A method for treating resistant genes in sewage by using a microalgae-fungal mycelium symbiotic system and application thereof

Abstract

The application belongs to the technical field of environmental protection, and discloses application of a bacterial agent in treatment of sewage containing resistance genes or preparation of a product for treating sewage containing resistance genes, wherein the bacterial agent comprises seawater microalgae and fungal hyphae, the resistance genes comprise at least one of integron, sulfonamides, fluoroquinolones and tetracyclines, and the salinity of the sewage is 1% to 4%. The method for treating sewage containing resistance genes can effectively remove ARGs in the sewage, has good removal effect on integron, sulfonamides, fluoroquinolones and tetracyclines in the sewage, and the total removal rate of ARGs reaches 97.66%. The method for treating sewage containing resistance genes is green, pollution-free, low in operation cost, simple to operate and short in treatment period.

Description

Technical Field

[0001] This invention belongs to the field of environmental protection technology, specifically relating to a method for treating resistance genes in wastewater using a microalgae-fungus hyphae symbiotic system and its application. Background Technology

[0002] my country boasts a long coastline and vast sea areas, providing ideal conditions for mariculture. During or after the aquaculture process, wastewater is discharged directly or indirectly into receiving sea areas. With advancements in technology and continuous development of aquaculture techniques, my country's aquaculture model has gradually shifted towards intensive farming. This model involves high stocking densities, which can easily lead to environmental degradation and frequent viral outbreaks. To prevent and treat bacterial diseases, farmers often use large amounts of antibiotics during the farming process. Antibiotics that cannot be fully absorbed or utilized by the farmed species remain in the wastewater. Furthermore, the pressure from antibiotics can induce the generation of antibiotic resistance genes (ARGs), potentially even leading to the emergence of "superbugs."

[0003] Statistics show that the usage and concentration of antibiotics in aquaculture wastewater in my country are significantly higher than in other countries. Tetracycline, norfloxacin, and sulfamethoxazole are frequently detected in aquaculture farms in the Pearl River Estuary and other areas of my country, and the abundance of related antibiotic resistance genes is also frequently detected. Currently used wastewater treatment methods such as physical, chemical, and advanced oxidation methods are not suitable for treating aquaculture wastewater with complex compositions and large volumes.

[0004] The technology of using algae-bacteria combined systems to remove nutrients from aquaculture wastewater is well-developed. However, there is currently no application of algae-bacteria combined systems to remove antibiotic resistance genes from marine aquaculture wastewater. Summary of the Invention

[0005] The present invention aims to at least solve the technical problems existing in the prior art. The present invention provides a method for treating resistance genes in wastewater using a microalgae-fungus hyphae symbiotic system and its application.

[0006] The first objective of this invention is to provide an application of a microbial agent in treating wastewater containing resistance genes or in preparing products for treating wastewater containing resistance genes.

[0007] A second aspect of the present invention is to provide a method for treating wastewater containing resistance genes.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the use of a microbial agent in treating wastewater containing resistance genes or in preparing a product for treating wastewater containing resistance genes.

[0009] In some embodiments of the present invention, the microbial agent includes marine microalgae and fungal hyphae.

[0010] In some embodiments of the present invention, the resistance gene includes at least one of integrons, sulfonamides, fluoroquinolones, and tetracyclines.

[0011] In some embodiments of the present invention, the integrinoid resistance gene includes intl1 and intl2 At least one of them.

[0012] In some embodiments of the present invention, the sulfonamide resistance gene includes sul1 and sul2 At least one of them.

[0013] In some embodiments of the present invention, the fluoroquinolone resistance gene includes qnrA , qnrB , qnrD , qnrS and aac(6’)-lb-cr At least one of them.

[0014] In some embodiments of the present invention, the tetracycline resistance gene includes tetA , tetB , tetC , tetG , tetM , tetO , tetB(P) , tetX and tetW At least one of them.

[0015] In some embodiments of the present invention, the salinity of the wastewater is 1% to 4%.

[0016] In some embodiments of the present invention, the marine microalgae include at least one of Chlorella vulgaris, Dunaliella salina, Subcoria scabra, Microcystis scabra and Chlorella pulvinata.

[0017] In some embodiments of the present invention, the fungal hyphae include at least one of Ganoderma lucidum hyphae, Monascus purpureus hyphae, Lentinus edodes hyphae, Aspergillus niger hyphae, and Pleurotus ostreatus hyphae.

[0018] In some embodiments of the present invention, the concentration ratio of the marine microalgae to fungi is (10~15) cells / mL: 1 spore / mL.

[0019] In some embodiments of the present invention, the concentration of the marine microalgae is 6 × 10⁻⁶. 6 cells / mL ~1.2×10 7cells / mL.

[0020] In some embodiments of the present invention, the concentration of the fungus is 6 × 10⁻⁶. 5 spores / mL ~8×10 5 spores / mL.

[0021] A second aspect of the present invention provides a method for treating wastewater containing resistance genes, the method comprising adding a microbial agent to the wastewater containing resistance genes for treatment, the microbial agent comprising marine microalgae and fungal hyphae.

[0022] In some embodiments of the present invention, the wastewater is high-salinity wastewater.

[0023] In some embodiments of the present invention, the salinity of the high-salinity wastewater is 1% to 4%.

[0024] In some embodiments of the present invention, the method further includes co-culturing marine microalgae and fungal hyphae to obtain a microalgae-fungus hyphae symbiotic system.

[0025] In some embodiments of the present invention, the culture conditions of the microalgae-fungus mycelium symbiotic system are: temperature 30±2℃, 140~160rpm, and light intensity 5000~8000lux.

[0026] In some embodiments of the present invention, the inoculum size of the microalgae in the marine microalgae-fungus hyphae symbiotic system is 6 × 10⁻⁶. 6 cells / mL ~1.2×10 7 cells / mL, fungal inoculum size 6 × 10⁻⁶ 5 spores / mL ~8×10 5 spores / mL.

[0027] In some embodiments of the present invention, the method for preparing the marine microalgae includes: culturing the algae in an artificial seawater F / 2 culture medium at a temperature of 20-35°C, a light intensity of 6000-8000 lux, and a light-dark cycle of (16-20) h (8-4) h, collecting the algal cells, and resuspending them in wastewater.

[0028] In some embodiments of the present invention, the algal cells are rinsed with wastewater to remove residual F / 2 culture medium and then resuspended in wastewater.

[0029] In some embodiments of the present invention, the artificial seawater F / 2 culture medium is composed of: NaCl, Na2SO4. 4、 MgCl2·6H2O, NaHCO3 3、 KCl, KBr, H3BO 3、NaF, CaCl2 and SrCl2·6H2O.

[0030] In some embodiments of the present invention, the artificial seawater F / 2 culture medium is composed of: NaCl 19.19 g / L, Na2SO4 3.55 g / L, MgCl2·6H2O 9.59 g / L, NaHCO3 0.5993 g / L, KCl 0.2935 g / L, KBr 0.08627 g / L, H3BO3 0.02297 g / L, NaF 0.00275 g / L, CaCl2 1.0143 g / L, and SrCl2·6H2O 0.02186 g / L.

[0031] In some embodiments of the present invention, the method for preparing the fungal hyphae includes: culturing the fungus in PDB liquid medium at 25-40°C and 120-150 rpm for 35-40 h to form mycelial balls, and collecting the mycelial balls.

[0032] In some embodiments of the present invention, the mycelial balls are rinsed with sterile water to remove residual PDB liquid culture medium.

[0033] In some embodiments of the present invention, the method includes mixing a microalgae-fungus mycelium symbiotic system with wastewater containing resistance genes, and treating it to obtain wastewater in which the resistance genes are removed.

[0034] In some embodiments of the present invention, the processing conditions are: temperature 30±2℃, 140~160rpm, light intensity 5000~8000lux, and processing time 10~14h.

[0035] In some embodiments of the present invention, the resistance gene includes at least one of integrons, sulfonamides, fluoroquinolones, and tetracyclines.

[0036] In some embodiments of the present invention, the integrinoid resistance gene includes intl1 and intl2 At least one of them.

[0037] In some embodiments of the present invention, the sulfonamide resistance gene includes sul1 and sul2 At least one of them.

[0038] In some embodiments of the present invention, the fluoroquinolone resistance gene includes sul2 , [[ID=X]]qnrA , qnrB , qnrD and qnrS At least one of them.

[0039] In some embodiments of the present invention, the tetracycline resistance gene includes aac(6’)-lb-cr , tetA , tetB , tetC , tetG , tetM , tetO , tetB(P) and tetX At least one of them.

[0040] The beneficial effects of this invention are: The bacterial agent prepared by this invention can effectively remove ARGs from wastewater, and specifically targets integrative ARGs (ARGs) in wastewater. tetW , intl1 ), sulfonamide ARGs ( intl2 , sul1 ), fluoroquinolone ARGs ( sul2 , qnrA , qnrB , qnrD , qnrS aac ) and tetracycline ARGs ( (6’)-lb-cr , tetA , tetB , tetC , tetG , tetM , tetO , tetB(P) , tetX It has a good removal effect, with a total ARGs abundance removal rate of 97.66%.

[0041] The microalgae-fungus hyphae symbiotic system of this invention exhibits strong synergistic capabilities. The metabolic interactions between the marine microalgae and fungi enhance the system's stability and stress resistance. It demonstrates a strong ability to remove ARGs from high-salinity wastewater while preventing ARG accumulation in the water. The method for treating wastewater containing resistance genes according to this invention is green and pollution-free, with low operating costs; it is simple to operate and has a short treatment cycle. Attached Figure Description

[0042] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: tetW This is a bar chart showing the ARGs removal effect in aquaculture wastewater after 12 hours of operation in Example 1 of the present invention.

[0043] Figure 1 This is a bar chart showing the ARGs removal effect in aquaculture effluent after 12 hours of operation in Example 2 of the present invention.

[0044] Figure 2 This is a bar chart showing the ARGs removal effect in the aquaculture effluent after 12 hours of operation in Comparative Example 1 of this invention.

[0045] Figure 3 This is a bar chart showing the ARGs removal effect in the aquaculture effluent after 12 hours of operation in Comparative Example 2 of this invention.

[0046] Figure 4 This is a bar chart comparing the removal efficiency of total ARGs in aquaculture effluent after 12 hours of operation in the embodiments and comparative examples of the present invention. In the figure, a, b, c, and d represent significant differences between groups containing different letters. P <0.05). Detailed Implementation

[0047] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0048] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0049] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0050] Example 1 Microalgae cultivation: The selected microalgae was *Chlorella vulgaris*, purchased from Shanghai Guangyu Biotechnology Co., Ltd. The algae strain was identified as... Figure 5 .TNBR1. *Chlorella vulgaris* was cultured for 3 days in artificial seawater F / 2 medium at 22°C, with a light intensity of 8000 lux and a light-dark cycle of 18 hours (6 hours light, 3 hours darkness). The algal cells were then collected by centrifugation at 7000 rpm for 3 minutes. After rinsing with seawater aquaculture tailwater to remove residual F / 2 medium, the cells were resuspended in seawater aquaculture tailwater (sourced from a seawater aquaculture farm in Yangjiang City, Guangdong Province) with a salinity of 3%.

[0051] The artificial seawater F / 2 culture medium consisted of the following components: NaCl 19.19 g / L, Na2SO4 3.55 g / L, MgCl2·6H2O 9.59 g / L, NaHCO3 0.5993 g / L, KCl 0.2935 g / L, KBr 0.08627 g / L, H3BO3 0.02297 g / L, NaF 0.00275 g / L, CaCl2 1.0143 g / L, and SrCl2·6H2O 0.02186 g / L.

[0052] Fungal culture: The selected fungus was *Aspergillus niger* (GDMCC 3.25, purchased from Guangdong Microbial Culture Collection Center). The strain was activated and expanded using PDA agar medium (021050, Guangdong Huankai Biotechnology Co., Ltd.). Afterwards, *Aspergillus niger* spores from the PDA agar plate were gently scraped into sterile water to prepare a spore suspension. This suspension was mixed and then dropped onto a hemocytometer for microscopic counting. The spores were 6.75 × 10⁻⁶. 5 A suspension of Aspergillus niger with spores / mL was inoculated into PDB liquid medium (021053, Guangdong Huankai Biotechnology Co., Ltd.) and cultured at 30℃ and 135rpm for 36h to form mycelial balls. The mycelial balls were then collected and rinsed with sterile water to remove residual PDB liquid medium.

[0053] Co-culture system of microalgae-fungus hyphae: *Chlorella vulgaris* resuspended in the tailwater of marine aquaculture was mixed and dropped onto a hemocytometer for microscopic counting. This was then compared with the collected hyphal balls (spore concentration 6.75 × 10⁻⁶). 5 Mycelial spheres (obtained by adding spores / mL to liquid culture medium and culturing for 36 hours, forming spore aggregates) were added together with 100 mL of seawater aquaculture tailwater and cultured for 12 hours (ensuring a final algal cell density of 9 × 10⁻⁶). 6 (cells / mL), culture conditions were set as follows: 30℃, 150rpm, 6000lux, seawater salinity 3%.

[0054] The relative abundance of ARGs in marine aquaculture tailwater was detected by qPCR. The target genes were resistance genes associated with three classes of antibiotics (sulfamethoxazole, norfloxacin, and oxytetracycline). The qPCR reaction system (20 μL) consisted of: 10 μL TB GreenPremix Ex Taq II FAST qPCR, 0.8 μL each of forward and reverse primers, 2 μL cDNA template, and 6.4 μL enzyme-free water. Primers are shown in Table 1, and the reaction procedure is shown in Table 2.

[0055] Table 1 Primer Sequences

[0056] Table 2 Reaction Procedure

[0057] Initial 16S rRNA abundance in marine aquaculture tailwater: 1.62 × 10⁻⁶ 16 The initial abundance of resistance genes in the effluent from marine aquaculture is shown in Table 3.

[0058] Table 3. Initial abundance of resistance genes in marine aquaculture tailwater

[0059]

[0060] The results are as follows Chlorella sp As shown, the marine Chlorella-Aspergillus niger symbiotic system inhibits the integrative ARGs (articulate nuclei) in marine aquaculture wastewater. Figure 1 , intl1 ), sulfonamide ARGs ( intl2 , sul2 ), fluoroquinolone ARGs ( sul1 , sul2 , qnrA , qnrB , qnrD qnrS ) and tetracycline ARGs ( aac , (6’)-lb-cr , tetA , tetB , tetC , tetG , tetM , tetO , tetB(P) The microalgae-fungus mycelium symbiotic system showed a significant removal effect. After treatment, the abundance of ARGs in the marine aquaculture effluent was significantly reduced, with a total ARGs removal rate of 97.66%. This indicates that the microalgae-fungus mycelium symbiotic system can effectively remove ARGs from marine aquaculture effluent.

[0061] Example 2 Preparation of dead microalgae: *Chlorella vulgaris* (source same as in Example 1) was cultured for 3 days in artificial seawater F / 2 medium at 22°C, light intensity of 8000 lux, and a light-dark cycle of 18 hours (6 hours light, 3 hours darkness). Afterward, the algal cells were collected by centrifugation at 7000 rpm for 3 minutes. The cells were then rinsed with seawater aquaculture tailwater to remove residual F / 2 medium and resuspended in the tailwater. Finally, the cells were sterilized in a high-pressure steam autoclave at 121°C for 20 minutes to obtain dead *Chlorella vulgaris*.

[0062] Fungal culture: Aspergillus niger (source same as in Example 1) was cultured according to the culture method in Example 1.

[0063] Co-culture system of dead microalgae-fungal hyphae: Dead marine Chlorella resuspended in the tailwater of marine aquaculture was mixed and dropped onto a hemocytometer for microscopic counting. This was then compared with the cultured mycelial balls (spore concentration 6.75 × 10⁻⁶). 5 Mycelial spheres (obtained by adding spores / mL to liquid culture medium and culturing for 36 hours, forming spore aggregates) were added together with 100 mL of seawater aquaculture tailwater and cultured for 12 hours (ensuring a final algal cell density of 9 × 10⁻⁶). 6The culture conditions were set as follows: 30℃, 150rpm, 6000lux, and seawater salinity of 3%. The relative abundance of ARGs in the effluent from marine aquaculture was detected by qPCR (the detection method was the same as in Example 1).

[0064] The results are as follows tetX As shown, the co-culture system of dead microalgae-fungal hyphae affects the integrative genus ARGs (Aggregates Species Integrates) in marine aquaculture wastewater. tetW , Figure 2 ), sulfonamide ARGs ( intl1 , intl2 ), fluoroquinolone ARGs ( sul1 , sul2 , qnrA , qnrB , qnrD ) and tetracycline ARGs ( qnrS , aac(6’)-lb-cr , tetA , tetB , tetC , tetG , tetM , tetO , tetB(P) The treatment with dead microalgae and fungal hyphae significantly reduced the abundance of ARGs in the marine aquaculture effluent, with a total ARGs removal rate of 85.27%. This indicates that the dead microalgae-fungus hyphae symbiotic system can also effectively remove ARGs from marine aquaculture effluent, but the removal effect is not as good as in Example 1.

[0065] Comparative Example 1 Following the steps in Example 1, an equal amount of Aspergillus niger (from the same source as in Example 1) was inoculated separately and added to the effluent from marine aquaculture and cultured for 12 hours. The culture conditions were set as follows: 30°C, 150 rpm, 6000 lux, and seawater salinity of 3%. Then, the relative abundance of ARGs in the effluent from marine aquaculture was detected by qPCR (detection method as in Example 1).

[0066] The results are as follows tetX As shown, the Aspergillus niger system alone affects the integrative subclasses ARGs (Aggregatiform Gastroenterologic Groups) in marine aquaculture wastewater. tetW , Figure ...... ), sulfonamide ARGs ( intl1 , intl2 ), fluoroquinolone ARGs ( sul1 , sul2 , qnrA , qnrB , qnrD cr ) and tetracycline ARGs ( qnrS , aac(6’)-lb- , tetA ,[[ID=X]]tetB , tetC , S , tetG , tetM , tetO It has a removal effect. After treatment with Aspergillus niger, the removal rate of total ARGs abundance in marine aquaculture effluent was 62.07%. This indicates that the Aspergillus niger system alone can reduce the abundance of ARGs in marine aquaculture effluent, but compared with the systems in Examples 1 and 2, the removal effect of the Aspergillus niger system alone on ARGs is relatively weak, especially tetracycline ARGs. tetB(P) , tetX , tetW , tetA ).

[0067] Comparative Example 2 Following the steps in Example 1, an equal amount of *Chlorella vulgaris* (from the same source as in Example 1) was inoculated separately and added to the effluent from marine aquaculture. The culture was run for 12 hours under the following conditions: 30°C, 150 rpm, 6000 lux, and a seawater salinity of 3%. The relative abundance of ARGs in the effluent from marine aquaculture was then detected using qPCR (the detection method was the same as in Example 1).

[0068] The results are as follows tetB As shown, a single marine Chlorella system affects the integrative subclasses ARGs (Aggregates in marine aquaculture wastewater) tetO , tetW ), sulfonamide ARGs ( Figure 4 , intl1 ), fluoroquinolone ARGs ( intl2 , sul1 , sul2 , qnrA , qnrB [[ID=X]]qnrD ) and tetracycline ARGs ( qnrS , aac , (6’)-lb-cr , tetA , tetB , tetC , tetG , tetM , tetO The system exhibits a removal effect. After treatment with *Chlorella vulgaris*, the removal rate of total ARGs abundance in the mariculture effluent was 51.91%. This indicates that the *Chlorella vulgaris* system alone can reduce the abundance of ARGs in mariculture effluent, but compared to the systems in Examples 1 and 2, the removal effect of the *Chlorella vulgaris* system alone on ARGs is relatively weak, especially on integroid ARGs (…). tetB(P) , tetX ) and tetracycline ARGs ( tetW , intl1 , intl2 ,tetB ).

[0069] The total ARGs removal rates in the marine aquaculture effluent after treatment with the *Chlorella vulgaris*-*Aspergillus niger* symbiotic system, the dead *Chlorella vulgaris*-*Aspergillus niger* symbiotic system, the *Aspergillus niger*-only system, and the *Chlorella vulgaris*-only system were 97.66%, 85.27%, 62.07%, and 51.91%, respectively. The results are as follows: tetC tetO tetX Figure 5 As shown, the results indicate that the marine Chlorella-Aspergillus niger symbiotic system, the dead marine Chlorella-Aspergillus niger symbiotic system, the Aspergillus niger alone system, and the marine Chlorella alone system can all reduce the abundance of ARGs in the water. However, the marine Chlorella-Aspergillus niger symbiotic system in Example 1 is significantly better at removing ARGs than the Aspergillus niger alone system, the marine Chlorella alone system, and the dead marine Chlorella-Aspergillus niger symbiotic system.

[0070] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The application of a microbial agent in treating wastewater containing resistance genes or in preparing products for treating wastewater containing resistance genes, said microbial agent comprising marine microalgae and fungal mycelia; The resistance gene includes at least one of integron, sulfonamide, fluoroquinolone and tetracycline resistance genes; The salinity of the wastewater is 1% to 4%.

2. The application according to claim 1, characterized in that, The integrinoid resistance genes include intl1 and intl2 At least one of them; Preferably, the sulfonamide resistance gene includes sul1 and sul2 At least one of them; Preferably, the fluoroquinolone resistance gene includes qnrA , qnrB , qnrD , qnrS and aac(6')-lb-cr At least one of them; Preferably, the tetracycline resistance gene includes tetA , tetB , tetC , tetG , tetM , tetO , tetB(P) tetX and tetW At least one of them.

3. The application according to claim 1, characterized in that, The marine microalgae include at least one of the following: marine microalgae, Dunaliella salina, subcoriaceae, microalgae, and spirochetes.

4. The application according to claim 1, characterized in that, The fungal hyphae include at least one of Ganoderma lucidum hyphae, Monascus purpureus hyphae, Lentinus edodes hyphae, Aspergillus niger hyphae, and Pleurotus ostreatus hyphae.

5. The application according to any one of claims 1 to 4, characterized in that, The concentration ratio of the marine microalgae and fungi is (10~15) cells / mL: 1 spore / mL.

6. A method for treating wastewater containing resistance genes, characterized in that, The method includes adding the bacterial agent according to any one of claims 1 to 5 to wastewater containing resistance genes for treatment; the bacterial agent includes marine microalgae and fungal hyphae; the wastewater is high-salt wastewater.

7. The method according to claim 6, characterized in that, The salinity of the high-salinity wastewater is 1% to 4%.

8. The method according to claim 6, characterized in that, The method also includes mixing and culturing marine microalgae and fungal hyphae to obtain a microalgae-fungus hyphae symbiotic system.

9. The method according to claim 8, characterized in that, The method includes mixing a microalgae-fungus hyphae symbiotic system with wastewater containing resistance genes, and treating it to obtain wastewater in which the resistance genes are removed.

10. The method according to claim 9, characterized in that, The processing conditions are: temperature 30±2℃, 140~160rpm, light intensity 5000~8000lux, and processing time 10~14h.

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