Caulis peristrophis for aquaculture tail water virus enrichment and disinfection and preparation method thereof

Abstract

This invention discloses a periclump biocarrier for virus enrichment and elimination in aquaculture wastewater and its preparation method. The periclump biocarrier has a core-shell structure, with a core consisting of a signal layer loaded with catalytic particles, and an outer shell consisting of a support layer. The signal layer is a gel dispersed with cyclic diguanosine monophosphate, the catalytic particles are nanocarriers loaded with sodium percarbonate and potassium ferrocyanide, and the support layer is a porous polymer. This invention optimizes the virus enrichment capacity of biofilms and enhances in-situ elimination efficiency by integrating signal molecule regulation and free radical synergistic inactivation mechanisms. This periclump biocarrier not only improves the targeting and response speed of virus treatment but also overcomes the environmental risks and cost bottlenecks of traditional methods, providing an innovative solution for the safe management of aquaculture water bodies, and can achieve a virus inactivation rate of ≥99.9%.

Description

Technical Field

[0001] This invention belongs to the field of water treatment technology, specifically relating to a periculture biological carrier for the enrichment and elimination of viruses in aquaculture tailwater and its preparation method, belonging to the interdisciplinary field of environmental functional materials and biofilm water treatment. Background Technology

[0002] Waterborne pathogens, including bacteria, viruses, fungi, and parasites, are widely present in aquaculture waters, posing a serious threat to the health of aquatic animals and the stability of ecosystems. Among them, viruses, due to their tiny size, extremely high infectivity, and tolerance to harsh environments, are among the most destructive pathogens. Viruses can not only cause large-scale infections at low concentrations, but also spread through water exchange, wastewater discharge, and diseased fish, leading to cross-regional and cross-species pathogen outbreaks and causing long-term harm to aquaculture systems and natural ecosystems. In areas with weak infrastructure, viral contamination is particularly prominent, threatening not only the economic benefits of the aquaculture industry but also potentially endangering human health.

[0003] Traditional virus treatment methods, such as chlorination, ozone oxidation, and ultraviolet irradiation, while effectively inactivating viruses, face challenges including byproduct generation, high energy consumption, and sensitivity to water quality conditions. Emerging methods like photocatalysis show promise, but are still limited by catalyst instability and low capture efficiency for low-concentration viruses.

[0004] Zhou Cong Biotechnology utilizes microorganisms and their secreted extracellular polymers to adsorb and degrade viruses, offering advantages such as low cost and environmental friendliness. However, natural biofilms have limited virus adsorption capacity, slow inactivation rates, and are easily disturbed in complex aquatic environments, leading to unstable treatment efficiency. Furthermore, traditional biofilms are insufficiently adaptable to high viral loads, making it difficult to meet the treatment needs of large-scale aquaculture water bodies. Summary of the Invention

[0005] To address the problems in the existing technologies mentioned above, this invention provides a periclump biocarrier for the enrichment and elimination of viruses in aquaculture wastewater based on signal molecule regulation and the synergistic effect of free radicals, and its preparation method. By integrating signal molecule regulation and the synergistic inactivation mechanism of free radicals, the virus enrichment capacity of the biofilm is optimized, and the in-situ elimination efficiency is enhanced. This periclump biocarrier not only improves the targeting and response speed of virus treatment, but also overcomes the environmental risks and cost bottlenecks of traditional methods, providing an innovative solution for the safe management of aquaculture water bodies.

[0006] The *Zhou Cong* biovector of this invention induces the formation of specific virus-binding sites in extracellular polymeric tissues via cyclic diguanylic acid (c-di-GMP), while simultaneously achieving radical-mediated in situ virus inactivation through a sodium percarbonate / potassium ferrocyanide (SPC-PB) catalytic system. This vector can achieve virus inactivation at temperatures as low as 10...2 Maintaining a virus removal rate of ≥99% at a virus concentration of copies / mL, with no toxic byproducts generated throughout the process, it provides technical support for the efficient and green treatment of aquaculture wastewater.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A periclump biological carrier for the enrichment and elimination of viruses in aquaculture wastewater, the periclump biological carrier having a core-shell structure, the core being a signal layer with catalytic particles loaded on its surface, and the outer shell being a support layer.

[0009] The signal layer is a gel dispersed with cyclic diguanylic acid (c-di-GMP), and the catalytic particles are nanocarriers loaded with sodium percarbonate (SPC) and potassium ferrocyanide (PB).

[0010] The support layer is a porous polymer.

[0011] Preferably, the support layer is polylactic acid (PLA).

[0012] Preferably, the average porosity of the support layer is 80-85%. A porosity within this range can meet the requirements for structural permeability and strength.

[0013] Preferably, the pore size of the support layer is 65~180μm. Within this range, the diffusion and adhesion functions can be satisfied.

[0014] Preferably, the nanocarrier is mesoporous silica nanoparticles.

[0015] Preferably, the amount of sodium percarbonate in the nanocarrier is 20 to 30 wt% of the mass of the nanocarrier.

[0016] Preferably, the potassium ferrocyanide is composed of nanoparticles with a particle size of 20–50 nm.

[0017] Preferably, the amount of potassium ferrocyanide is 5-10 wt% of the mass of the nanocarrier. The potassium ferrocyanide is fixed to the carrier surface by electrostatic adsorption.

[0018] Preferably, the mesoporous silica has a pore size of 2–5 nm and a specific surface area of ​​500 m². 2 / g or more.

[0019] Sodium percarbonate and potassium ferrocyanide work together to catalyze the generation of hydrogen peroxide (H2O2) under the action of catalase. The hydrogen peroxide then reacts with potassium ferrocyanide to produce hydroxyl radicals (·OH) and high-valent iron oxide Fe(IV)=O.

[0020] Preferably, the gel is sodium alginate gel.

[0021] Preferably, the content of cyclic diguanosine monophosphate in the signal layer is 0.002 to 0.004 wt%, calculated as a percentage of the mass of cyclic diguanosine monophosphate to the mass of sodium alginate gel.

[0022] Preferably, the diameter of the periclump biological carrier is 2-4 mm. The specific surface area of ​​the periclump biological carrier is 300-400 m². 2 / g. The density of the periclump biological carrier is 1.0~1.5g / cm³. 3 .

[0023] The second objective of this invention is to provide a method for preparing the peritrichous biological vector for the enrichment and disinfection of viruses in aquaculture tailwater, comprising the following steps:

[0024] (1) Mix the solution of cyclic diguanylic acid with the gel solution evenly, and then solidify to form gel microspheres;

[0025] (2) The sodium percarbonate solution was impregnated into the nanocarrier using the initial wet impregnation method to obtain a sodium percarbonate-loaded nanocarrier (SPC@SiO2); then the sodium percarbonate-loaded nanocarrier was mixed with a cationic polymer solution containing potassium ferrocyanide (PB) nanoparticles, and the potassium ferrocyanide nanoparticles were fixed on the surface of the sodium percarbonate-loaded nanocarrier by electrostatic adsorption. After cleaning, the catalytic particles were obtained.

[0026] (3) The catalyst particles are suspended in water to obtain a suspension, and then the gel microspheres obtained in step (1) are immersed in the suspension and ultrasonically treated to obtain the core;

[0027] (4) The core is immersed in a polymer solution, so that the polymer solution is uniformly attached to the surface of the core, and then the solvent is removed to obtain the Zhou Cong biological carrier.

[0028] Preferably, the concentration of the cyclic diguanosine monophosphate solution in step (1) is 0.05-0.1 mM.

[0029] Preferably, the gel solution in step (1) is a sodium alginate solution, and the curing method is: adding calcium chloride solution dropwise.

[0030] Preferably, the concentration of the sodium percarbonate solution in step (2) is 30 wt%.

[0031] Preferably, step (2) further includes drying the sodium percarbonate-loaded nanocarrier. The drying conditions are: vacuum drying at 60°C for 6 hours.

[0032] Preferably, the cationic polymer mentioned in step (2) is polydiallyldimethylammonium chloride (PDDA).

[0033] Preferably, the mass concentration of the cationic polymer solution in step (2) is 0.5–1 mg / mL. This is used to stabilize and disperse the electrostatic interaction between the PB nanoparticles and the surface of the nanocarrier, thereby achieving uniform adsorption of PB on the carrier surface and forming effective catalytic particles.

[0034] Preferably, the solvent removal method in step (4) is as follows: first, the solvent is evaporated at room temperature, then the core is pre-frozen at -40°C for 2 hours to quickly set the surface film, and then freeze-dried at -80°C for 48 hours.

[0035] Preferably, the polymer solution in step (4) is an 8-12 wt% polylactic acid (PLA) solution.

[0036] The present invention also provides the application of the above-mentioned periculture biological carrier for the enrichment and disinfection of viruses in aquaculture tailwater.

[0037] The application includes virus disinfection in aquaculture wastewater.

[0038] Specifically, the application includes the following steps:

[0039] (1) Virus enrichment stage: The aforementioned periwinkle biological carrier was added to the aquaculture wastewater. c-di-GMP induced microorganisms to secrete β-glucan (EPS content ≥65%), which specifically adsorbed virus particles through the hydrophobic interaction of the α-helical structure, with an adsorption capacity of up to 10. 10 pfu / g;

[0040] (2) In situ inactivation stage: The endogenous catalase of the biofilm catalyzes the decomposition of sodium percarbonate to release H2O2. H2O2 reacts with PB to generate hydroxyl radicals (·OH) and ferric oxygen (Fe(IV)=O), which attack the cysteine ​​residues of the viral capsid protein (oxidation rate ≥90%) and the genomic DNA (fracture rate ≥95%), respectively, to achieve a virus inactivation rate ≥99.9%.

[0041] Furthermore, the application also includes the regeneration of the periclump biological carrier.

[0042] Preferably, the regeneration of the periclump biocarrier includes: soaking the periclump biocarrier in 0.1M NaOH solution to dissolve the inactivated biofilm and restore the β-glucan adsorption activity, and the number of cycles is ≥15.

[0043] The obtained signal layer was detected by circular dichroism spectroscopy at the Cotton effect peak at 220 nm, and the proportion of α-helical structure was optimized to be ≥70%.

[0044] Furthermore, the method for verifying the virus inactivation effect includes:

[0045] Electron paramagnetic resonance (EPR) was used to detect the ·OH signal intensity (≥5×10⁻⁶). 16 spins / g);

[0046] The characteristic peak of Fe(IV)=O was analyzed by Mössbauer spectroscopy (δ=0.12mm / s).

[0047] The residual viral nucleic acid was determined by RT-qPCR, and the inactivation rate was ≥99.9%.

[0048] The beneficial effects of this invention are as follows:

[0049] (1) This invention develops a dual-function 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 using c-di-GMP, virus-specific adsorption is achieved (adsorption rate ≥99%). Combined with the in-situ generation of reactive oxygen species (·OH) and high-valence iron (Fe(IV)=O) by the SPC-PB catalytic system, viral capsid proteins and the genome are simultaneously destroyed, achieving an inactivation efficiency ≥99.9%. This chemical-biological synergistic mechanism overcomes the efficiency bottleneck of traditional separation-based treatment techniques.

[0050] (2) The present invention optimizes the carrier function through layered loading and pH-responsive release technology: the sodium 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 operational stability.

[0051] (3) The porous PLA support layer of the present invention has both high mechanical strength (≥8MPa) and permeability (porosity 80%), maintains structural integrity under high-speed water flow (1.5m / s), and allows free radicals to diffuse to the surface of the biofilm, ensuring the full progress of the virus inactivation reaction. Attached Figure Description

[0052] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention as described above or otherwise will become clearer.

[0053] Figure 1 This is a schematic diagram of the structure of the peritrichous biological carrier described in this invention.

[0054] Figure 2 This is a flowchart illustrating the process of enriching and eliminating viruses using the periphery biological carrier described in this invention.

[0055] Figure 3This is a comparison chart showing the effects of different methods in Example 1 on treating viruses in aquaculture wastewater.

[0056] Figure 4 It is a fitted curve showing the change in virus concentration with treatment time in Example 1.

[0057] Among them, 1 is cyclic diguanosine monophosphate, 2 is gel, 3 is nanocarrier, 4 is potassium ferrocyanide, 5 is sodium percarbonate, 6 is support layer, 7 is pore, 8 is intact virus particle, 9 is viral capsid, and 10 is viral fragment. Detailed Implementation

[0058] The present invention can be better understood from the following embodiments.

[0059] Example 1

[0060] (1) Mix a 0.05-0.1mM solution of cyclic diguanosine monophosphate with a sodium alginate solution, then add 2-4wt% calcium chloride solution dropwise to crosslink and solidify to form gel microspheres.

[0061] (2) Using the initial wet impregnation method, a 30 wt% sodium percarbonate solution is impregnated into mesoporous silica nanoparticles (pore size 2-5 nm, specific surface area ≥500 m²). 2 Potassium ferrocyanide nanoparticles (SPC@SiO2) were vacuum dried at 60℃ for 6 h. These nanoparticles were then mixed with a 0.5–1 mg / mL solution of polydiallyldimethylammonium chloride (PDDA) and then introduced into SPC@SiO2. PB was immobilized on the surface via electrostatic adsorption, forming catalytic particles. The loading amounts were 20–30 wt% SPC and 5–10 wt% PB, calculated based on the mass of SiO2.

[0062] (3) The catalyst particles are suspended in water to obtain a suspension, and then the gel microspheres obtained in step (1) are immersed in the suspension and ultrasonically treated for 10-20 minutes to obtain the core;

[0063] (4) The core gel sphere was immersed in 8-12 wt% polylactic acid (PLA) solution, pre-frozen (-40℃) for 2 hours, and then freeze-dried at -80℃ to form a periclump biological carrier with a porous support layer (pore size 50-200 μm, porosity 82%).

[0064] (5) The aforementioned cyclophosphamide carrier was added to the aquaculture wastewater containing white spot syndrome virus, and the virus concentration in the water before treatment was 10. 6 copies / mL (Ct value ≤25 as detected by RT-qPCR).

[0065] like Figure 2 As shown, after the introduction of this cyclophosphamide carrier, c-di-GMP induces microorganisms to secrete β-glucan, which specifically adsorbs virus particles through the hydrophobic interaction of the α-helix structure, achieving an adsorption capacity of 10. 10 pfu / g; intact virus particles are adsorbed and aggregated; subsequently, the virus enters the contact catalytic particles under fluid drive, and the catalytic components release ·OH and Fe(IV)=O for in-situ inactivation, attacking cysteine ​​residues (oxidation rate ≥90%) and genomic DNA (fragmentation rate ≥95%) of the viral capsid protein, respectively. The viral capsid gradually breaks down under oxidation; finally, the viral 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.

[0066] After treatment, the residual virus in the water is ≤10. 2 Copies / mL (Ct value ≥ 35), inactivation efficiency ≥ 99.99% after 24 hours. Throughout the process, the carrier structure maintains a stable laminar flow environment, ensuring that each reaction stage proceeds in an orderly manner.

[0067] The fitted curve of virus concentration changing with treatment time is as follows: Figure 4 As shown: The experiment was conducted at 25±1℃, and the initial virus concentration in the tailwater was approximately 10. 6 The viral load was 0.5 g / L, with a volume of 5 L water and a pH maintained at 7.0. Water samples were collected at 0, 2, 4, 6, 12, and 24 hours during the experiment. Virus concentration was detected using RT-qPCR. Data are expressed as logarithmic values. 10 The results, in formal terms, show that the virus concentration decreases exponentially over time. Fitting analysis indicates that the inactivation process conforms to a first-order reaction kinetic model, and the fitting equation is as follows:

[0068] ln(C / C0) = -kt,

[0069] Where C represents the virus concentration at any time point, C0 represents the initial virus concentration, and k is the reaction rate constant. Calculated via nonlinear regression, k = 0.543 h. -1 Correlation coefficient R 2 = 0.993. The results show that the vector of the present invention has a stable time-dependent and predictable inactivation process of viruses in aquaculture effluent, and the inactivation efficiency increases significantly with reaction time.

[0070] (6) Three parallel experiments were set up: a chemical treatment group, a UV treatment group, and the group using the *Zygophyllum commune* biological carrier of this invention, all treated with the same initial concentration (10... 6 The treatment volume for each type of wastewater containing white spot syndrome virus (copies / mL) was 5 L.

[0071] Chemical treatment group: Add 0.5 mM H2O2 + 0.1 mM FeSO4, react for 30 minutes, and adjust the pH to 6.5.

[0072] Ultraviolet treatment group: Insert a 254 nm wavelength ultraviolet lamp (36 W power) into the water body, with the lamp tube 5 cm away from the bottom of the water, and place it vertically in the central area for continuous irradiation for 60 minutes. At the same time, set up magnetic stirring to maintain the slow flow of the water body.

[0073] Carrier treatment group: The circulatory biological carriers prepared in steps (1) to (4) were added at 0.5 g / L, the reaction temperature was 25℃, the pH was 7.0, and the water was slowly stirred.

[0074] like Figure 3 As shown, under the same initial virus concentration conditions (10 6 The three treatment methods showed significantly different trends in virus concentration reduction over 24 hours. The *Cyclocarya paliurus* bio-vector group described in this invention exhibited a rapid decrease in virus concentration after 6 hours, reaching the detection limit (approximately 10 copies / mL) by 24 hours, with an inactivation rate ≥99.99%. This effect is comparable to the ultraviolet treatment group, but the *Cyclocarya paliurus* bio-vector, while achieving a high inactivation rate, also possesses virus enrichment capabilities, providing a possibility for subsequent separation. In contrast, the chemical treatment group had significantly lower inactivation efficiency, with the virus concentration in the effluent still exceeding 10 copies / mL after 24 hours. 4 copies / mL, inactivation rate less than 90%.

[0075] like Figure 4 As shown, the virus concentration (log) of the treatment group in this invention 10 The change with treatment time conforms to a first-order reaction kinetic model, with a fitting constant of k = 0.543 h. -1 Correlation coefficient R 2 = 0.993, indicating that the vector system exhibits a clear time dependence and mechanistic stability in the virus inactivation process. The model fitting curve closely matches the measured value, further supporting the reproducibility and predictability of the virus inactivation process.

Claims

1. A periculture biological vector for the enrichment and disinfection of viruses in aquaculture wastewater, characterized in that, The pericarp biocarrier has a core-shell structure, with the core being a signal layer on which catalytic particles are loaded, and the outer shell being a support layer. The signal layer is a gel dispersed with cyclic diguanosine monophosphate, and the catalytic particles are nanocarriers loaded with sodium percarbonate and potassium ferrocyanide. The support layer is polylactic acid; the gel is sodium alginate gel.

2. The periclump biological carrier according to claim 1, characterized in that, The pore size of the support layer is 65~180μm.

3. The periclump biological carrier according to claim 1, characterized in that, The nanocarrier is mesoporous silica nanoparticles.

4. The periclump biological carrier according to claim 1, characterized in that, The amount of sodium percarbonate in the nanocarrier is 20-30 wt% of the mass of the nanocarrier.

5. The periclump biological carrier according to claim 1, characterized in that, The amount of potassium ferrocyanide is 5 to 10 wt% of the mass of the nanocarrier.

6. The periclump biological carrier according to claim 1, characterized in that, In the signal layer, the content of cyclic diguanosine monophosphate is 0.002-0.004 wt%, calculated as a percentage of the mass of cyclic diguanosine monophosphate to the mass of sodium alginate gel.

7. The method for preparing the peritrichous biological carrier for virus enrichment and disinfection in aquaculture tailwater as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Mix the solution of cyclic diguanylic acid with the gel solution evenly, and then solidify to form gel microspheres; (2) The sodium percarbonate solution is impregnated into the nanocarrier by the initial wet impregnation method to obtain the sodium percarbonate loaded nanocarrier; then the sodium percarbonate loaded nanocarrier is mixed with a cationic polymer solution containing potassium ferrocyanide nanoparticles, and the potassium ferrocyanide nanoparticles are fixed on the surface of the sodium percarbonate loaded nanocarrier by electrostatic adsorption. After cleaning, the catalyst particles are obtained. (3) The catalyst particles are suspended in water to obtain a suspension, and then the gel microspheres obtained in step (1) are immersed in the suspension and ultrasonically treated to obtain the core; (4) The core is immersed in a polymer solution, so that the polymer solution is uniformly attached to the surface of the core, and then the solvent is removed to obtain the Zhou Cong biological carrier; the polymer solution is an 8-12 wt% polylactic acid solution.

8. The preparation method according to claim 7, characterized in that, The cationic polymer mentioned in step (2) is polydiallyldimethylammonium chloride.

9. The application of the peritrichous biological carrier for virus enrichment and disinfection in aquaculture tailwater as described in any one of claims 1-6.

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