Rapid recovery device and method for free viruses in activated sludge

Abstract

The invention belongs to the field of sewage treatment, and particularly relates to a device and a method for quickly recovering free viruses in activated sludge. Aiming at the problems that the existing method for recovering free viruses in activated sludge is poor in suitability and low in efficiency, viruses are easy to inactivate and free nucleic acid interferes with subsequent analysis, the invention provides a novel method for recovering free viruses. The method comprises the following steps: primarily separating an activated sludge sample by a paper membrane filtering unit to remove most solid particle impurities; and finally, the nucleic acid is treated by the nucleic acid pretreatment unit to complete recovery. The method does not need to add additional chemical reagents, realizes accurate and efficient separation of free viruses and sludge impurities through the synergistic effect of physical separation and low-temperature protection, guarantees the integrity of the viruses, effectively removes free nucleic acids, improves the subsequent analysis accuracy, is simple and convenient to operate, is green and efficient, and has a wide application prospect. A feasible technical path is provided for deep research and function development of the free viruses in the activated sludge.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of sewage treatment, and particularly relates to a device and method for rapid recovery of free viruses in activated sludge. BACKGROUND

[0002] The activated sludge method is the most widely used biological technology in the field of municipal sewage and industrial wastewater treatment at present, and its core is to metabolically degrade organic pollutants in water through complex microbial communities (including bacteria, fungi, protozoa, etc.) in activated sludge. Viruses in activated sludge also play an indispensable important function.

[0003] Viruses in activated sludge exist in various forms such as free viruses and adsorbed viruses. Among them, free viruses are a key component of microbial communities in activated sludge, which not only exhibit significant global diversity and biogeographic characteristics, but also participate in the regulation of the stability of the bacterial community structure by regulating the growth and reproduction of host microorganisms. As the main group of viruses, phages have important application potential in key process links such as sludge bulking and foam control. For example, phages can be used as biological control agents to effectively lyse filamentous bacteria such as Corynebacteria and Haliscomenobacter that cause sludge bulking, thereby improving sludge settling performance, and are more environmentally friendly than traditional chemical treatment methods. In addition, in-depth analysis of the community composition, abundance and dynamic change characteristics of free viruses in activated sludge can also provide core basis for revealing the epidemiological model of viruses, optimizing wastewater biological treatment processes, and exploring efficient functional virus resources. Therefore, efficient and rapid recovery of free viruses in activated sludge is a prerequisite and key to carrying out functional research on viruses and promoting their application in wastewater treatment process optimization.

[0004] Currently, methods for recovering viruses from activated sludge are mainly based on modifications of traditional wastewater virus extraction technologies, and a specialized process solution tailored to the characteristics of activated sludge systems has not yet been developed. Existing methods are ill-suited to the high solids content, high viscosity, and complex composition of activated sludge systems, and struggle to simultaneously ensure the efficiency, completeness, and accuracy of free virus recovery and subsequent analysis. The core idea of ​​existing technologies is primarily to disrupt the activated sludge structure through physical and chemical means, causing viruses to desorb from sludge solid particles or extracellular polymers, followed by separation and concentration to obtain a viral solution. Specifically, this can be categorized as follows: First, physical methods, such as ultrasonic disruption, use mechanical force to break down the sludge floc structure, releasing encapsulated or adsorbed viruses, which are then concentrated through filtration or centrifugation. While this method is relatively simple to operate, existing centrifugation methods related to concentration may fail to achieve efficient separation of free viruses from fine sludge particles due to factors such as increased temperature during centrifugation and insufficient centrifugation speed, further exacerbating the problems of low recovery efficiency and poor quality. Secondly, there is the chemical method, which uses solutions such as FeCl3 and MgCl2 to adjust the ionic strength of the system, weakening the binding force between the virus and sludge particles to achieve desorption, followed by virus concentration through filtration. While this method can improve virus desorption efficiency, it easily introduces impurities, interfering with subsequent virus detection or analysis. More importantly, the elution efficiency during membrane elution significantly affects virus recovery efficiency. Furthermore, existing methods generally lack the crucial step of pretreatment with DNase I and RNase A to remove free nucleic acids before subsequent analyses such as viral gene sequencing, severely limiting in-depth research on their function and community characteristics.

[0005] Therefore, developing a rapid recovery method for free viruses in activated sludge that is easy to operate, has high recovery efficiency, short cycle, good stability, and includes low-temperature high-speed centrifugation ultrafiltration concentration and pretreatment before sequencing has become an urgent technical problem to be solved in this field, and has important practical significance and application value. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid method for recovering free viruses from activated sludge.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: a method for recovering free viruses from activated sludge, the method comprising the following steps:

[0008] (1) The activated sludge is filtered through a paper membrane to remove solid particulate impurities; the pore size of the paper membrane is 25 μm.

[0009] (2) The activated sludge treated in step (1) is concentrated by low-temperature high-speed centrifugal ultrafiltration. The operating conditions for low-temperature high-speed centrifugal ultrafiltration concentration are: 2-4℃, rotation speed of 3000-4500g, and ultrafiltration membrane pore size of 50kDa.

[0010] (3) The activated sludge treated in step (2) is enzymatically hydrolyzed with DNase I and / or RNase A to remove free nucleic acids. The hydrolysis temperature is 37℃.

[0011] Accordingly, an apparatus for recovering free viruses from activated sludge is provided. The apparatus includes a paper membrane filtration unit, a low-temperature high-speed centrifugal ultrafiltration concentration unit, and a nucleic acid pretreatment unit, which are sequentially connected and used. The paper membrane filtration unit is equipped with a paper membrane material suitable for wastewater filtration. The low-temperature high-speed centrifugal ultrafiltration concentration unit is a device capable of high-speed centrifugation at a controllable temperature. The nucleic acid pretreatment unit is a sealed chamber capable of enzymatic hydrolysis, and enzymes for enzymatically hydrolyzing free nucleic acids in wastewater can be automatically or manually released inside. The nucleic acid pretreatment unit includes a temperature control unit and a stirring unit.

[0012] Accordingly, the device is used to recover free viruses from activated sludge.

[0013] This invention offers the following advantages: It provides a rapid recovery device and method for free viruses from activated sludge using a paper-membrane coupled ultrafiltration system, with an additional nucleic acid pretreatment step. The system and method provided by this invention require no additional chemical reagents. Through the synergistic effect of physical separation and low-temperature protection, it not only achieves precise and efficient separation of free viruses from sludge impurities, ensuring the integrity of the recovered viruses, but also effectively removes free nucleic acid impurities, improving the accuracy of subsequent sequencing analysis. This method simplifies the operation process, achieving green and efficient operation, and provides a practical technical path for in-depth research and functional development of free viruses in activated sludge. Attached Figure Description

[0014] Figure 1 This is a flowchart illustrating an embodiment of the present invention;

[0015] Figure 2 A schematic diagram illustrating the activity identification of free viral substitutes T4 (DNA virus) and MS2 (RNA virus);

[0016] Figure 3 A schematic diagram of the standard curve for (RT)-qPCR molecular quantification of free viral substitutes T4 and MS2;

[0017] Figure 4 This is a schematic diagram showing the comparison of the recovery rates of T4 and MS2 in activated sludge using various concentration methods. Different lowercase letters indicate significant differences between different concentration methods (P < 0.05).

[0018] Figure 5This is a schematic diagram of the orthogonal experiment results for DNase I enzyme. Different uppercase letters indicate significant differences between different concentrations at the same temperature (P < 0.05), and different lowercase letters indicate significant differences between different temperatures at the same concentration (P < 0.05).

[0019] Figure 6 This is a schematic diagram of the orthogonal experimental results of DNase I enzyme in pure nucleic acid (a, b) and mixed virus particles and nucleic acid (c, d) systems. Different lowercase letters indicate significant differences between different concentrations at the same temperature (P < 0.05).

[0020] Figure 7 This is a schematic diagram of the orthogonal experiment results for RNase A enzyme. Different uppercase letters indicate significant differences between different concentrations at the same temperature (P < 0.05), and different lowercase letters indicate significant differences between different temperatures at the same concentration (P < 0.05).

[0021] Figure 8 This is a schematic diagram of the orthogonal experimental results of RNase A enzyme in pure nucleic acid (a,b) and a mixed system of virus particles and nucleic acid. ** indicates that there are significant differences between different concentrations at the same temperature (P<0.05). Detailed Implementation

[0022] This invention provides a device for rapidly recovering free viruses from activated sludge. The device includes a paper membrane filter unit, a low-temperature high-speed centrifugal ultrafiltration concentration unit, and a nucleic acid pretreatment unit, which are sequentially connected and used in sequence. The paper membrane filter unit uses a paper membrane material suitable for wastewater filtration, preferably a paper membrane material with a pore size of 25 μm. The low-temperature high-speed centrifugal ultrafiltration concentration unit is a device capable of high-speed centrifugation at a controlled temperature, a mature existing technology. For example, a low-temperature centrifugal concentrator can be used; another example is the use of Vivaspin 20 ultrafiltration centrifuge tubes (protein cutoff (MWCO) of 10 kDa, PES material) and a temperature-controlled centrifuge. The nucleic acid pretreatment unit is a sealed chamber capable of enzymatic hydrolysis, with automatic or manual release of enzymes, such as DNase I and RNase A, for enzymatic hydrolysis of free nucleic acids in wastewater. The nucleic acid pretreatment unit also includes a temperature control unit and a stirring unit to provide a suitable enzymatic hydrolysis temperature and ensure thorough and uniform hydrolysis. Both the temperature control unit and the stirring unit are mature existing technologies: the temperature control unit may consist of, for example, a cooling rod, a heating rod and a thermometer, and the stirring unit may be an electric stirring shaft.

[0023] This invention also provides a method for rapidly recovering free viruses from activated sludge, specifically including the following steps:

[0024] 1. The activated sludge sample (sludge-water mixture or supernatant after activated sludge sedimentation) is first separated by paper membrane filtration to remove most of the solid particulate impurities (e.g., by flowing through a paper membrane filtration unit).

[0025] 2. The activated sludge treated in step 1 is then subjected to low-temperature high-speed centrifugal ultrafiltration for virus concentration (e.g., by flowing through a low-temperature high-speed centrifugal ultrafiltration concentration unit). After filtration, the filtrate is discarded. The activated sludge sample is added back in, and centrifugation continues until the sample concentration is complete and the target substance is retained on the surface of the ultrafiltration membrane. The membrane surface is then repeatedly agitated with 1 mL of PBS for elution. The operating conditions for low-temperature high-speed centrifugal ultrafiltration concentration can be selected as follows: temperature controlled at 2–4℃, centrifugation speed at 3000–4500 g, and ultrafiltration membrane pore size of 50 kDa.

[0026] 3. Finally, the free nucleic acid is removed by enzymatic hydrolysis, for example, by passing it through a nucleic acid pretreatment unit. In this unit, DNase I and RNase A are added, the hydrolysis temperature is 37°C, and the hydrolysis time can be selected as 30 min for each.

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0028] Example

[0029] The specific operation process of this embodiment is as follows: Figure 1 As shown.

[0030] 1. Establish a quantitative system for viral activity.

[0031] T4 (AB 2015375, CCTCC) was selected as the DNA virus surrogate, and MS2 (15597-B1™, ATCC) was selected as the RNA virus surrogate. The surrogates were enriched overnight in LB liquid medium containing the host (AB 206126, CCTCC and 15597™, ATCC) (37℃, 150 rpm). The enriched solutions were then centrifuged (5000 rpm, 10 min), and the supernatant was purified using a 0.22 μm sterile polyethersulfone (PES) membrane. The recovered solutions were homogenized, aliquoted, and stored at -80℃ for later use.

[0032] In addition, according to the agar plate method, 1 mL of homogenized T4 and MS2 stock solutions were taken and serially diluted to 10⁻⁶. -10Using the droplet method, 10 μL of different concentrations of dilution were inoculated onto LB agar plates containing host bacteria and incubated overnight. Plaque counting was then performed. Results are as follows: Figure 2 As shown, the abundance of T4 is 4.67 × 10⁻⁶. 7 (±5.77×10 6 The abundance of PFU / mL and MS2 was 2.6 × 10⁻⁶. 10 (±1.2×10 10 )PFU / mL.

[0033] 2. Establish a quantitative system for viral molecules.

[0034] (RT)-qPCR technology offers advantages such as precise detection and short reaction time, allowing for real-time monitoring of fluorescence signals. Accurate quantification of MS2 and T4 phages using a standard curve provides an efficient and accurate basis for screening virus recovery methods. Therefore, (RT)-qPCR technology was used to quantify T4 and MS2. A standard curve was prepared using positive control materials in the form of T4 and MS2 gene fragments ordered from Sangon Biotech (Shanghai) Co., Ltd. Information on T4 and MS2 primers, probe sequences, and cycling conditions is shown in Table 1. In Table 1, MM is 2XTaqMan Fast qPCR (NO. B639274), containing Taq DNA polymerase, dNTPs, etc., purchased from Sangon Biotech (Shanghai) Co., Ltd.; RT is RevertAid Reverse Transcriptase (EP0441), purchased from Thermoscientific.

[0035] Table 1 Primers, probes, and PCR reaction parameters for (RT)-qPCR systems of T4 and MS2.

[0036]

[0037] Constructing (RT)-qPCR standard curves for T4 and MS2 phages (real-time quantitative PCR instrument, CFX384, BIO-RAD, USA), as detailed below. Figure 3 As shown. The results show that the T4 phage standard curve is within the concentration range of 10. 1 ~10 8 The linear relationship within copies / mL was good, with a correlation coefficient R² of 0.996 and an amplification efficiency of 99.3%; the MS2 phage standard curve showed good linearity within 10 copies / mL. 2 ~10 9Significant linear correlation was also observed within the copy / mL concentration range, with an R² of 0.979 and an amplification efficiency of 102.2%. The slopes of the two phage standard curves were -3.338 and -3.27, respectively, and the intercepts were 41.261 and 38.037, respectively, both meeting the requirements for (RT)-qPCR quantitative analysis. This indicates that the established standard curves can be used for accurate quantification of T4 and MS2 phages in actual samples, providing a reliable quantitative benchmark for the subsequent screening of virus recovery methods in activated sludge systems.

[0038] 3. Demonstration of the free virus recovery effect of the activated sludge system.

[0039] In this embodiment, the concentrated volume of activated sludge was based on the minimum specified volume in USEPA-1615 (2012), and the recommended volume settings in EPA-464.2 (2003) and USEPA Water Reuse Guidelines (2012). Using an activated sludge concentrated volume of 100 mL, (RT)-qPCR was employed to compare the optimal recovery rates of T4 and MS2 in activated sludge using three methods: PEG, paper-coated ultrafiltration (PFC-UF), and virus adsorption-elution (VIRADEL). The specific experimental procedures for the three free virus recovery methods are as follows:

[0040] (1) PEG method. Following the US ISO protocol 15216-2 standard, a 5× polyethylene glycol (PEG) solution (500 g / L PEG 8000, 1.5 mol / L NaCl) was prepared. 100 mL of activated sludge was centrifuged at 4700 g for 30 min at 4 °C. The supernatant was collected and evenly dispersed into centrifuge tubes. PEG solution was added to bring the final PEG concentration to 10% and the NaCl concentration to 0.3 M. The mixture was vortexed for 10 min and then incubated at 4 °C and 150 rpm for 2 h. After incubation, the mixture was centrifuged at 12000 g at 4 °C for 100 min, and the supernatant was removed, leaving the residual precipitate. The precipitate was resuspended in <1 mL of PBS to obtain the concentrated virus sample.

[0041] (2) PFC-UF method. A filtration device was built using experimental equipment such as a glass filter funnel, filter bottle, vacuum pump and rubber tubing. 100 mL of activated sludge sample was filtered through a paper membrane (1004-047, Whatman, UK) with a pore size of 25 μm and a diameter of 47 mm. The sample was then centrifuged at 2-4℃ and 3000-4500 g (HC-3016R, Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.) using a Vivispin 20 (MWCO 10 KDa, Sartorius, Germany) ultrafiltration centrifuge tube to concentrate 100 mL to 1 mL. The membrane surface of the ultrafiltration centrifuge tube was then thoroughly cleaned by blowing and the concentrate was recovered to complete the concentration of free virus.

[0042] (3) VIRADEL method. MgCl2 was added to 100 mL of activated sludge sample to obtain a final concentration of 25 mL MgCl2. A vacuum filtration device (SHB-Ⅲ, Zhengzhou Great Wall Science & Industry Co., Ltd.) was set up and its airtightness was checked. A mixed cellulose ester membrane (MCE, HAWP04700, Merck Millipore, Billerica, MA, USA) with a pore size of 0.45 μm and a diameter of 47 mm was selected as the virus adsorption carrier. The membrane was stably embedded in the membrane support of the filter funnel and compacted and fixed. The sample containing MgCl2 was slowly poured into the vacuum filtration device. After the sample was completely filtered, the MCE membrane with adsorbed free virus particles was taken out and transferred to a 50 mL sterile centrifuge tube.

[0043] After concentrating the free virus using the three methods described above, the mixed viral nucleic acid was extracted using the RNeasy Mini Kit (50) (74104, QIAGEN, USA), and then quantified by (RT)-qPCR to calculate the recovery rate. The formula for calculating the recovery rate (%) is as follows:

[0044]

[0045] GC sample This refers to the GC value corresponding to the concentrated viral sample. ck This represents the GC value for a virus sample that has not undergone any processing.

[0046] The results are as follows Figure 4 As shown in the figure. The results show that, for activated sludge systems, the PFC-UF method has a better recovery rate for MS2 bacteriophage and T4 bacteriophage than the PEG method and the VIRADEL method.

[0047] 4. Demonstration of the effects of DNase I and RNase A pretreatment.

[0048] (1) Optimization of DNase I pretreatment conditions

[0049] DNase I was purchased from Sangon Biotech (Shanghai) Co., Ltd. (B618252, 1 KU). This enzyme can hydrolyze single-stranded and double-stranded DNA at 37°C. Currently, there is no universally applicable concentration of DNase I for treating environmental virus samples, and the reaction is generally stopped at 65°C after DNase I hydrolysis. However, 65°C may cause free virus particles in activated sludge to lose their activity, and therefore may not be suitable for the method of this invention.

[0050] In this embodiment, the effects of different conditions on MS2 and T4 viral particles were investigated within three reaction termination temperatures (55, 60, and 65°C) and four DNase I enzyme concentrations (0, 1, 5, and 10 U / mL). 0.5 mL of homogenized T4 and MS2 solutions (potency: 10) were taken respectively. 5 As a sample of free virus particles (PFU / mL), DNase I enzyme at the four concentrations mentioned above was added. After incubation at 37°C for 30 min, 1 μL of 50 mM EDTA was immediately added to terminate the reaction. The samples were then incubated at the three temperatures mentioned above for 10 min each to terminate the reaction, yielding DNase I pretreated samples. Viral nucleic acid was extracted from the pretreated samples and identified by (RT)-qPCR. The residual nucleic acid percentage (%) was calculated. Results are as follows: Figure 5 As shown, the conditions with the least impact on MS2 and T4 viral particles are 1, 5, and 10 U / mL at 55℃ and 1 U / mL at 60℃.

[0051] To further verify the four experimental conditions selected by the orthogonal experiment, a pure nucleic acid environment and a mixed system of virus particles and nucleic acid were constructed to simulate the actual viral sample environment. The specific experimental design is shown in Table 2.

[0052] Table 2 Orthogonal experimental design table for pretreatment of RNase I and RNase A

[0053]

[0054] Based on the experimental results, the optimal conditions were determined to be 55℃ and 1U / mL ( Figure 6 Under these optimal conditions, the degradation effect on free nucleic acids in pure nucleic acid systems is good, while the damage to virus particles in mixed systems of virus particles and nucleic acids is minimal. It can effectively remove free nucleic acids and protect the integrity of virus particles to the greatest extent.

[0055] The formula for calculating residual percentage (%) is as follows:

[0056]

[0057] GC sample The GC values ​​are the corresponding values ​​of the samples after DNase I and RNase A pretreatment. ck This represents the GC value for a virus sample that has not undergone any processing.

[0058] (2) Optimization of RNase A pretreatment conditions

[0059] RNase A was also purchased from Sangon Biotech (Shanghai) Co., Ltd. (B500474, 1 mL). Similarly, through orthogonal experiments, the effects of different RNase A concentrations on MS2 and T4 virus particles were investigated at three temperatures (55, 60, and 65℃) and four concentration ranges (0, 1, 5, and 10 U / mL). The two experimental conditions with the least impact on MS2 and T4 virus particles were screened out. The results were: 1 U / mL at 55℃ and 1 U / mL at 65℃ (… Figure 7 ).

[0060] Subsequently, a hybrid system of pure nucleic acid and viral particles plus nucleic acid was constructed to simulate the actual environment, further validating the conditions screened by orthogonal experiments. Figure 8 It is evident that under conditions of 65℃ and 1 U / mL, RNase A significantly degrades free RNA in pure nucleic acid systems while causing minimal damage to viral particles in mixed systems containing both viral particles and nucleic acids. This demonstrates efficient removal of free RNA while maximally preserving the integrity of viral particles. Under these conditions, RNase A efficiently degrades free RNA, reducing its interference with virus detection and analysis, while minimizing damage to MS2 and T4 viral particles. This contrasts with DNase I, which primarily targets free DNA, exhibiting different target groups and application scenarios. Furthermore, RNase A complements DNase I in free DNA degradation and its corresponding experimental conditions (55℃, 1 U / mL). Both target different types of free nucleic acids, providing more comprehensive and reliable pretreatment technology support for the utilization of free viral resources in activated sludge, ensuring the quality of subsequent experimental samples and the accuracy of research results.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for recovering free viruses from activated sludge, characterized in that: The method includes the following steps: (1) The activated sludge is filtered through a paper membrane to remove solid particulate impurities; (2) The activated sludge treated in step (1) is concentrated by low-temperature high-speed centrifugation and ultrafiltration; (3) The activated sludge treated in step (2) is enzymatically hydrolyzed to remove free nucleic acids.

2. The method according to claim 1, characterized in that: The pore size of the paper film in step (1) is 25 μm.

3. The method according to claim 1, characterized in that: The operating conditions for low-temperature high-speed centrifugal ultrafiltration concentration in step (2) are: 2-4℃, rotation speed of 3000-4500g, and ultrafiltration membrane pore size of 50kDa.

4. The method according to claim 1, characterized in that: In step (3), DNase I and / or RNase A enzymes are used for enzymatic hydrolysis.

5. The method according to claim 4, characterized in that: The enzymatic hydrolysis temperature is 37℃.

6. A device for recovering free viruses from activated sludge, characterized in that: The device includes a paper membrane filtration unit, a low-temperature high-speed centrifugal ultrafiltration concentration unit, and a nucleic acid pretreatment unit that are connected and used in sequence. The paper membrane filtration unit is equipped with a paper membrane material that can be used for wastewater filtration. The low-temperature high-speed centrifugal ultrafiltration concentration unit is a device that can perform high-speed centrifugation at a controllable temperature. The nucleic acid pretreatment unit is a sealed box that can perform enzymatic hydrolysis, and enzymes for enzymatic hydrolysis of free nucleic acids in wastewater can be released automatically or manually inside.

7. The apparatus according to claim 6, characterized in that: The nucleic acid pretreatment unit includes a temperature control unit and a stirring unit.

8. The application of the apparatus of claim 6 or 7 in the recovery of free viruses from activated sludge.

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