A method for enriching microplastics in a fixed body of water using microorganisms

Abstract

The application discloses a method for enriching and fixing microplastics in a water body by microorganisms and belongs to the technical field of microplastics biology. The method comprises the following steps: (1) concentrated bacterial solution preparation: Escherichia coli is picked into LB liquid culture medium for culturing, centrifuged, the upper liquid is poured out, M9 culture medium is added, and the prepared concentrated bacterial solution is uniformly mixed and distributed; and (2) co-incubation of microorganisms and microplastics: microplastic solution and the concentrated bacterial solution of step (1) are mixed and incubated. The method has the beneficial effects that the concentrated bacterial solution is prepared by culturing Escherichia coli, and then the concentrated bacterial solution is mixed and co-incubated with the microplastic solution, so that the microplastics are enriched and fixed, the operation is simple, the processing efficiency is high, and a new method for enriching and fixing microplastics in a water body is provided.

Description

Technical Field

[0001] This invention relates to the field of microplastic biotechnology, and more specifically to a method for microbial enrichment and fixation of microplastics in water. Background Technology

[0002] The concept of microplastics was first proposed in 2004, referring to plastic particles or fragments with a diameter of less than 5 mm. Compared with general non-degradable plastics, they have smaller particle sizes, larger specific surface areas, and stronger ability to adsorb pollutants, leading to more serious environmental pollution problems. Microplastics mainly include polyethylene, polypropylene, polystyrene, and polyvinyl chloride, and are widely distributed, found in the ocean, on land, and in the atmosphere. Because they are high-molecular-weight organic materials, microplastics are difficult to degrade, and nanoscale microplastics can cross physical barriers to enter organisms, accumulating and cycling through the food chain, making them one of the key pollutants of concern in the environmental field.

[0003] Microplastics were first detected in the marine environment. It is estimated that Americans release approximately 263 tons of polyethylene microplastics into the environment annually through the use of everyday toiletries. Simultaneously, large plastic fragments in the environment undergo a series of physicochemical reactions to degrade, producing secondary microplastics that also enter the environment. Due to their small particle size and low density, these microplastics are dispersed by ocean currents to various parts of the marine environment and are widely ingested by various marine organisms. To date, microplastics have been found in soil, seawater, and seabed sediments. Furthermore, they have been detected in seagulls, various marine fish, shellfish, and marine crustaceans. Even more concerning, some microplastics have drifted with the seawater to the Arctic region and become trapped in Arctic icebergs. As the concentration of microplastics in the environment increases year by year, their adverse effects on organisms also increase annually. Currently, the enrichment and recycling of microplastics has attracted attention, but biological methods for enriching and fixing microplastics in water bodies are still lacking.

[0004] Escherichia coli (scientific name: *Escherichia coli*, commonly abbreviated as *E. coli*) belongs to the class Gamma-Proteobacteria and is a bacterium found in the intestines of humans and animals. *E. coli* is a motile, non-spore-forming, Gram-negative, facultative anaerobic, rod-shaped bacterium of the genus *Escherichia coli*, commonly found in the lower small intestine as a warm-blooded organism. Most strains of *E. coli* are harmless, but certain serotypes (EPEC, ETEC, etc.) can cause severe food poisoning in their hosts and occasionally lead to food contamination incidents resulting in product recalls. Harmless strains are part of the normal flora in the human gut, producing vitamin K, preventing the growth of other pathogenic bacteria in the gut, and are beneficial to humans. This bacterium can be easily and inexpensively grown and cultured in a laboratory setting and has been extensively studied for over 60 years. *E. coli* is the most widely studied prokaryotic model organism and an important species in biotechnology and microbiology, as well as the most common model microorganism.

[0005] Currently, microorganisms have been shown to enrich organic materials and heavy metals. For example, Chinese patent application CN115739023A discloses an iron-nitrogen co-doped biocarbon material based on microbial enrichment, its preparation method, and its application; and Chinese patent application CN115572699A discloses a bacterium capable of enriching heavy metals and its application. However, there is currently no existing technology for using microorganisms to enrich and fix microplastics in water. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a new method for microbial enrichment and fixation of microplastics in water.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] A method for microbial enrichment and fixation of microplastics in water includes the following steps:

[0009] (1) Preparation of concentrated bacterial culture: Select a single colony of Escherichia coli and culture it in LB liquid medium. Centrifuge, discard the supernatant, add M9 medium, mix well and dispense for use.

[0010] (2) Co-incubation of microorganisms and microplastics: The microplastic solution and the concentrated bacterial solution from step (1) are mixed and incubated.

[0011] Beneficial effects: This invention prepares a concentrated bacterial solution by culturing Escherichia coli, and then mixes it with a microplastic solution for co-incubation, thereby enriching and fixing microplastics. The operation is simple and the processing efficiency is high, providing a new method for microbial enrichment and fixation of microplastics in water.

[0012] Preferably, the Escherichia coli is Escherichia coli OP50, Escherichia coli BL21, Escherichia coli Nissle 1917, etc.

[0013] Preferably, the LB liquid culture medium is formulated as follows: 1g NaCl, 1g peptone and 0.5g yeast powder are added to every 100mL of ultrapure water.

[0014] Preferably, the culture includes pre-culture and expansion culture, wherein the pre-culture is: pre-shaking the bacteria in a shaker at 37°C and 220 rpm for 14 hours; wherein the expansion culture is: adding the mother liquor obtained from the pre-culture to new LB liquid medium (the volume ratio of mother liquor to new LB liquid medium is 2.5:100) and continuing to expand the culture in a shaker at 37°C and 220 rpm for 48 hours.

[0015] Preferably, the centrifugation speed is 4000-6000 rpm and the centrifugation time is 10-20 min.

[0016] Preferably, the M9 culture medium is formulated as follows: 2.4g KH2PO4, 4g NaCl, and 12.1g NA2HPO4·12H2O are added to every 800mL of ultrapure water, and then 800μL of MgSO4 is added after high-temperature sterilization.

[0017] Preferably, in step (1), the volume ratio of LB liquid culture medium to M9 culture medium is 60:0.5-2.

[0018] Preferably, the microplastic is a polystyrene microsphere with a diameter of 1-20 μm.

[0019] Preferably, the microplastic solution is an 80-150 μg / L polystyrene microsphere solution.

[0020] Preferably, the microplastic solution is a 100 μg / L polystyrene microsphere solution.

[0021] Preferably, in step (2), the volume ratio of the microplastic solution to the concentrated bacterial solution is 80-120:1.

[0022] The advantages of this invention are:

[0023] This invention provides a novel method for enriching and immobilizing microplastics in water by culturing Escherichia coli to prepare a concentrated bacterial solution, which is then mixed with a microplastic solution and co-incubated. The method is simple to operate, highly efficient, and provides a novel method for enriching and immobilizing microplastics in water by microorganisms. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the process for microbial enrichment of microplastics in water in Embodiment 1 of the present invention;

[0025] Figure 2 This is a characterization diagram of the microbial enrichment of microplastics in water in Example 1 of the present invention;

[0026] Figure 3 This is a comparison diagram of the microbial enrichment of microplastics in water (i.e., the microbial and microplastic co-incubation group) in Example 1 of the present invention with Comparative Example 1 (microplastic group) and Comparative Example 2 (microbial group);

[0027] Figure 4 This is a diagram showing the enrichment process of microorganisms in water bodies from the first to the tenth day in an embodiment of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1:

[0030] A method for microbial enrichment and fixation of microplastics in water includes the following steps:

[0031] (1) Preparation of concentrated bacterial culture: Single clones of GFP Escherichia coli OP50 were selected and placed into LB liquid medium (formulation: 100 mL ultrapure water, 1 g NaCl, 1 g peptone, 0.5 g yeast extract), and pre-cultured at 37℃ and 220 rpm for 14 hours. Then, the stock solution was added to fresh LB liquid medium at a ratio of 2.5:100 and cultured at 37℃ and 220 rpm for another 48 hours. The bacterial culture after two days of culture was poured into 50 mL sterile centrifuge tubes and centrifuged at 4℃ and 5000 rpm for 15 minutes in a low-temperature refrigerated centrifuge. The supernatant was discarded, and the bottom layer was the concentrated bacterial culture. Add M9 to a 50 mL centrifuge tube at a ratio of 60:1 (the formula is an 800 mL system, consisting of 2.4 g KH2PO4, 4 g NaCl, 12.1 g NA2HPO4·12H2O sterilized at high temperature, followed by the addition of 800 μL MgSO4). Mix well and dispense into 2 mL centrifuge tubes. Measure the absorbance (OD600) of the fresh bacterial culture at 600 nm using a spectrophotometer. The OD value is 3.6. Store at 4 °C for later use.

[0032] (2) Co-incubation of microorganisms and microplastics: In a 30 mm culture dish, a microplastic solution with a microplastic content of 100 μg / m² was prepared using M9 as the substrate. The microplastics were fluorescent polystyrene microspheres with a diameter of 1 μm. 30 μL of concentrated bacteria were added for mixed incubation.

[0033] Figure 1 This is a schematic diagram of the process for microbial enrichment of microplastics in water in this embodiment.

[0034] Observe the microplastic enrichment and fixation using a fluorescence microscope:

[0035] Observation was performed using a fluorescence microscope from day one until day ten. The specific steps for using an inverted fluorescence microscope are as follows: Use a 1000 μL pipette to evenly pipette 10-15 times, randomly aspirate the liquid and place it on a slide for observation, and take GFP images under bright field and 488 nm excitation light respectively under a fluorescence microscope, and merge them with Las x to observe the enrichment and fixation.

[0036] The results are as follows Figure 4 As shown in the figure, on the second day, mycelia began to appear on the microplastics. On the third and fourth days, the microplastics began to accumulate, and by the tenth day, there were no more suspended microplastics in the water.

[0037] Characterization was performed using optical-thermal infrared (O-PTIR) technology:

[0038] On day 5, individual polystyrene microspheres were characterized by O-PTIR, and the results are as follows: Figure 2 As shown in Figure B, six points were randomly selected on the surface of the microspheres for testing. The results showed that the background of the microspheres was polystyrene, but microorganisms were attached to some of the points. This confirms that microorganisms enrich and immobilize the microplastics through a process of gradual adsorption onto the microplastics.

[0039] Ten days later, a membrane-like substance appeared in the culture dish and sank to the bottom. It was characterized using O-PTIR technology, and the results were as follows: Figure 2 As shown in Figure A, three points were randomly selected on the membrane surface for detection. The membrane was determined to be a biological membrane, and the presence of protein peaks confirmed it to be a microbial membrane.

[0040] Comparative Example 1 (Microplastics Group):

[0041] The difference between this comparative example and Example 1 is that concentrated bacteria are not added in step (2), while the other steps are the same as in Example 1.

[0042] Comparative Example 2 (Microbiome):

[0043] The difference between this comparative example and Example 1 is that: no microplastic solution is added in step (2), while the other steps are the same as in Example 1.

[0044] Figure 3This is a comparison diagram of the microbial enrichment of microplastics in water (i.e., the microbial and microplastic co-incubation group) in Example 1 of the present invention with Comparative Example 1 (microplastic group) and Comparative Example 2 (microbial group). As can be seen from the figure, on the tenth day, the microbial group will be enriched and the microplastics will show slight aggregation. The microorganisms in the incubation group will enrich and fix the microplastics, forming a microbial membrane network, which directly proves that microorganisms can enrich and fix microplastics.

[0045] Example 2:

[0046] A method for microbial enrichment and fixation of microplastics in water includes the following steps:

[0047] (1) Preparation of concentrated bacterial culture: Single clones of GFP Escherichia coli BL21 were selected and placed into LB liquid medium (formulation: 100 mL ultrapure water, 1 g NaCl, 1 g peptone, 0.5 g yeast extract), and pre-cultured at 37℃ and 220 rpm for 14 hours. Then, the stock solution was added to fresh LB liquid medium at a ratio of 2.5:100 and cultured at 37℃ and 220 rpm for another 48 hours. The bacterial culture after two days of culture was poured into 50 mL sterile centrifuge tubes and centrifuged at 4000 rpm for 20 min at 4℃ in a low-temperature refrigerated centrifuge. The supernatant was discarded, and the bottom layer was the concentrated bacterial culture. Add M9 to a 50 mL centrifuge tube according to the ratio of LB volume to M9 (formulation is 800 mL system, 2.4 g KH2PO4, 4 g NaCl, 12.1 g NA2HPO4·12H2O sterilized at high temperature and then 800 μL MgSO4) at 60:0.5, mix well and dispense into 2 mL centrifuge tubes. Measure the absorbance (OD600) of the fresh bacterial solution at 600 nm using a spectrophotometer. The OD value is 3.6. Store at 4℃ for later use.

[0048] (2) Co-incubation of microorganisms and microplastics: In a 30 mm culture dish, 2.4 mL of microplastic solution with a microplastic content of 80 μg / m² was prepared using M9 as the substrate. The microplastics were fluorescent polystyrene microspheres with a diameter of 5 μm. 30 μL of concentrated bacteria were added for mixed incubation.

[0049] Example 3:

[0050] A method for microbial enrichment and fixation of microplastics in water includes the following steps:

[0051] (1) Preparation of concentrated bacterial culture: Single clones of GFP Escherichia coli Nissle 1917 were picked and placed into LB liquid medium (formulation: 100 mL ultrapure water, 1 g NaCl, 1 g peptone, 0.5 g yeast extract), and pre-cultured at 37℃ and 220 rpm for 14 hours. Then, the stock solution was added to fresh LB liquid medium at a ratio of 2.5:100 and cultured for another 48 hours at 37℃ and 220 rpm. The bacterial culture after two days of culture was poured into 50 mL sterile centrifuge tubes and centrifuged at 4℃ and 6000 rpm for 10 min in a low-temperature refrigerated centrifuge. The supernatant was discarded, and the bottom layer was the concentrated bacterial culture. Add M9 to a 50 mL centrifuge tube according to the LB volume and M9 ratio (the formula is an 800 mL system, 2.4 g KH2PO4, 4 g NaCl, 12.1 g NA2HPO4·12H2O sterilized at high temperature and then 800 μL MgSO4) at a ratio of 60:2. Mix well and dispense into 2 mL centrifuge tubes. Measure the absorbance (OD600) of the fresh bacterial solution at 600 nm using a spectrophotometer. The OD value is 3.6. Store at 4 °C for later use.

[0052] (2) Co-incubation of microorganisms and microplastics: In a 30 mm culture dish, 3.6 mL of microplastic solution with a microplastic content of 150 μg / m² was prepared using M9 as the substrate. The microplastics were fluorescent polystyrene microspheres with a diameter of 20 μm. 30 μL of concentrated bacteria were added for mixed incubation.

[0053] The enrichment effects achieved in Examples 2 and 3 are similar to those in Example 1.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for microbial enrichment and fixation of microplastics in water, characterized in that, Includes the following steps: (1) Preparation of concentrated bacterial culture: Select a single colony of Escherichia coli and culture it in LB liquid medium. Centrifuge, discard the supernatant, add M9 medium, mix well and dispense for use; the Escherichia coli is Escherichia coli OP50, Escherichia coli BL21 or Escherichia coli Nissle 1917. (2) Co-incubation of microorganisms and microplastics: M9 culture medium, microplastic solution and concentrated bacterial solution from step (1) are mixed and incubated; the microplastics are polystyrene microspheres with a diameter of 1-20 μm.

2. The method for microbial enrichment and fixation of microplastics in water according to claim 1, characterized in that, The LB liquid culture medium is formulated as follows: 1g NaCl, 1g peptone and 0.5g yeast powder are added to every 100mL of ultrapure water.

3. The method for microbial enrichment and fixation of microplastics in water according to claim 1 or 2, characterized in that, The culture includes pre-culture and expansion culture, wherein the pre-culture is: pre-shaking the bacteria in a shaker at 37°C and 220 rpm for 14 hours; wherein the expansion culture is: adding the mother liquor obtained from the pre-culture to new LB liquid medium and continuing to expand the culture in a shaker at 37°C and 220 rpm for 48 hours.

4. The method for microbial enrichment and fixation of microplastics in water according to claim 3, characterized in that, The centrifugation speed is 4000-6000 rpm, and the centrifugation time is 10-20 min.

5. The method for microbial enrichment and fixation of microplastics in water according to claim 4, characterized in that, The formula for the M9 culture medium is as follows: 2.4g KH2PO4, 4g NaCl, and 12.1g Na2HPO4·12H2O are added to every 800mL of ultrapure water, and then 800μL of MgSO4 is added after high-temperature sterilization.

6. The method for microbial enrichment and fixation of microplastics in water according to claim 1, characterized in that, In step (1), the volume ratio of LB liquid culture medium to M9 culture medium is 60:0.5-2.

7. The method for microbial enrichment and fixation of microplastics in water according to claim 1, characterized in that, The microplastic is a polystyrene microsphere with a diameter of 1 μm.

8. The method for microbial enrichment and fixation of microplastics in water according to claim 1, characterized in that, The microplastic solution is an 80-150 μg / L polystyrene microsphere solution.

9. The method for microbial enrichment and fixation of microplastics in water according to claim 8, characterized in that, The microplastic solution is a 100 μg / L polystyrene microsphere solution.

10. The method for microbial enrichment and fixation of microplastics in water according to claim 1, characterized in that, In step (2), the volume ratio of microplastic solution to concentrated bacterial solution is 80-120:1.

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