Application of lactobacillus plantarum p101 in adsorption and removal of nano-plastic in environmental water

Abstract

This invention belongs to the field of microbial technology and discloses the application of *Lactobacillus plantarum* P101 in the adsorption and removal of nanoplastics in environmental water. The *Lactobacillus plantarum* P101 strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2021108, deposit date January 19, 2021. This strain exhibits an adsorption rate of ≥85% for nanoplastics with a particle size of 100-1000 nm. The strain was isolated from fermented sauerkraut. The *Lactobacillus plantarum* P101 provided by this invention demonstrates significant adsorption capacity for nanoplastics in both live and inactivated states, which is of great significance for the environmental remediation of nanoplastics.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the application of Lactobacillus plantarum P101 in the adsorption and removal of nanoplastics in environmental water. Background Technology

[0002] Nanoplastics are plastic particles with a diameter of less than 1 μm, widely found in water, soil, and the atmosphere. Due to their persistence and non-degradability, nanoplastics have become a global environmental pollution problem. Studies have shown that nanoplastics not only cause direct harm to ecosystems, such as affecting the feeding and reproduction of aquatic organisms, but may also accumulate through the food chain, ultimately threatening human health. The surface of nanoplastics readily adsorbs toxic chemicals (such as heavy metals and organic pollutants), further exacerbating their ecotoxicity.

[0003] Currently, technologies for remediating nanoplastic pollution mainly include physical methods (such as filtration and adsorption), chemical methods (such as oxidative degradation), and biological methods (such as microbial degradation). However, physical and chemical methods suffer from high costs and significant risks of secondary pollution, making large-scale application difficult. In contrast, biological methods have become a research hotspot due to their environmental friendliness and sustainability. Among these, the technology of utilizing microorganisms to adsorb or degrade nanoplastics has attracted considerable attention. Microorganisms interact with nanoplastics through special structures on their cell surfaces (such as extracellular polymers and hydrophobic proteins) or metabolites (such as enzymes), thereby achieving adsorption or degradation. In recent years, studies have shown that certain bacteria, fungi, and algae can adsorb or degrade nanoplastics, but the efficiency is generally low, and there is a lack of strains with strong specificity and broad adaptability. *Lactobacillus plantarum* is a lactic acid bacterium widely found in the natural environment and fermented foods, possessing good environmental adaptability and biosafety. Its cell surface is rich in hydrophobic proteins and polysaccharides, which may give it a high adsorption capacity for hydrophobic nanoplastics. Furthermore, the application potential of *Lactobacillus plantarum* in environmental remediation has not been fully explored, especially in the field of nanoplastic pollution remediation, where research is almost nonexistent.

[0004] Therefore, using Lactobacillus plantarum to adsorb nanoplastics and applying it to environmental remediation can not only improve the efficiency of microbial adsorption of nanoplastics, but also provide an efficient and environmentally friendly biological solution for the treatment of nanoplastic pollution. Summary of the Invention

[0005] To address the problems of low degradation efficiency, poor adaptability, and environmental unfriendliness in existing technologies, this invention provides the application of *Lactobacillus plantarum* P101 in the adsorption and removal of nanoplastics in environmental water bodies. The strain was isolated from fermented sauerkraut. The *Lactobacillus plantarum* P101 provided by this invention exhibits significant nanoplastic adsorption capacity in both live and inactivated states, which is of great significance for the environmental remediation of nanoplastics. To achieve the above objectives, this invention provides, in one aspect, the application of *Lactobacillus plantarum* P101 in the adsorption and removal of nanoplastics in environmental water, wherein *Lactobacillus plantarum* P101 (… Lactiplantibacillus plantarum P101) is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2021108 and deposit date of January 19, 2021.

[0006] Further, the method for preparing the live bacterial suspension of *Lactobacillus plantarum* P101 is as follows: *Lactobacillus plantarum* P101 is inoculated into MRS medium and cultured under facultative anaerobic conditions at 30-37℃ for 18-48 h. After culture, the culture is centrifuged, washed with sterile PBS buffer, and then resuspended with 1 / 50 volume of the original bacterial suspension in buffer until the bacterial concentration is 1×10⁻⁶. 10 ~1×10 11 The result is CFU / mL.

[0007] Furthermore, the live bacteria suspension is added to water containing nanoplastics at a concentration of 10-50 mL / L, and reacted at 25-37°C for 2-8 hours. The removal rate of nanoplastics with a particle size of 100-1000 nm in the environmental water is ≥80%.

[0008] Further, the method for preparing the inactivated bacterial suspension of *Lactobacillus plantarum* P101 is as follows: *Lactobacillus plantarum* P101 is inoculated into MRS medium and cultured under facultative anaerobic conditions at 30-37℃ for 18-48 h. After culture, the culture is centrifuged, washed with sterile PBS buffer, and then resuspended with 1 / 50 volume of the original bacterial suspension in buffer until the bacterial concentration is 1×10⁻⁶. 10 ~1×10 11 CFU / mL is obtained by inactivating the bacteria through boiling, chemical methods, or ultra-high pressure sterilization.

[0009] Furthermore, the inactivated bacterial suspension is added to water containing nanoplastics at a concentration of 10-50 mL / L, and reacted at 25-37°C for 2-8 hours. The removal rate of nanoplastics with a particle size of 100-1000 nm in the environmental water is ≥60%.

[0010] Furthermore, the environmental water body includes one or more of the following: surface water, groundwater, effluent from domestic sewage treatment plants, effluent from secondary treatment of industrial wastewater, and aquaculture tailwater.

[0011] Furthermore, the nanoplastic is selected from one or more of polystyrene (PS), polyethylene (PE), polypropylene (PP), and polyethylene terephthalate (PET).

[0012] A second aspect of the present invention provides a microbial adsorbent for the removal of nanoplastics from environmental water bodies. The effective component of the adsorbent is the live Lactobacillus plantarum P101 bacteria described in the present invention, and it also includes a freeze-dried protective carrier and a stabilizer. The freeze-dried protective carrier is one or more of maltodextrin, lactose, and soluble starch, and the stabilizer is one or more of skim milk powder, vitamin C, and trehalose.

[0013] Furthermore, the content of *Lactobacillus plantarum* P101 in the adsorbent is ≥1×10⁻⁶. 10 CFU / g.

[0014] The third aspect of the present invention provides a method for removing nanoplastics from environmental water bodies, comprising the following steps: adding the live bacterial suspension of *Lactobacillus plantarum* P101 described in the present invention to the environmental water body containing nanoplastics to be treated, and reacting for 2 to 12 hours at 15 to 40°C and a stirring speed of 50 to 150 r / min to complete the adsorption and removal of nanoplastics from the water body.

[0015] Furthermore, the dosage of the *Lactobacillus plantarum* P101 live bacterial suspension is 10~50 mL / L of the water to be treated; when the initial concentration of nanoplastics in the water to be treated is ≤100 μg / L, the removal rate of nanoplastics in the water after the reaction is ≥80%.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: This invention fills a gap in the application of *Lactobacillus plantarum* in the environmental remediation of nanoplastics, particularly in the treatment of nanoplastic pollution, providing a highly efficient and environmentally friendly biological solution for nanoplastic pollution control. This invention demonstrates good efficacy, is environmentally friendly, cost-effective, and has positive significance for environmental remediation. Attached Figure Description

[0017] Figure 1 These are experimental photos of Lactobacillus rhamnosus LGG and Lactobacillus plantarum P101 adsorbing polystyrene nanoplastics.

[0018] Figure 2 The adsorption rate of polystyrene nanoplastics by *Lactobacillus rhamnosus* LGG and *Lactobacillus plantarum* P101 in the live bacterial state is shown.

[0019] Figure 3 The adsorption rates of Lactobacillus rhamnosus LGG and Lactobacillus plantarum P101 on polystyrene nanoplastics in the inactivated state are shown. Detailed Implementation

[0020] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] 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, not all, of the embodiments of the present invention. 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.

[0022] Activation and cell preparation of existing Lactobacillus plantarum P101 1. Experimental strains: Lactobacillus plantarum P101 and Lactobacillus rhamnosus LGG used in this example. Lactobacillus rhamnosus strain GG, ATCC 53103 is a commercially available standard strain (purchased from the China Center for Type Culture Collection, a publicly available and legally accessible strain with no intellectual property disputes).

[0023] 2. Culture medium preparation: MRS liquid culture medium (per liter): 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g triammonium citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 1.0 mL Tween 80, with the remainder being distilled water. Adjust the pH to 6.5±0.2, autoclave at 121℃ for 15 min, and cool for later use. MRS solid culture medium is prepared by adding 15.0 g / L agar to the above formula.

[0024] 3. Strain activation: The glycerol-containing bacteria preserved at low temperature were streaked onto MRS solid medium plates and cultured in a facultative anaerobic environment at 37°C for 48 h. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C and 100 r / min for 24 h to obtain the primary seed culture. The primary seed culture was then transferred to fresh MRS liquid medium at an inoculation rate of 3% (v / v) and cultured under the same conditions for 24 h to obtain the secondary seed culture.

[0025] 4. Preparation of live bacterial suspension: Centrifuge the secondary seed culture at 8000 r / min for 5 min, discard the supernatant, wash twice with PBS, and then resuspend in 1 / 50 volume of buffer solution to adjust the bacterial concentration to 1×10⁻⁶. 10 ~1×10 11CFU / mL, refrigerated at 4℃ for later use in adsorption experiments.

[0026] 5. Preparation of inactivated bacterial suspension: Centrifuge the secondary seed culture at 8000 r / min for 5 min, discard the supernatant, wash twice with PBS, and then resuspend the original bacterial culture in 1 / 50 volume of buffer solution to adjust the bacterial concentration to 1×10⁻⁶. 10 ~1×10 11 The concentration of CFU / mL was then placed in boiling water and heated for 30 min, and then refrigerated at 4°C for later use in subsequent adsorption experiments. Example 1: Adsorption of 250 nm polystyrene nanoplastics in a live bacterial state Experimental groups: The experiment was divided into two groups: Lactobacillus rhamnosus LGG control group and Lactobacillus plantarum P101 group.

[0028] Bacterial pretreatment: Centrifuge the activated bacterial culture (8000 rpm, 5 min), discard the supernatant, wash twice with PBS, and then resuspend in 1 / 50 volume of the original bacterial culture buffer to adjust the bacterial concentration to 1×10⁻⁶. 10 ~1×10 11 CFU / mL, take 20 μL of bacterial suspension and add it to 1 mL of polystyrene nanoplastic solution containing 200 μg / mL red fluorescent label (excitation wavelength 365nm, emission wavelength 600nm).

[0029] Adsorption efficiency determination: After incubation at 37℃ and 200 rpm for 4 h, centrifugation at 200 ×g for 10 min, standing at room temperature for 30 min, and then centrifugation at 2000 ×g for 10 min. 100 μL of the supernatant was taken to determine its fluorescence value. The calculation formula is as follows: Adsorption rate (%) = [(Initial fluorescence intensity - Supernatant fluorescence intensity) / Initial fluorescence intensity] × 100% Experimental results: such as Figure 2 As shown, in its viable state, *Lactobacillus plantarum* P101 exhibited an adsorption rate of 96.9% for polystyrene nanoplastics, significantly higher than the adsorption rate (37.7%) of the control group LGG under the same conditions. This result demonstrates that *Lactobacillus plantarum* possesses excellent nanoplastic adsorption capacity.

[0030] Example 2: Adsorption of polystyrene nanoplastics in a sterile state Experimental groups: The experiment was divided into two groups: Lactobacillus rhamnosus LGG control group and Lactobacillus plantarum P101 group.

[0031] Bacterial cell pretreatment: Centrifuge the activated bacterial culture (8000 rpm, 5 min), discard the supernatant, wash twice with PBS, and then resuspend the original bacterial culture in 1 / 50 volume of buffer to a bacterial concentration of 1×10⁻⁶. 10 ~1×10 11 The bacterial suspension was inactivated by boiling in water for 30 min at a concentration of CFU / mL. After cooling, the suspension was resuspended, and 20 μL of the suspension was added to 1 mL of polystyrene nanoplastic solution containing 200 μg / mL of red fluorescent labeling (excitation wavelength 365 nm, emission wavelength 600 nm).

[0032] Adsorption efficiency determination: After incubation at 37℃ and 200 rpm for 4 h, centrifugation at 200 ×g for 10 min, standing at room temperature for 30 min, and then centrifugation at 2000 ×g for 10 min. Take 100 μL of the supernatant to measure the fluorescence of the solution. The calculation formula is: Adsorption rate (%) = [(Initial fluorescence intensity - Supernatant fluorescence intensity) / Initial fluorescence intensity] × 100% Experimental results: such as Figure 3 As shown, under the dead bacterial state, *Lactobacillus plantarum* P101 exhibited an adsorption rate of 62.5% for polystyrene nanoplastics, significantly higher than the adsorption rate of LGG nanoplastics (25.7%) under the same conditions. This result indicates that *Lactobacillus plantarum* still possesses excellent nanoplastic adsorption rates under dead bacterial conditions.

[0033] like Figure 1 As shown, the adsorption comparison between Examples 1 and 2 is illustrated.

[0034] Example 3: Effects of different environmental conditions on the adsorption performance of the strain This embodiment uses 0.1μm PS nanoplastics, which are most common in environmental water bodies, as the adsorption target to investigate the influence of key factors in the actual water environment on the adsorption performance of the strain and to verify the environmental adaptability of the strain. Three parallel samples were set up for each experimental group.

[0035] Effect of pH on adsorption performance: The pH of PBS buffer was adjusted with hydrochloric acid and sodium hydroxide, and seven gradients (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) were set up. Adsorption experiments were conducted according to the method in Example 1. The results showed that the adsorption rate was 84.3% at pH 3.0, 86.2% at pH 9.0, and remained stable at over 92.6% within the pH range of 4.0 to 8.0. The adsorption rate reached a peak of 95.1% to 96.3% at pH 6.0 to 7.0, indicating that the strain has stable adsorption performance over a wide pH range and is suitable for water bodies with different acidity and alkalinity.

[0036] Effect of temperature on adsorption performance: Six temperature gradients were set at 15℃, 25℃, 30℃, 37℃, 40℃, and 45℃. Adsorption experiments were conducted according to the method in Example 1. The results showed that the adsorption rate was 82.6% at 15℃ and 81.2% at 45℃. The adsorption rate remained stable at over 87.9% within the range of 25~40℃, indicating that the strain can adapt to the environmental water temperature conditions in different regions and seasons in my country.

[0037] Effect of salinity on adsorption performance: Sodium chloride was used to adjust the salinity of the water body, and five salinity gradients were set at 0‰, 1‰, 3‰, 5‰ and 8‰. Adsorption experiments were carried out according to the method in Example 1. The results showed that when the salinity was ≤5‰, the adsorption rate of the strain was ≥81.6%, and the adsorption rate was the highest at 0‰, reaching 91.1%, indicating that the strain can adapt to the salinity conditions of most freshwater environments.

[0038] Effect of reaction time on adsorption performance: Five gradients of reaction time were set at 2h, 4h, 6h, 8h, and 12h. Adsorption experiments were conducted according to the method in Example 1. The results showed that the adsorption rate reached 83.0% after 2h of reaction, and reached adsorption equilibrium at 4h with an adsorption rate of 91.4%. Extending the reaction time to 12h did not significantly decrease the adsorption rate, indicating that the strain can quickly achieve adsorption of nanoplastics and the adsorption effect is stable.

[0039] Example 4: Verification of the removal effect of the strain on nanoplastics in actual environmental water bodies Water sampling: Four types of actual environmental water bodies were collected: surface water from urban rivers, groundwater, secondary effluent from domestic sewage treatment plants, and aquaculture tailwater. After collection, the water samples were filtered through a 0.22μm filter membrane to remove native microorganisms and impurities, and stored at 4℃ for later use.

[0040] Preparation of simulated water samples containing nanoplastics: 0.1 μm PS fluorescent nanoplastics were added to the above four filtered actual water samples to adjust the initial concentration of nanoplastics in the water to 50 μg / L (which is consistent with the concentration level of nanoplastics in most polluted water bodies in my country).

[0041] Treatment Experiment: 250 mL of each of the simulated water samples was placed in an Erlenmeyer flask, and *Lactobacillus plantarum* P101 live bacterial suspension was added at concentrations of 10 mL / L, 30 mL / L, and 50 mL / L, respectively. A blank control group (without bacterial suspension) was set up, and three replicates were prepared for each group. The Erlenmeyer flasks were placed in a constant-temperature shaker at 25℃ and 100 rpm for 6 hours. After the reaction, the water samples were filtered through a 0.22 μm filter membrane, and the number of nanoplastics on the filter membrane was counted under a fluorescence microscope to calculate the removal rate of nanoplastics in the water.

[0042] Experimental results are shown in the table below. The results show that the *Lactobacillus plantarum* P101 used in this invention can achieve efficient removal of nanoplastics in different types of actual environmental water bodies. When the dosage is ≥10 mL / L, the removal rate of nanoplastics is ≥81.6%, and when the dosage is ≥30 mL / L, the removal rate is ≥90.8%, which fully meets the application requirements for the treatment of nanoplastic pollution in environmental water bodies.

[0043] Example 5: Validation of the effect of simulated water treatment on a large scale Experimental design: A 5L simulated reactor was used. Actual surface water from urban rivers was taken and 0.1μm PS nanoplastics were added to adjust the initial concentration to 50μg / L. Two reactors were set up, namely the experimental group and the blank control group, with two replicates in each group.

[0044] Treatment process: The live bacterial suspension of Example 1 was added to the reactor of the experimental group at a dosage of 50 mL / L, while no adsorbent was added to the blank control group. The temperature of both reactors was controlled at 25℃ and the stirring speed was 80 r / min. The reaction was carried out continuously for 8 hours. Samples were taken at 2h, 4h, 6h and 8h of the reaction to determine the concentration of residual nanoplastics in the water and calculate the removal rate.

[0045] Experimental results: After 2 hours of reaction, the removal rate of nanoplastics in the experimental group reached 80.5%; after 4 hours, the removal rate reached 91.0%; and after 8 hours, the removal rate reached 90.2%. The concentration of nanoplastics in the water of the blank control group showed no significant change. These results indicate that the microbial adsorbent described in this invention can maintain a high efficiency in removing nanoplastics in a large-scale treatment system, demonstrating its feasibility for engineering applications.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and not to limit them; although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of this application or equivalent substitutions can be made to some technical features, all of which should be covered within the scope of the technical solutions claimed in this application.

Claims

1. The application of *Lactobacillus plantarum* P101 in the adsorption and removal of nanoplastics in environmental water, characterized in that... The Lactobacillus plantarum P101 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2021108 and deposit date of January 19, 2021.

2. The application according to claim 1, characterized in that, The method for preparing the live bacterial suspension of *Lactobacillus plantarum* P101 is as follows: *Lactobacillus plantarum* P101 is inoculated into MRS medium and cultured at 30-37℃ for 18-48 h under facultative anaerobic conditions. After culture, the culture is centrifuged, washed with sterile PBS buffer, and then resuspended with 1 / 50 volume of the original bacterial suspension in buffer until the bacterial concentration is 1×10⁻⁶. 10 ~1×10 11 The result is CFU / mL.

3. The application according to claim 2, characterized in that, The live bacteria suspension was added to water containing nanoplastics at a concentration of 10-50 mL / L and reacted at 25-37°C for 2-8 hours. The removal rate of nanoplastics with a particle size of 100-1000 nm in the environmental water was ≥80%.

4. The application according to claim 1, characterized in that, The method for preparing the inactivated bacterial suspension of *Lactobacillus plantarum* P101 is as follows: *Lactobacillus plantarum* P101 is inoculated into MRS medium and cultured at 30-37℃ for 18-48 h under facultative anaerobic conditions. After culture, the culture is centrifuged, washed with sterile PBS buffer, and then resuspended with 1 / 50 volume of the original bacterial suspension in buffer until the bacterial concentration is 1×10⁻⁶. 10 ~1×10 11 CFU / mL is obtained by inactivating the bacteria through boiling, chemical methods, or ultra-high pressure sterilization.

5. The application according to claim 4, characterized in that, The inactivated bacterial suspension was added to water containing nanoplastics at a concentration of 10-50 mL / L and reacted at 25-37°C for 2-8 hours. The removal rate of nanoplastics with a particle size of 100-1000 nm in the environmental water was ≥60%.

6. The application according to claim 1, characterized in that, The environmental water bodies include one or more of the following: surface water, groundwater, effluent from domestic sewage treatment plants, effluent from secondary treatment of industrial wastewater, and aquaculture tailwater.

7. The application according to claim 1, characterized in that, The nanoplastics are selected from one or more of polystyrene, polyethylene, polypropylene, and polyethylene terephthalate.

8. A microbial adsorbent for the removal of nanoplastics from environmental water, characterized in that, The effective component of the adsorbent is the live Lactobacillus plantarum P101 bacteria as described in claim 1, and it also includes a freeze-dried protective carrier and a stabilizer; the freeze-dried protective carrier is one or more of maltodextrin, lactose, and soluble starch, and the stabilizer is one or more of skim milk powder, vitamin C, and trehalose.

9. A method for removing nanoplastics from environmental water bodies, characterized in that, Includes the following steps: Add the live Lactobacillus plantarum P101 suspension as described in claim 1 to the water containing nanoplastics to be treated, and react for 2 to 12 hours at 15 to 40°C and stirring speed of 50 to 150 r / min to complete the adsorption and removal of nanoplastics in the water.

10. The removal method according to claim 9, characterized in that, The dosage of the *Lactobacillus plantarum* P101 live bacterial suspension is 10~50 mL / L of the water to be treated; when the initial concentration of nanoplastics in the water to be treated is ≤200 μg / L, the removal rate of nanoplastics in the water after the reaction is ≥80%.

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