A method for reducing microplastics in wastewater using pseudomonas aeruginosa biofilm

Abstract

The application discloses a method for removing microplastics in sewage by using Pseudomonas aeruginosa biofilm, and belongs to the technical field of microplastic pollution prevention and control.The application aims to solve the problems of high energy consumption, easy clogging, and the need for secondary treatment of the separated plastic concentrate, which is easy to form "pollution transfer", and the problems of harsh reaction conditions and higher toxicity of by-products of the chemical method.Method: I. strain recovery and enrichment; II. preparation of biofilm; III. removal of microplastics.The method can effectively control the microplastics in the water environment.The removal rate of the method for different concentrations of microplastics has no significant difference, and the highest microplastic removal rate can reach 92%.

Description

Technical Field

[0001] This invention belongs to the field of microplastic pollution control technology, specifically relating to a method for reducing microplastics in wastewater using Pseudomonas aeruginosa biofilm. Background Technology

[0002] Microplastic (particle size <5mm) pollution has become one of the most serious environmental problems of the 21st century. These recalcitrant polymer particles can accumulate through the food chain, leading to reproductive disorders, immunosuppression, and genotoxicity in organisms, ultimately threatening human health. In terrestrial ecosystems, microplastics spread widely through soil infiltration and atmospheric deposition, with their pollution range far exceeding expectations. However, studies show that typical urban wastewater treatment plants have low microplastic removal rates, and their interception efficiency for particles <100μm is significantly reduced. Treated effluent still carries large amounts of microplastics into surface water bodies, becoming a major point source of microplastic pollution in rivers, lakes, and coastal areas. Current wastewater treatment technologies for microplastic removal face significant limitations. For example, physical methods (such as sand filtration and membrane filtration) can intercept plastic particles ≥5μm, but they are energy-intensive, prone to clogging, and the separated plastic concentrate requires secondary treatment, easily leading to "pollution transfer." Chemical methods (such as photocatalysis and ozone oxidation) can degrade some plastics, but the reaction conditions are harsh, and the byproducts (such as microplastic fragments and halogenated hydrocarbons) are even more toxic. Summary of the Invention

[0003] The purpose of this invention is to address the problems of existing physical methods for removing microplastics, such as high energy consumption, easy clogging, and the need for secondary treatment of the separated plastic concentrate, which can easily lead to "pollution transfer," and chemical methods, such as harsh reaction conditions and higher toxicity of byproducts. The invention provides a method for reducing microplastics in wastewater using Pseudomonas aeruginosa biofilm.

[0004] A method for reducing microplastics in wastewater using Pseudomonas aeruginosa biofilm is specifically carried out according to the following steps:

[0005] I. Strains' recovery and enrichment:

[0006] Pseudomonas aeruginosa bacterial suspension was inoculated into LB liquid medium and cultured in a constant temperature shaking incubator for a period of time to allow the strain to recover and obtain a Pseudomonas aeruginosa suspension.

[0007] II. Preparation of biomembranes:

[0008] First, the microfluidic reactor is immersed in an ethanol solution for a period of time, and then rinsed several times with sterile water. The Pseudomonas aeruginosa suspension obtained in step one is injected into the chip of the microfluidic reactor and cultured statically at 25℃~27℃ for a period of time. The LB liquid culture medium is introduced by a micro-injection pump at a flow rate of 45μL / min~55μL / min and cultured at 25℃~27℃ for a period of time to form a mature biofilm.

[0009] III. Removal of microplastics:

[0010] Polystyrene microplastics were added to LB liquid culture medium to obtain a polystyrene microplastic solution. The flow rate of the microinjection pump was controlled to pass the polystyrene microplastic solution into a microfluidic reactor. The reactor was run continuously at 25℃~27℃ for a period of time to remove the polystyrene microplastics, thus completing a method for reducing microplastics in wastewater using Pseudomonas aeruginosa biofilm.

[0011] The *Pseudomonas aeruginosa* bacterial culture in step one of this invention is an existing strain, specifically *Pseudomonas aeruginosa* derived from […]. 15442 TM Purchased from the China Industrial Microbial Culture Collection Center;

[0012] The microfluidic reactor mentioned in step two of this invention is the microfluidic reactor disclosed in Chinese patent application publication number CN 117106572, application number 202310953663.4, entitled "In-situ Evaluation and Disinfection Device and Method Based on Quorum Sensing Strains".

[0013] The principle of this invention:

[0014] This invention addresses the aforementioned bottlenecks by showcasing the unique potential of biofilm technology based on microbial extracellular polymers. *Pseudomonas aeruginosa*, a typical biofilm-forming bacterium, secretes polysaccharide-protein complexes that efficiently adsorb microplastic particles. The extracellular polymers secreted by *P. aeruginosa* form a three-dimensional network structure, effectively capturing microplastics in the environment. Esterases and laccases secreted by the bacteria can break the ester / urethane bonds of plastics, decomposing them into oligomer monomers. Once the biofilm is immobilized on a carrier, it can maintain continuous operation for a relatively long period, and carrier regeneration consumes low energy (requiring only gentle ultrasonic cleaning), avoiding the limitations of traditional processes. Compared to existing methods, this invention's method for reducing microplastics in wastewater using *P. aeruginosa* biofilms offers a breakthrough in terms of high environmental compatibility, the absence of any chemical additives throughout the process, and reduced operating costs. This technology can be coupled with existing secondary sedimentation tanks / advanced treatment units in wastewater treatment plants without large-scale infrastructure modifications, providing a new approach and method for controlling microplastic pollution.

[0015] The present invention has the following beneficial effects:

[0016] This invention discloses a method for reducing microplastics in wastewater using Pseudomonas aeruginosa biofilm, which can effectively control microplastics in the aquatic environment; the method shows no significant difference in removal rate for microplastics of different concentrations, and the microplastic removal rate can reach up to 92%. Detailed Implementation

[0017] Specific Implementation Method 1: This implementation method describes a method for reducing microplastics in wastewater using Pseudomonas aeruginosa biofilm, which is specifically carried out according to the following steps:

[0018] I. Strains' resuscitation and enrichment:

[0019] Pseudomonas aeruginosa bacterial suspension was inoculated into LB liquid medium and cultured in a constant temperature shaking incubator for a period of time to allow the strain to recover and obtain a Pseudomonas aeruginosa suspension.

[0020] II. Preparation of biomembranes:

[0021] First, the microfluidic reactor is immersed in an ethanol solution for a period of time, and then rinsed several times with sterile water. The Pseudomonas aeruginosa suspension obtained in step one is injected into the chip of the microfluidic reactor and cultured statically at 25℃~27℃ for a period of time. The LB liquid culture medium is introduced by a micro-injection pump at a flow rate of 45μL / min~55μL / min and cultured at 25℃~27℃ for a period of time to form a mature biofilm.

[0022] III. Removal of microplastics:

[0023] Polystyrene microplastics were added to LB liquid culture medium to obtain a polystyrene microplastic solution. The flow rate of the microinjection pump was controlled to pass the polystyrene microplastic solution into a microfluidic reactor. The reactor was run continuously at 25℃~27℃ for a period of time to remove the polystyrene microplastics, thus completing a method for reducing microplastics in wastewater using Pseudomonas aeruginosa biofilm.

[0024] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that: the volume ratio of *Pseudomonas aeruginosa* bacterial suspension to LB liquid culture medium in step one is 1:100; the concentration of *Pseudomonas aeruginosa* bacterial suspension in step one is 10... 8 CFU / mL. Other steps are the same as in Specific Implementation Method 1.

[0025] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that the temperature of the constant-temperature oscillating shaker described in step one is 35℃~37℃, and the rotation speed is 150rpm~160rpm. Other steps are the same as in Specific Implementation Method One or Two.

[0026] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that the cultivation time in step one is 20 to 24 hours. The other steps are the same as in Specific Implementation Methods One to Three.

[0027] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that: in step two, the microfluidic reactor is first immersed in a 70%–80% ethanol solution for 2–3 hours, and then rinsed 3–5 times with sterile water. The other steps are the same as in Specific Implementation Methods One to Four.

[0028] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that: in step two, the *Pseudomonas aeruginosa* suspension obtained in step one is injected into the chip of the microfluidic reactor and incubated statically at 25°C to 27°C for 2 to 3 hours. The other steps are the same as in Specific Implementation Methods One to Five.

[0029] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One to Six in that: in step two, the micro-injection pump is controlled to introduce LB liquid culture medium at a flow rate of 45 μL / min to 55 μL / min, and cultured at 25℃ to 27℃ for 45h to 50h to form a mature biofilm. Other steps are the same as in Specific Implementation Methods One to Six.

[0030] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods One to Seven in that: the concentration of the polystyrene microplastic solution in step three is 200 mg / L to 500 mg / L; and the flow rate of the micro-injection pump in step three is 45 μL / min to 55 μL / min. The other steps are the same as in Specific Implementation Methods One to Seven.

[0031] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Methods One to Eight in that: the particle size of the polystyrene microplastics mentioned in step three is 8μm to 12μm; the continuous operation time mentioned in step three is 30h to 40h. Other steps are the same as in Specific Implementation Methods One to Eight.

[0032] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Methods One to Nine in that the preparation method of the LB liquid culture medium described in steps one, two, and three is as follows: dissolve yeast powder, tryptone, and NaCl in deionized water, and then adjust the pH of the solution to 7.0–7.5 to obtain the LB liquid culture medium. The concentration of yeast powder in the LB liquid culture medium is 5 g / L, the concentration of tryptone is 10 g / L, and the concentration of NaCl is 10 g / L. Other steps are the same as in Specific Implementation Methods One to Nine.

[0033] The beneficial effects of the present invention are verified using the following embodiments:

[0034] Example 1: A method for reducing microplastics in wastewater using Pseudomonas aeruginosa biofilm, characterized in that the method is specifically carried out according to the following steps:

[0035] I. Strains' recovery and enrichment:

[0036] The Pseudomonas aeruginosa bacterial suspension was inoculated into LB liquid medium and placed in a constant temperature shaking incubator at 35°C and 150 rpm for 24 h to revive the strain and obtain a Pseudomonas aeruginosa suspension.

[0037] The *Pseudomonas aeruginosa* bacterial culture in step one is an existing strain, specifically *Pseudomonas aeruginosa* derived from... 15442 TM Purchased from the China Industrial Microbial Culture Collection Center;

[0038] The volume ratio of Pseudomonas aeruginosa bacterial suspension to LB liquid culture medium in step one is 1:100;

[0039] The concentration of the *Pseudomonas aeruginosa* bacterial suspension mentioned in step one is 10. 8 CFU / mL;

[0040] II. Preparation of biomembranes:

[0041] First, the microfluidic reactor was immersed in a 75% ethanol solution for 2 hours, and then rinsed three times with sterile water. The Pseudomonas aeruginosa suspension obtained in step one was injected into the chip of the microfluidic reactor and cultured statically at 25°C for 2 hours. LB liquid culture medium was then introduced at a flow rate of 50 μL / min using a microinjection pump and cultured at 25°C for 48 hours to form a mature biofilm.

[0042] The microfluidic reactor mentioned in step two is the microfluidic reactor disclosed in Chinese patent application publication number CN 117106572, application number 202310953663.4, invention title "In-situ evaluation disinfection device and method based on quorum sensing strains";

[0043] III. Removal of microplastics:

[0044] Polystyrene microplastics were added to LB liquid culture medium to obtain a polystyrene microplastic solution. The flow rate of the microinjection pump was controlled at 50 μL / min, and the polystyrene microplastic solution was introduced into the microfluidic chip. The solution was continuously run at 25℃~27℃ for 36 hours to remove the polystyrene microplastics, thus completing a method for reducing microplastics in wastewater using Pseudomonas aeruginosa biofilm.

[0045] The concentration of polystyrene microplastics in the polystyrene microplastic solution mentioned in step three is 200 mg / L;

[0046] The polystyrene microplastics mentioned in step three have a particle size of 10 μm;

[0047] The preparation method of LB liquid culture medium described in steps one, two and three is as follows: dissolve yeast powder, tryptone and NaCl in deionized water, and then adjust the pH of the solution to 7.0 to obtain LB liquid culture medium. The concentration of yeast powder in LB liquid culture medium is 5 g / L, the concentration of tryptone is 10 g / L, and the concentration of NaCl is 10 g / L.

[0048] In Example 1, the removal rate of polystyrene microplastics was 87%.

[0049] Example 2: The difference between this example and Example 1 is that the concentration of polystyrene microplastics in the polystyrene microplastic solution in step three is 500 mg / L. All other steps and parameters are the same as in Example 1.

[0050] In Example 2, the removal rate of polystyrene microplastics was 92%.

[0051] The above research results indicate that biofilms prepared using *Pseudomonas aeruginosa* can effectively remove microplastics from the aquatic environment. Under low and high concentrations of microplastic exposure conditions, the removal rates of this method for 10-micron polystyrene microplastics were 87% and 92%, respectively. This invention is of great significance for the development of technologies for the reduction and control of microplastics in wastewater.

Claims

1. A method for reducing microplastics in wastewater using Pseudomonas aeruginosa biofilm, characterized in that... The method is specifically implemented according to the following steps: I. Strains' recovery and enrichment: Pseudomonas aeruginosa bacterial suspension was inoculated into LB liquid medium and cultured in a constant temperature shaking incubator for a period of time to allow the strain to recover and obtain a Pseudomonas aeruginosa suspension. The *Pseudomonas aeruginosa* in the bacterial culture solution mentioned in step one is an existing strain. Pseudomonas aeruginosa Purchased from China Industrial Microbial Culture Collection Center, product code 0693-CRM; II. Preparation of biomembranes: First, the microfluidic reactor is immersed in an ethanol solution for a period of time, and then rinsed several times with sterile water. The Pseudomonas aeruginosa suspension obtained in step one is injected into the chip of the microfluidic reactor and cultured statically at 25℃~27℃ for a period of time. The LB liquid culture medium is then introduced by a micro-injection pump at a flow rate of 45μL / min~55μL / min and cultured at 25℃~27℃ for a period of time to form a mature biofilm. III. Removal of microplastics: Polystyrene microplastics were added to LB liquid culture medium to obtain a polystyrene microplastic solution. The flow rate of the microinjection pump was controlled to pass the polystyrene microplastic solution into a microfluidic reactor. The reactor was run continuously at 25℃~27℃ for a period of time to remove the polystyrene microplastics, thus completing a method for reducing microplastics in wastewater using Pseudomonas aeruginosa biofilm.

2. The method for reducing microplastics in wastewater using *Pseudomonas aeruginosa* biofilm according to claim 1, characterized in that... The volume ratio of *Pseudomonas aeruginosa* bacterial suspension to LB liquid medium in step one is 1:100; the concentration of *Pseudomonas aeruginosa* bacterial suspension in step one is 10. 8 CFU / mL.

3. The method for reducing microplastics in wastewater using *Pseudomonas aeruginosa* biofilm according to claim 1, characterized in that... The temperature of the constant temperature oscillating shaker mentioned in step one is 35℃~37℃, and the rotation speed is 150rpm~160rpm.

4. The method for reducing microplastics in wastewater using *Pseudomonas aeruginosa* biofilm according to claim 1, characterized in that... The incubation time described in step one is 20h~24h.

5. The method for reducing microplastics in wastewater using *Pseudomonas aeruginosa* biofilm according to claim 1, characterized in that... In step two, the microfluidic reactor is first immersed in a 70% to 80% ethanol solution for 2 to 3 hours, and then rinsed with sterile water 3 to 5 times.

6. The method for reducing microplastics in wastewater using *Pseudomonas aeruginosa* biofilm according to claim 1, characterized in that... In step two, the Pseudomonas aeruginosa suspension obtained in step one is injected into the chip of the microfluidic reactor and incubated at 25℃~27℃ for 2h~3h.

7. The method for reducing microplastics in wastewater using *Pseudomonas aeruginosa* biofilm according to claim 1, characterized in that... In step two, the micro-injection pump is controlled to introduce LB liquid culture medium at a flow rate of 45 μL / min to 55 μL / min, and cultured at 25℃ to 27℃ for 45h to 50h to form a mature biofilm.

8. The method for reducing microplastics in wastewater using *Pseudomonas aeruginosa* biofilm according to claim 1, characterized in that... The concentration of the polystyrene microplastic solution mentioned in step three is 200 mg / L to 500 mg / L; the flow rate of the micro-injection pump mentioned in step three is 45 μL / min to 55 μL / min.

9. The method for reducing microplastics in wastewater using *Pseudomonas aeruginosa* biofilm according to claim 1, characterized in that... The particle size of the polystyrene microplastics mentioned in step three is 8μm~12μm; the continuous operation time mentioned in step three is 30h~40h.

10. A method for reducing microplastics in wastewater using *Pseudomonas aeruginosa* biofilm according to claim 1, characterized in that... The preparation method of LB liquid culture medium described in steps one, two and three is as follows: dissolve yeast powder, tryptone and NaCl in deionized water, and then adjust the pH of the solution to 7.0~7.5 to obtain LB liquid culture medium. The concentration of yeast powder in LB liquid culture medium is 5g / L, the concentration of tryptone is 10g / L, and the concentration of NaCl is 10g / L.