insufficient stability of catalysts and low capture efficiency for low-concentration viruses.

[0004] The periphyton biotechnology uses microorganisms and their secreted extracellular polymers to adsorb and degrade viruses, with the advantages of low cost and environmental friendliness. However, the virus adsorption capacity of natural biofilms is limited, the inactivation rate is slow, and it is easily disturbed in complex water environments, resulting in unstable treatment efficiency. In addition, traditional biofilms have insufficient adaptability to high virus loads and are difficult to meet the treatment requirements of large-scale aquaculture water bodies. Summary of the Invention

[0005] Aiming at the problems in the above-mentioned existing technologies, the present invention provides a periphyton biological carrier for virus enrichment and disinfection of aquaculture tail water based on the synergistic effect of signal molecule regulation and free radicals and a preparation method thereof. By integrating the signal molecule regulation and free radical synergistic inactivation mechanism, the virus enrichment ability of the biofilm is optimized, and the in-situ disinfection efficiency is enhanced. This periphyton biological carrier not only improves the pertinence and response speed of virus treatment, but also overcomes the environmental risks and cost bottlenecks of traditional methods, providing an innovative solution for the safety management of aquaculture water bodies.

[0006] The periphyton biological carrier of the present invention uses cyclic diguanylate (c-di-GMP) to directionally induce the formation of specific virus binding sites on the extracellular polymers of the biofilm, and at the same time couples a sodium percarbonate / potassium ferrocyanide (SPC-PB) catalytic system to achieve free radical-mediated in-situ inactivation of viruses. This carrier can be as low as 102 At a virus concentration of copies / mL, a removal rate of ≥99% is maintained, and no toxic by-products are generated throughout the process, providing technical support for the efficient and green treatment of aquaculture tail water.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows: A periphyton carrier for virus enrichment and disinfection of aquaculture tail water, the periphyton carrier having a core-shell structure, the core being a signal layer with catalytic particles loaded on the surface, and the shell being a support layer. The signal layer is a gel in which cyclic diguanosine monophosphate (c-di-GMP) is dispersed, and the catalytic particles are nano-carriers loaded with sodium percarbonate (SPC) and potassium ferrocyanide (PB). The support layer is a polymer containing pores.

[0008] Preferably, the support layer is polylactic acid (PLA).

[0009] Preferably, the average porosity of the support layer is 80-85%. The porosity within this range can meet the requirements of structural permeability and strength.

[0010] Preferably, the pore diameter of the support layer is 65-180 μm. Within this range, the requirements for diffusion and attachment functions can be met.

[0011] Preferably, the nano-carrier is mesoporous silica nanoparticles.

[0012] Preferably, the amount of sodium percarbonate in the nano-carrier is 20-30 wt% of the mass of the nano-carrier.

[0013] Preferably, the potassium ferrocyanide is nano-particles with a particle size of 20-50 nm.

[0014] Preferably, the amount of potassium ferrocyanide is 5-10 wt% of the mass of the nano-carrier. Potassium ferrocyanide is fixed on the surface of the carrier by electrostatic adsorption.

[0015] Preferably, the pore diameter of the mesoporous silica is 2-5 nm, and the specific surface area of the mesoporous silica is 500 m 2 / g or more.

[0016] Sodium percarbonate and potassium ferrocyanide catalyze synergistically to generate hydrogen peroxide (H2O2) under the action of catalase, and then react with potassium ferrocyanide to produce hydroxyl radicals (·OH) and high-valent iron oxide Fe(IV)=O.

[0017] Preferably, the gel is sodium alginate gel.

[0018] Preferably, in the signal layer, the content of cyclic diguanosine monophosphate is 0.002-0.004 wt%, calculated as the percentage of the mass of cyclic diguanosine monophosphate in the mass of sodium alginate gel.

[0019] Preferably, the diameter of the periphyton carrier is 2-4 mm. The specific surface area of the periphyton carrier is 300-400 m 2 / g. The density of the periphyton carrier is 1.0-1.5 g / cm 3 .

[0020] The second object of the present invention is to provide a preparation method of the periphyton carrier for virus enrichment and disinfection of aquaculture tail water, comprising the following steps: (1) Uniformly mix the solution of cyclic diguanosine monophosphate with the gel solution, and then solidify to form gel beads; (2) Adopt the incipient wetness impregnation method to impregnate the sodium percarbonate solution into the nano-carrier to obtain the nano-carrier loaded with sodium percarbonate (SPC@SiO2); subsequently, mix the nano-carrier loaded with sodium percarbonate with the solution of cationic polymer dispersed with potassium ferrocyanide (PB) nanoparticles, and fix the potassium ferrocyanide nanoparticles on the surface of the nano-carrier loaded with sodium percarbonate through electrostatic adsorption, and obtain the catalytic particles after washing; (3) Suspend the catalytic particles in water to obtain a suspension, and then immerse the gel beads obtained in step (1) in the suspension and perform ultrasonic treatment to obtain the core; (4) Put the core into the polymer solution, make the polymer solution uniformly adhere to the surface of the core, and then remove the solvent to obtain the periphyton carrier.

[0021] Preferably, in step (1), the concentration of the solution of cyclic diguanosine monophosphate is 0.05-0.1 mM.

[0022] Preferably, in step (1), the gel solution is a sodium alginate solution, and the solidification method is: dropwise add calcium chloride solution.

[0023] Preferably, in step (2), the concentration of the sodium percarbonate solution is 30 wt%.

[0024] Preferably, step (2) further includes a step of drying the nano-carrier loaded with sodium percarbonate. The drying conditions are: vacuum drying at 60 °C for 6 h.

[0025] Preferably, in step (2), the cationic polymer is poly(diallyldimethylammonium chloride) (PDDA).

[0026] Preferably, the mass concentration of the cationic polymer solution in step (2) is 0.5 - 1 mg / mL. It is used to stabilize the electrostatic interaction between PB nanoparticles and the surface of the nanocarrier, achieve uniform adsorption of PB on the carrier surface, and form effective catalytic particles.

[0027] Preferably, the method for removing the solvent in step (4) is as follows: First, dry the solvent at room temperature, then pre-freeze the core at -40°C for 2 hours to rapidly shape the surface film layer, and then transfer it to -80°C for freeze-drying for 48 hours.

[0028] Preferably, the polymer solution in step (4) is a 8 - 12 wt% polylactic acid (PLA) solution.

[0029] The present invention also provides the application of the above-mentioned periphyton carrier for virus enrichment and disinfection in aquaculture tail water.

[0030] The application includes virus disinfection in aquaculture tail water.

[0031] Specifically, the application includes the following steps: (1) Virus enrichment stage: Add the periphyton carrier to aquaculture tail water, c-di-GMP induces microorganisms to secrete β-glucan (EPS accounts for ≥65%), and specifically adsorbs virus particles through the hydrophobic effect of the α-helical structure, with an adsorption capacity of 10 10 pfu / g; (2) In-situ inactivation stage: Endogenous catalase in the biofilm catalyzes the decomposition of sodium percarbonate to release H2O2, and H2O2 reacts with PB to generate hydroxyl radicals (·OH) and ferryl oxygen (Fe(IV)=O), which attack the cysteine residues of the virus capsid protein (oxidation rate ≥90%) and genomic DNA (fragmentation rate ≥95%) respectively, achieving a virus inactivation rate ≥99.9%.

[0032] Furthermore, the application also includes the regeneration of the periphyton carrier.

[0033] Preferably, the regeneration of the periphyton sub-carrier includes: soaking the periphyton carrier with 0.1M NaOH solution to dissolve the inactivated biofilm and restore the β-glucan adsorption activity, and the number of recycling times ≥15 times.

[0034] The obtained signal layer is detected by circular dichroism spectroscopy for the Cotton effect peak at 220 nm, and the proportion of the α-helical structure is optimized to be ≥70%.

[0035] Furthermore, the method for verifying the virus inactivation effect includes: Using electron paramagnetic resonance (EPR) to detect the ·OH signal intensity (≥5×10 16spins / g); Analyze the characteristic peak of Fe(IV)=O (δ = 0.12 mm / s) by Mössbauer spectroscopy; Use RT-qPCR to measure the residual amount of viral nucleic acid, and the inactivation rate is ≥ 99.9%.

[0036] The beneficial effects of the present invention are as follows: (1) The present invention develops a dual-functional virus treatment system based on signal regulation and free radical synergy. By inducing the formation of a high-affinity β-glucan helical structure in the biofilm through c-di-GMP, specific adsorption of viruses is achieved (adsorption rate ≥ 99%), and in-situ generation of reactive oxygen species (·OH) and high-valent iron (Fe(IV)=O) is combined with the SPC-PB catalytic system to simultaneously destroy the viral capsid protein and genome, and the inactivation efficiency is ≥ 99.9%. The chemical-biological synergy mechanism breaks through the efficiency bottleneck of traditional separation treatment technologies.

[0037] (2) The present invention optimizes the carrier function through hierarchical loading and pH-responsive release technology: the alginate signal layer continuously releases c-di-GMP under near-neutral conditions (pH 6.5 - 7.5) to maintain the helical conformation of β-glucan; the catalytic layer precisely controls the release of H2O2 under the action of catalase to avoid biofilm damage caused by excessive generation of free radicals and ensure long-term operation stability.

[0038] (3) The porous PLA support layer of the present invention has both high mechanical strength (≥ 8 MPa) and permeability (porosity 80%), maintains structural integrity under high-speed water flow (1.5 m / s), and at the same time allows free radicals to diffuse to the biofilm surface to ensure the full progress of the virus inactivation reaction. Description of the Drawings

[0039] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above or other advantages of the present invention will become clearer.

[0040] Figure 1 It is a schematic structural diagram of the periphyton carrier described in the present invention.

[0041] Figure 2 It is a flowchart of the function of the periphyton carrier described in the present invention for enriching and killing viruses.

[0042] Figure 3 It is a comparison chart of the effects of treating viruses in aquaculture tail water by each method in Example 1.

[0043] Figure 4 It is a fitting curve of the virus concentration changing with the treatment time in Example 1.

[0044] Among them, 1 is cyclic diguanosine monophosphate, 2 is the gel, 3 is the nanocarrier, 4 is potassium ferrocyanide, 5 is sodium percarbonate, 6 is the support layer, 7 is the pore, 8 is the intact virus particle, 9 is the virus capsid, and 10 is the virus fragment. Detailed implementation mode

[0045] The present invention can be better understood according to the following embodiments.

[0046] Example 1 (1) A solution of 0.05 - 0.1 mM cyclic diguanosine monophosphate is uniformly mixed with a sodium alginate solution, and then a 2 - 4 wt% calcium chloride solution is added dropwise to crosslink and solidify to form gel beads.

[0047] (2) Using the incipient wetness impregnation method, a 30 wt% sodium percarbonate solution is impregnated into mesoporous silica nanoparticles (pore size 2 - 5 nm, specific surface area ≥ 500 m 2 / g), and vacuum dried at 60 °C for 6 h to obtain sodium percarbonate-loaded nanoparticles (SPC@SiO2). Potassium ferrocyanide nanoparticles with a particle size of 20 - 50 nm are added to a 0.5 - 1 mg / mL poly(diallyldimethylammonium chloride) (PDDA) solution and mixed, and then SPC@SiO2 is introduced. PB is fixed on its surface through electrostatic adsorption to form catalytic particles. The loading amounts are 20 - 30 wt% for SPC and 5 - 10 wt% for PB, calculated based on the mass of SiO2.

[0048] (3) The catalytic particles are suspended in water to obtain a suspension, and then the gel beads obtained in step (1) are impregnated in the suspension and ultrasonicated for 10 - 20 min to obtain the core; (4) The core gel sphere is impregnated in an 8 - 12 wt% polylactic acid (PLA) solution, pre-frozen (-40 °C) for 2 hours, and then freeze-dried at -80 °C to form a periphyton biocarrier with a porous support layer (pore size 50 - 200 μm, porosity 82%).

[0049] (5) The periphyton biocarrier is added to the aquaculture tail water containing white spot syndrome virus. The virus concentration in the water before treatment is 10 6 copies / mL (Ct value ≤ 25 detected by RT-qPCR).

[0050] As Figure 2 shown, after adding the periphyton biocarrier, c-di-GMP induces microorganisms to secrete β-glucan, which specifically adsorbs virus particles through the hydrophobic action of the α-helical structure, and the adsorption capacity reaches 10 10pfu / g; intact virus particles are adsorbed and aggregated; subsequently, the virus enters the contact catalytic particles under fluid drive, and the catalytic component releases ·OH and Fe(IV)=O for in-situ inactivation, attacking the cysteine residues of the virus capsid protein (oxidation rate ≥90%) and genomic DNA (fragmentation rate ≥95%) respectively. The virus capsid gradually ruptures under oxidation; finally, the virus fragments with completely destroyed genetic material are discharged from the porous structure of the support layer, realizing the whole process of disinfection from directional adsorption, catalytic cleavage to complete inactivation.

[0051] After treatment, the residual virus in the water body ≤10 2 copies / mL (Ct value ≥35), and the inactivation efficiency in 24 hours ≥99.99%. During the whole process, the carrier structure maintains a stable laminar flow environment to ensure the orderly progress of each reaction stage.

[0052] The fitting curve of virus concentration changing with treatment time is as Figure 4 shown: The experiment was carried out under the condition of 25±1°C. The initial virus concentration in the tail water was about 10 6 copies / mL (Ct value ≤25), the carrier dosage concentration was 0.5 g / L, the water body volume was 5 L, and the pH was maintained at 7.0. During the experiment, water samples were collected at 0, 2, 4, 6, 12, and 24 hours, and the virus concentration was detected by RT-qPCR method. The data was expressed in the form of log 10 . The results showed that the virus concentration decreased exponentially with time. After fitting analysis, it was shown that this inactivation process conforms to the first-order reaction kinetic model, and the fitting equation is as follows: ln(C / C0) = -kt, where C is the virus concentration at any time point, C0 is the initial virus concentration, and k is the reaction rate constant. Through nonlinear regression calculation, k = 0.543 h -1 , and the correlation coefficient R 2 = 0.993. The results indicate that the carrier described in the present invention has a stable time dependence and predictability for the inactivation process of viruses in aquaculture tail water, and the inactivation efficiency increases significantly with the reaction time.

[0053] (6) Set three groups of parallel experiments, namely the chemical treatment group, the ultraviolet treatment group, and the periphyton bio-carrier group of the present invention, to treat the tail water containing white spot syndrome virus with the same initial concentration (10 6 copies / mL), and the treatment volume is 5 L for all.

[0054] Chemical treatment group: Add 0.5 mM H2O2 + 0.1 mM FeSO4, react for 30 minutes, and adjust the pH to 6.5.

[0055] UV treatment group: Insert a UV lamp with a wavelength of 254 nm (power 36 W) into the interior of the water body. The lamp tube is 5 cm away from the bottom of the water and is vertically placed in the central area. Irradiate continuously for 60 minutes, and at the same time, set magnetic stirring to maintain slow water flow.

[0056] Carrier treatment group: Add the periphyton carrier prepared in steps (1) to (4) at a dosage of 0.5 g / L. The reaction temperature is 25°C, pH is 7.0, and the water body is slowly stirred.

[0057] As Figure 3 shown, under the condition of the same initial virus concentration (10 6 copies / mL), the trends of virus concentration decline of the three treatment methods within 24 hours are significantly different. For the periphyton carrier group described in the present invention, the virus concentration drops rapidly after the 6th hour and drops to the detection limit (about 10 copies / mL) at the 24th hour, with an inactivation rate ≥ 99.99%. This effect is comparable to that of the UV treatment group, but while achieving a high inactivation rate, the periphyton carrier has the function of virus enrichment, providing the possibility for subsequent separation. The inactivation efficiency of the chemical treatment group is significantly lower, and the virus concentration in the tail water is still higher than 10 4 copies / mL after 24 hours, with an inactivation rate of less than 90%.

[0058] As Figure 4 shown, the change of the virus concentration (log 10 ) of the treatment group of the present invention with the treatment time conforms to the first-order reaction kinetic model, and the fitting constant is k = 0.543 h -1 , and the correlation coefficient R 2 = 0.993, indicating that the carrier system has a clear time-dependence and mechanism stability for the virus inactivation process. The model fitting curve is highly consistent with the measured values, further supporting the repeatability and predictability of the virus inactivation process.

Claims

1. A periphyton biological carrier for virus enrichment and disinfection of aquaculture tail water, characterized in that The periphyton biological carrier has a core-shell structure, with the core being a signal layer loaded with catalytic particles on the surface and the shell being a support layer. The signal layer is a gel in which cyclic diguanosine monophosphate is dispersed, and the catalytic particles are nano-carriers loaded with sodium percarbonate and potassium ferrocyanide. The support layer is a polymer containing pores; preferably, the support layer is polylactic acid.

2. The periphytic organism carrier according to claim 1, wherein The pore size of the support layer is 65 - 180 μm.

3. The periphytic organism carrier according to claim 1, wherein The nano-carrier is mesoporous silica nanoparticles.

4. The periphyton carrier according to claim 1, wherein The amount of sodium percarbonate in the nano-carrier is 20 - 30 wt% of the mass of the nano-carrier.

5. The periphytic organism carrier according to claim 1, characterized in that, The amount of potassium ferrocyanide is 5 - 10 wt% of the mass of the nano-carrier.

6. The periphyton carrier according to claim 1, characterized in that, The gel is sodium alginate gel.

7. The periphyton carrier according to claim 1, wherein In the signal layer, the content of cyclic diguanosine monophosphate is 0.002 - 0.004 wt%, calculated as the percentage of the mass of cyclic diguanosine monophosphate in the mass of sodium alginate gel.

8. The preparation method of the periphyton carrier for virus enrichment and disinfection of aquaculture tail water according to any one of claims 1-7, characterized in that, It includes the following steps: (1) Uniformly mix the solution of cyclic diguanosine monophosphate with the gel solution, and then solidify to form gel beads. (2) Using the incipient wetness impregnation method, impregnate the sodium percarbonate solution into the nano-carrier to obtain a nano-carrier loaded with sodium percarbonate; subsequently, mix the nano-carrier loaded with sodium percarbonate with a solution of a cationic polymer in which potassium ferrocyanide nanoparticles are dispersed, and fix the potassium ferrocyanide nanoparticles on the surface of the nano-carrier loaded with sodium percarbonate through electrostatic adsorption, and obtain the catalytic particles after washing. (3) Suspend the catalytic particles in water to obtain a suspension, and then immerse the gel beads obtained in step (1) in the suspension and perform ultrasonic treatment to obtain the core. (4) Put the core into the polymer solution, make the polymer solution uniformly adhere to the surface of the core, and then remove the solvent to obtain the periphyton biological carrier.

9. The preparation method according to claim 8, characterized in that, The cationic polymer described in step (2) is poly(diallyldimethylammonium chloride).

10. Application of the periphyton biological carrier for virus enrichment and disinfection in aquaculture tail water according to any one of claims 1 - 7.

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