Composite microbial agent for degrading micro-plastics in seawater and preparation method of composite microbial agent

Abstract

The invention discloses a composite microbial agent for degrading micro-plastics in seawater and a preparation method thereof, the microbial agent is composed of effective viable bacteria and auxiliary components, the effective viable bacteria are marine-derived halomonas, acinetobacter and bacillus and pseudomonas with high affinity with the micro-plastics, the effective viable bacteria are compounded according to a specific viable bacteria number ratio, and the microplastics are degraded by the auxiliary components. And function collaboration is realized. The auxiliary components comprise a protective agent consisting of trehalose, skimmed milk, glycerol and cane sugar, and a nutritional agent consisting of glucose and yeast extract. The preparation method comprises the following steps: respectively fermenting, centrifugally concentrating, compounding in proportion, mixing with auxiliary components, and finally drying to prepare powder. The invention further discloses application of the microbial inoculum to degradation of microplastics in beach intertidal zones and ship wastewater systems. The microbial agent is high in pertinence, good in stability in a high-salt seawater environment and high in degradation efficiency, and an effective solution is provided for in-situ bioremediation of marine micro-plastic pollution.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology and marine pollution control, specifically relating to a method for preparing a composite microbial agent for degrading microplastics in seawater. Background Technology

[0002] In the field of marine environmental protection, the remediation of microplastic pollution in seawater has become a global challenge. Microplastics, due to their stable chemical properties, large specific surface area, and tendency to adsorb toxic substances, pose a long-term threat to marine ecosystems. Developing biotechnologies capable of efficiently and specifically degrading these polymers is a key approach to achieving in-situ remediation of marine microplastic pollution.

[0003] Currently, the use of composite microbial agents for biodegradation is considered an environmentally friendly and sustainable solution. Existing research on composite microbial agents largely focuses on the degradation of specific organic pollutants. For example, prior art document 1 (CN111304128A) discloses a composite microbial agent for treating kitchen wastewater, whose core consists of Bacillus, Citrobacter, and Pseudomonas. While it performs well in oil degradation, its bacterial composition and functional targeting are entirely specific to oil pollutants, failing to address microplastics as a target pollutant or consider the agent's survival and activity in high-salinity seawater environments. Similarly, prior art document 2 (CN111961624A) provides a highly efficient composite microbial agent for nitrogen and phosphorus degradation, composed of specific Bacillus, Pseudomonas, and Acinetobacter. Its application is strictly limited to nitrogen and phosphorus removal from food industry wastewater, similarly failing to address the specific needs of seawater substrate and microplastic degradation.

[0004] These existing technological approaches face several core contradictions and limitations when applied to the degradation of microplastics in seawater: First, functional mismatch: the enzyme systems and metabolic pathways of existing bacterial agents are designed for oils or nitrogen and phosphorus, exhibiting almost no degradation activity against microplastics such as polyethylene (PE) and polypropylene (PP). Second, insufficient environmental adaptability: strains from conventional freshwater or wastewater sources struggle to maintain high activity and competitiveness in the high-salt, high-pressure, and low-nutrient marine environment. Third, poor stability of the bacterial agents: simple bacterial solution formulations, when introduced into the ocean, are prone to rapid inactivation of live bacteria due to environmental stress, failing to achieve long-term, stable degradation effects.

[0005] In addition, existing technologies focus on the combination of strains themselves, while neglecting to design matching bacterial agent forms (such as stress-resistant powders) for specific application scenarios (such as open sea areas) and engineered application methods for synergistic effects, making it difficult to translate laboratory results into on-site treatment effectiveness. Summary of the Invention

[0006] In order to overcome the core defects of existing microbial agents in marine microplastic degradation, such as functional mismatch, insufficient environmental adaptability and poor long-term stability, as pointed out in the background art, this invention discloses a composite microbial agent for microplastic degradation in seawater and its preparation method, aiming to provide a comprehensive solution that integrates specific degradation function, high-salt environment adaptability and long-term storage stability.

[0007] To achieve the above objectives, the present invention includes the following technical solutions.

[0008] A composite microbial agent for the degradation of microplastics in seawater comprises effective live bacteria and auxiliary components; the effective live bacteria are a compound of marine-derived Halomonas, Acinetobacter, Bacillus and Pseudomonas, with a live bacteria ratio of (1-3):(1-3):(2-4):(1-2); the auxiliary components include a protectant and a nutrient, wherein the protectant is composed of trehalose, skim milk, glycerol and sucrose, and the nutrient is composed of glucose and yeast extract.

[0009] Furthermore, in the above-mentioned compound microbial agent, the ratio of the number of viable live bacteria is 2:2:3:1.

[0010] Furthermore, in the above-mentioned compound microbial agent, the halomonas is selected from Halomonas elongata, the Acinetobacter is selected from Acinetobacter venetianus, the Bacillus is selected from Bacillus subtilis, and the Pseudomonas is selected from Pseudomonas putida.

[0011] Furthermore, based on the total weight of the above-mentioned compound microbial agent, the amount of trehalose added is 5%-10%, the amount of skim milk added is 3%-8%, the amount of glycerol added is 1%-3%, the amount of sucrose added is 2%-5%, the amount of glucose added is 1%-3%, and the amount of yeast extract added is 0.5%-2%.

[0012] Furthermore, the above-mentioned compound microbial agent is in the form of a powder obtained by freeze-drying or spray drying, and its total viable count is not less than 1.0×10^10 CFU / g.

[0013] Furthermore, in the above-mentioned compound microbial agent, after the powder is sealed and stored at 25°C for 6 months, the survival rate of live bacteria is not less than 70%.

[0014] This invention also discloses a method for preparing the above-mentioned composite microbial agent, comprising the following steps:

[0015] S1: Separate fermentation: Halomonas, Acinetobacter, Bacillus and Pseudomonas were fermented at high density in suitable culture media and cultured to the late logarithmic growth stage to obtain four single bacterial cultures;

[0016] S2: Centrifugation and concentration: The four single bacterial suspensions obtained in step S1 are centrifuged respectively, the bacterial sludge is collected, and the suspension is resuspended with sterile physiological saline to obtain a high concentration of single bacterial suspension.

[0017] S3: Combination and protection: According to the proportions described in claim 2, measure the four high-concentration bacterial suspensions obtained in step S2 and mix them. Then, add the protectant and nutrient to the mixed bacterial solution and mix thoroughly to obtain a compound bacterial agent mixture.

[0018] S4: Drying and shaping: The compound microbial agent mixture obtained in step S3 is dried to obtain the powdered microbial agent.

[0019] Furthermore, prior to step S1 (separate fermentation), a pre-acclimatization culture step is included: commercially available *Haloxysporum*, *Acinetobacter*, *Bacillus*, and *Pseudomonas* are inoculated separately into a liquid culture medium using polyethylene or polypropylene microplastics as the sole carbon source, and cultured continuously for multiple generations (e.g., 3-5 generations) under suitable conditions, allowing the strains to gradually adapt to the metabolic environment using microplastics as the carbon source. The acclimatized strains are then preserved as seed strains for subsequent fermentation. This pre-acclimatization treatment can further improve the survival rate and degradation initiation efficiency of the inoculum in the target degradation system.

[0020] The present invention also discloses the application of the composite microbial agent described herein in the degradation of polyethylene (PE) or polypropylene (PP) microplastics.

[0021] This invention also discloses the application of the above-mentioned composite microbial agent in degrading microplastics in nearshore beaches or seawater. The invention is characterized by mixing the agent with an inert carrier to form a solid agent, which is then spread on the intertidal zone of a beach contaminated with microplastics. The agent is then allowed to fully contact and degrade the microplastics through tidal action.

[0022] This invention also discloses the application of the above-mentioned composite microbial agent in a ship wastewater microplastic treatment system, characterized in that the agent is fixed on the biological packing material of a bioreactor for treating microplastics in ship ballast water or domestic sewage; the bioreactor is used in conjunction with an ultraviolet photocatalytic unit to form a synergistic treatment system of ultraviolet pretreatment-biodegradation.

[0023] Compared with the prior art, the present invention has the following outstanding advantages:

[0024] First, by scientifically combining carefully selected marine sources with highly compatible bacterial strains, a synergistic degradation effect is achieved, enabling the effective degradation of typical microplastics such as polyethylene and polypropylene. Second, the bacterial agent exhibits excellent adaptability to seawater environments, maintaining superior activity over a wide salinity range compared to ordinary bacterial agents, overcoming the problem of easy inactivation of ordinary bacterial agents in marine environments. Third, a unique composite protective agent system endows the bacterial agent with an ultra-long shelf life and stability, ensuring consistent performance from production to practical application. Finally, the combination of built-in nutrients and specific application methods ensures that the bacterial agent can quickly activate and maintain its degradation function after being introduced into a real marine environment. In summary, this invention provides reliable technical support for achieving efficient and stable biodegradation of marine microplastics. Attached Figure Description

[0025] Figure 1 Comparison of microplastic degradation efficiency in Test Example 1;

[0026] Figure 2 Comparison of degradation efficiency at different salinities in Test Example 2;

[0027] Figure 3 Results of accelerated storage stability of the microbial agent in Test Example 3 (survival rate %). Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and 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] The microbial strains used in this invention are all commercially available mature strains. Other raw materials and reagents used in the preparation process are as follows:

[0030] 1. High-salt LB liquid medium:

[0031] Tryptone: 10.0 g / L

[0032] Yeast extract: 5.0 g / L

[0033] Sodium chloride: 30.0 g / L

[0034] pH adjustment: Adjust the pH value to 7.2 ± 0.2 using sodium hydroxide or hydrochloric acid solution.

[0035] 2. Solvent: Deionized water.

[0036] Sterilization conditions: 121°C, 20 minutes.

[0037] 3. Sterile saline solution:

[0038] Sodium chloride: 8.5 g / L

[0039] Solvent: Deionized water.

[0040] Sterilization conditions: 121°C, 20 minutes.

[0041] 4. Artificial seawater (for experiments):

[0042] Prepare according to ASTM D1141 standard, or use commercially available sea salt and deionized water to prepare a salinity of 3.5%.

[0043] The strains used in this invention are derived from Table 1, but similar commercial strains can also be used; this is not a limitation.

[0044] Table 1. Public Information on Strains

[0045] Strain name (Latin name) Main sources of purchase / suppliers strain information Halomonas elongata Beijing Bio-Bio Biotechnology ATCC 33173 Acinetobacter venetianus Beijing Bio-Bio Biotechnology ATCC 31012 Bacillus subtilis Wuhan Huana Biotechnology Co., Ltd. ATCC 6633 Pseudomonas putida Wuhan Warner Biotechnology Business Number: SHBCC D18015 AS 1.1130

[0046] Example 1

[0047] A composite microbial agent for the degradation of microplastics in seawater, consisting of effective live bacteria and auxiliary components.

[0048] The effective live bacteria are compounded from the following commercially available strains in the following proportions: 2 parts Halomonaselongata, 2 parts Acinetobacter venetianus, 3 parts Bacillus subtilis, and 1 part Pseudomonasputida.

[0049] The auxiliary ingredients include protectants and nutrients. Based on the total weight of the final microbial agent powder, the added amounts of each ingredient are: trehalose 6%, skim milk 5%, glycerin 1%, sucrose 3%, glucose 2%, and yeast extract 1%.

[0050] The preparation method of the compound microbial agent includes the following steps:

[0051] S1: Separate fermentation: The four strains were inoculated separately into high-salt LB liquid medium and cultured with shaking at 35°C and 180 rpm for 24-36 hours until the bacterial culture OD reached the target concentration. 600 By reaching a pH of 1.2-1.5, four different bacterial cultures were obtained.

[0052] S2: Centrifugal Concentration: The four single bacterial suspensions obtained in step S1 were centrifuged at 4℃ and 8000 rpm for 10 minutes. The supernatant was discarded, the bacterial sludge was collected, and the suspensions were resuspended in sterile physiological saline. The concentration of each bacterial suspension was adjusted to 1.0 × 10⁻⁶. 10 CFU / mL was used to obtain a high-concentration single bacterial suspension.

[0053] S3: Combination and Protection: According to the above-mentioned proportion of live bacteria, measure and mix the high-concentration bacterial suspensions. Then add the protective agent (trehalose, skim milk, glycerin, sucrose) and nutrients (glucose, yeast extract) to the mixed bacterial solution, and stir thoroughly until completely dissolved and mixed to obtain a compound bacterial agent mixture.

[0054] S4: Freeze-drying: The compound bacterial agent mixture obtained in step S3 was dispensed into freeze-drying bottles and pre-frozen at -40℃ for 4 hours. Then, it was transferred to a freeze dryer and subjected to primary drying for 24 hours at a cold trap temperature of -50℃ and a vacuum degree below 10 Pa to obtain the powdered bacterial agent. The total viable count of the powder was found to be 5.8 × 10⁻⁶. 10 CFU / g.

[0055] Example 2

[0056] A composite microbial agent for the degradation of microplastics in seawater, consisting of effective live bacteria and auxiliary components.

[0057] The effective live bacteria are compounded from the following commercially available strains in the following proportions: 1 part Halomonaselongata, 3 parts Acinetobacter venetianus, 2 parts Bacillus subtilis, and 2 parts Pseudomonasputida.

[0058] The auxiliary ingredients include protectants and nutrients. Based on the total weight of the final microbial agent powder, the added amounts of each ingredient are: trehalose 8%, skim milk 7%, glycerin 2%, sucrose 4%, glucose 1.5%, and yeast extract 1.5%.

[0059] The preparation method is the same as in Example 1, and the total viable count of the powder obtained is 4.9 × 10⁻⁶. 10 CFU / g.

[0060] Example 3

[0061] A composite microbial agent for the degradation of microplastics in seawater, consisting of effective live bacteria and auxiliary components.

[0062] The effective live bacteria are compounded from the following commercially available strains in the following proportions: 3 parts Halomonaselongata, 1 part Acinetobacter venetianus, 4 parts Bacillus subtilis, and 1.5 parts Pseudomonasputida.

[0063] The auxiliary ingredients include protectants and nutrients. Based on the total weight of the final microbial agent powder, the added amounts of each ingredient are: trehalose 10%, skim milk 3%, glycerin 3%, sucrose 2%, glucose 3%, and yeast extract 0.5%.

[0064] The preparation method is the same as in Example 1, and the total viable count of the powder obtained is 6.5 × 10⁻⁶. 10 CFU / g.

[0065] Example 4

[0066] Pre-acclimatization treatment of the strain (other aspects are the same as in Example 1)

[0067] To improve the adaptability and degradation efficiency of the microbial agent in an environment where microplastics are the sole carbon source, the microbial strain was pre-acclimatized before conventional fermentation. The specific steps are as follows:

[0068] Prepare an acclimatization culture medium with microplastics as the sole carbon source: artificial seawater (salinity 3.5%), with polyethylene (PE) (particle size 100-500 μm) added as the sole carbon source at a concentration of 1 g / L, supplemented with trace amounts of nitrogen and phosphorus inorganic nutrients (0.1% NH4Cl, 0.05% KH2PO4).

[0069] Commercially available Halomonas elongata, Acinetobacter venetianus, Bacillus subtilis, and Pseudomonas putida were inoculated into the above-mentioned acclimatization medium and cultured at 30°C and 150 rpm for 7-10 days with shaking.

[0070] Transfer the culture medium to fresh acclimatization medium and subculture for 5 generations.

[0071] The final generation of bacterial culture was purified by streak plate separation and stored in glycerol tubes at -80℃ as seed culture for subsequent fermentation.

[0072] Comparative Example 1

[0073] A formulation comprising the following strains in a specific ratio of live bacteria: 2 parts Acinetobacter venetianus, 3 parts Bacillus subtilis, and 1 part Pseudomonas putida (excluding marine-derived Halomonas elongata).

[0074] The types and amounts of auxiliary ingredients are the same as in Example 1.

[0075] The preparation method is the same as in Example 1.

[0076] Comparative Example 2

[0077] A formulation comprising the following strains in a specific ratio of live bacteria: 2 parts Halomonas elongata, 3 parts Bacillus subtilis, and 1 part Pseudomonas putida (excluding Acinetobacter venetianus).

[0078] The types and amounts of auxiliary ingredients are the same as in Example 1.

[0079] The preparation method is the same as in Example 1.

[0080] Comparative Example 3

[0081] A formulation having the same composition and proportion of effective live bacteria as in Example 1.

[0082] The auxiliary ingredients contain only protectants (trehalose, skim milk, glycerin, sucrose), and no nutrients (glucose, yeast extract).

[0083] The preparation method is the same as in Example 1.

[0084] Comparative Example 4

[0085] A formulation having the same composition and proportion of effective live bacteria as in Example 1.

[0086] The auxiliary ingredients only contain nutrients (glucose, yeast extract), without any added preservatives (trehalose, skim milk, glycerin, sucrose). The S4 freeze-drying step is omitted in its preparation method; it is directly stored at 4°C as a liquid inoculum.

[0087] Comparative Example 5

[0088] A formulation having the same composition and proportion of effective live bacteria as in Example 1.

[0089] No auxiliary ingredients are added (neither protective agents nor nutrients are included). The preparation method involves directly freeze-drying the high-concentration mixed bacterial suspension obtained in S2.

[0090] Test Example 1

[0091] Comparison of microplastic degradation efficiency

[0092] Objective: To verify the degradation effect of composite microbial agents on polyethylene (PE) microplastics and to examine the performance differences between the complete formulation and the comparative formulation with missing components.

[0093] method:

[0094] Construction of degradation system:

[0095] Artificial seawater (salinity 3.5%, pH 8.0) was used as the medium.

[0096] PE microplastic powder with a particle size of 100-500 μm was added to an Erlenmeyer flask at a concentration of 1 g / L.

[0097] The bacterial agents prepared in Examples 1-3 and Comparative Examples 1-5 were inoculated at an amount of 0.1% (w / v, based on powder).

[0098] A PE microplastic group without inoculation agent was set up as a blank control.

[0099] Culture conditions: Place the conical flask in a shaker at 25°C and 150 rpm and culture in the dark for 60 days.

[0100] Detection indicators: After cultivation, microplastics were quantitatively recovered and repeatedly vortexed with sterile phosphate-buffered saline (PBS) containing trace amounts of surfactant (0.05% Tween-80) to remove surface-attached bacteria. The mixture was then rinsed thoroughly with ultrapure water and finally dried at 60°C to constant weight. The weight loss rate was calculated.

[0101] Results: See Table 2 below and Figure 1 As shown.

[0102] Table 2: Comparison of microplastic degradation efficiency.

[0103] Group 60-day PE weight loss rate (%) Blank control 0.2 Example 1 28.5 Example 2 25.8 Example 3 31.2 Comparative Example 1 12.4 Comparative Example 2 10.7 Comparative Example 3 19.6 Comparative Example 4 16.3 Comparative Example 5 14.1

[0104] The degradation rate of PE microplastics by the bacterial agent in this embodiment of the invention is significantly higher than that of all comparative examples. Comparative examples 1 and 2 (lacking the core strain) have the lowest degradation rates, indicating that the synergistic effect of the four strains is crucial. The degradation rates of comparative examples 3-5 (with incomplete auxiliary components) show that the nutrients and protectants together ensure the efficient and stable degradation performance of the bacterial agent.

[0105] Test Example 2

[0106] Adaptability testing under different salinity environments

[0107] Objective: To investigate the degradation activity of the microbial agent in different salinity environments and compare it with key comparative examples.

[0108] method:

[0109] Salinity and bacterial agent settings: Artificial seawater media with salinities of 1.0%, 3.5% (standard seawater), and 5.0% were prepared. These were then inoculated into Example 1, Comparative Example 1 (lacking marine bacteria), and Comparative Example 4 (lacking protectant, liquid bacterial agent).

[0110] Degradation experiment: 1 g / L of PE microplastics and 0.1% bacterial agent were added to media of various salinities and cultured at 25°C and 150 rpm for 30 days.

[0111] Detection index: The weight loss rate of PE was measured after the culture was completed.

[0112] Results: See Table 3 below. Figure 2 As shown.

[0113] Table 3: Comparison of degradation efficiency under different salinities.

[0114] Group 1.0% Salinity degradation rate (%) 3.5% Salinity degradation rate (%) 5.0% Salinity degradation rate (%) Example 1 18.4 27.3 21.9 Comparative Example 1 8.5 11.2 9.1 Comparative Example 4 5.1 7.5 3.8

[0115] Conclusion: The bacterial agent of Example 1 of this invention maintains high degradation capacity across a wide salinity range, with optimal performance, especially at standard seawater salinity. Comparative Example 1 (lacking marine bacteria) showed significantly reduced efficiency at different salinity levels, demonstrating that marine-derived strains are key to adapting to high-salinity environments. Comparative Example 4 (liquid formulation) suffered severe activity loss due to lack of protection, exhibiting the worst performance and highlighting the superiority of the powder formulation.

[0116] Test Example 3

[0117] Accelerated Storage Stability Test of Microbial Agents

[0118] Objective: To evaluate the activity retention rate of powdered microbial agents after long-term storage and compare it with unstable formulations.

[0119] method:

[0120] Accelerated storage conditions: The bacterial agents prepared in Example 1, Comparative Example 4 (liquid bacterial agent), and Comparative Example 5 (lyophilized powder without protectant) were sealed and placed in a 37°C constant temperature incubator for accelerated storage.

[0121] Viable count: At the end of the 0th, 3rd and 6th month of storage, the number of viable bacteria was determined by plate counting and the survival rate was calculated.

[0122] Results: See Table 4 below. Figure 3 As shown.

[0123] Table 4: Results of Accelerated Storage Stability of Microbial Agents (Survival Rate %)

[0124] Group 0-month survival rate (%) March survival rate (%) June survival rate (%) Example 1 100 81.0 70.7 Comparative Example 4 100 15.3 2.1 Comparative Example 5 100 35.6 10.8

[0125] Conclusion: The bacterial agent (complete formulation) of Example 1 of this invention exhibited a survival rate far exceeding conventional requirements (>50%) after 6 months of accelerated storage. In contrast, the survival rates of Comparative Example 4 (liquid) and Comparative Example 5 (without protectant) decreased sharply, demonstrating the indispensability of the composite protectant system and process for ensuring the shelf life and application effectiveness of the bacterial agent.

[0126] Test Example 4

[0127] Degradation verification in real seawater environment

[0128] Objective: To verify the degradation ability of the bacterial agent in a real and complex seawater environment and compare it with key comparative examples.

[0129] method:

[0130] Seawater and bacterial agent: Taken from nearshore waters and filtered through a 0.45 μm filter membrane. Inoculated into Example 1, Comparative Example 1 (lacking marine bacteria), and Comparative Example 3 (lacking nutrients), respectively.

[0131] Experimental setup: PE microplastics (1 g / L) and 0.1% bacterial agent were added to filtered real seawater. A blank control was used without bacterial agent inoculation.

[0132] Cultivation and detection: After culturing at 25°C and 150 rpm for 45 days, the weight loss rate of the recovered PE microplastics was determined.

[0133] The results are shown in Table 5:

[0134] Table 5: Comparison of degradation efficiency in real seawater environment

[0135] Group 45-day PE weight loss rate (%) Blank control 0.3 Example 1 22.4 Comparative Example 1 8.5 Comparative Example 3 15.6

[0136] Conclusion: In a real seawater environment characterized by complex composition, low nutrient levels, and competition from native microorganisms, the bacterial agent of this invention still exhibits significant degradation efficiency. Although slightly lower than that of the optimized artificial seawater system, its improvement over the blank control and comparative examples is still very significant. The low efficiency of Comparative Example 1 (lacking marine bacteria) highlights the colonization and competitive advantage of native marine strains in the real environment. The decreased efficiency of Comparative Example 3 (lacking nutrients) indicates that internal nutrients are crucial for the rapid initiation of metabolism of the bacterial agent in oligotrophic seawater.

[0137] Test Example 5

[0138] Degradation spectrum of polypropylene (PP) microplastics by bacterial agents

[0139] Objective: To verify the degradation ability of the microbial agent on different types of microplastics and to examine its broad applicability.

[0140] method:

[0141] Substrate replacement: PP microplastic powder (100-500 μm) was used as the sole carbon source at a concentration of 1 g / L.

[0142] Degradation experiment: In artificial seawater with a salinity of 3.5%, 0.1% of the bacterial agent of Example 1 and Comparative Example 2 (without Acinetobacter) was inoculated and cultured for 60 days at 25°C and 150 rpm.

[0143] Test indicator: Determine the weight loss rate of PP.

[0144] The results are shown in Table 6:

[0145] Table 6: Comparison of degradation efficiency of polypropylene (PP) microplastics.

[0146] Group 60-day PP weight loss rate (%) Example 1 18.5 Comparative Example 2 7.4

[0147] The bacterial agent of this invention also exhibits significant degradation ability against PP microplastics, which are more difficult to degrade. The degradation efficiency of Comparative Example 2 (lacking Acinetobacter) was significantly reduced, indicating that Acinetobacter plays an irreplaceable role in the PP degradation chain, further demonstrating the necessity of the four-strain combination strategy for achieving broad-spectrum degradation.

[0148] Test Example 6

[0149] Comparison of degradation performance between pre-acclimatized strains and original strains

[0150] Objective: To verify whether the survival rate and degradation efficiency of strains pre-acclimatized in the degradation system are improved.

[0151] Methods: A compound bacterial agent was prepared using the pre-domesticated strain of Example 4 (other conditions were the same as in Example 1), and compared with the bacterial agent prepared by the undomesticated original strain. Microplastic degradation experiments were carried out under the same conditions.

[0152] Results: The pre-acclimatization agent in Example 4 increased the degradation initiation rate by about 20% in the first 7 days after inoculation and the total degradation rate by about 6.5% after 60 days (compared to Example 1).

[0153] Application Example 1

[0154] Validating the storage stability of microbial agents for beach restoration

[0155] 1. Objective: To verify whether the bacterial agent, after long-term storage, still maintains high efficiency in degrading microplastics (especially PE) in a real beach environment.

[0156] 2. Scenario: Simulating the intertidal zone of a coastal tourist beach polluted by PE microplastics.

[0157] 3. Method:

[0158] Preparation of microbial agent: A batch of the compound microbial agent powder prepared in Example 1 was sealed and stored for 9 months under dry and light-protected conditions at 25°C. Before use, the viable bacteria survival rate was tested by plate counting method and found to be 71%.

[0159] Site setup: Mark several 1m × 1m experimental areas on the beach. Pre-weighed PE microplastic fragments (2-5mm in diameter) labeled with Rhodamine B are then introduced at a rate of 1g / m³.2 The density is evenly mixed into the top 5cm of sand.

[0160] Application of microbial agent: Mix the microbial agent stored for 9 months with dry fine sand at a weight ratio of 1:5 to make a solid microbial agent, and then spread it evenly on the surface of the treatment area.

[0161] Process monitoring: Relying on natural tidal action, without any human intervention. A region without added microbial agents was set up as a blank control.

[0162] Effect evaluation: On days 0, 30, 60 and 90 after application, sand samples were quantitatively collected from each experimental area. The pre-embedded marked PE fragments were recovered by density flotation combined with fluorescence screening / specific wavelength optical detection. After being washed with ultrapure water, the fragments were dried at 60°C to constant weight, and the weight loss rate was accurately calculated.

[0163] 4. Results:

[0164] The weight of PE fragments in the blank control area did not change significantly throughout the experiment (loss rate <3%).

[0165] In the experimental area where the microbial agent was applied after 9 months of storage, the average weight loss rate of PE fragments reached 48% after 90 days.

[0166] Conclusion: This application example demonstrates that even after 9 months of storage, the microbial agent of this invention can still fully meet the stability requirements, and maintains efficient and stable degradation performance on polyethylene (PE) microplastics in a real intertidal beach environment, fully demonstrating the product's excellent storage stability and practical application value.

[0167] Application Example 2

[0168] Application of microbial agents in simulated ship wastewater treatment systems

[0169] 1. Purpose

[0170] The study verified the synergistic degradation effect of the composite microbial agent of the present invention on microplastics (taking PP as an example) in a simulated ship ballast water treatment system when used in conjunction with an ultraviolet photocatalytic unit.

[0171] 2. Scene

[0172] The simulation of a ship's ballast water treatment system targets polypropylene (PP) microplastics as the pollutant.

[0173] 3. Methods

[0174] 3.1 System Construction: Establish a synergistic treatment system of "ultraviolet pretreatment - biofilm reactor".

[0175] Ultraviolet unit: Low-pressure mercury lamp (main wavelength 254 nm), light intensity 40 mJ / cm² 2 .

[0176] Biological unit: a bioreactor filled with porous polyethylene suspended packing material.

[0177] 3.2 Microbial agent immobilization:

[0178] Take the compound microbial inoculant powder prepared in Example 1 and reconstitute it with sterile physiological saline.

[0179] The reconstituted bacterial solution is pumped into the bioreactor for 48 hours, allowing the bacterial agent to naturally form a biofilm on the packing material, thus creating a stable biofilm.

[0180] 3.3 Experimental Execution:

[0181] Experimental group: Simulated ship ballast water (salinity 3.5%) containing PP microplastic powder (particle size 100-500 μm, initial concentration 30 mg / L) was first pumped into the UV unit for pretreatment, and then flowed into the bioreactor with attached biofilm. The hydraulic retention time (HRT) was 24 hours.

[0182] Control group 1: Only UV pretreatment was performed, without passing through a bioreactor.

[0183] Control group 2: The samples were introduced directly into the bioreactor without UV pretreatment.

[0184] Blank control: No processing performed.

[0185] 3.4 Detection and Analysis:

[0186] The system ran continuously for 30 days. After the operation, all packing material in the reactor and suspended solids in the effluent were accurately collected, and the same "weight loss method" as in Test Example 1 was used for final evaluation. That is, all PP microplastics were thoroughly washed and recovered with PBS containing surfactant and ultrapure water, dried to constant weight, and the total weight loss rate of the entire system was calculated.

[0187] 4. Results

[0188] The final degradation effect measured by the weight loss method is shown in Table 7 below.

[0189] Table 7: Degradation effect of simulated ship wastewater system on PP microplastics (weight loss rate)

[0190] Group 30-day PP weight loss rate (%) Blank control 0.8 Control group 1 (UV only) 4.5 Control group 2 (biological only) 16.3 Experimental group (UV + biological) 28.7

[0191] 5. Conclusion

[0192] This application example demonstrates that immobilizing the composite microbial agent described in this invention onto a biological packing material and combining it with a UV photocatalytic unit can constitute a highly efficient synergistic treatment system (UV pretreatment-biodegradation). Using a rigorous weight loss method for evaluation, the system achieved a final degradation rate of 28.7% for PP microplastics, significantly higher than the simple sum of UV pretreatment alone (4.5%) and biodegradation alone (16.3%) (20.8%), demonstrating a clear synergistic enhancement effect.

[0193] The performance of this invention was systematically verified through a series of test examples. Test example 1 shows that the degradation efficiency of the bacterial agent in this embodiment for polyethylene microplastics is significantly higher than that of all comparative examples lacking core strains or auxiliary components, demonstrating the synergistic necessity of the complete formulation. Test example 2 shows that the bacterial agent maintains superior degradation ability compared to the comparative bacterial agent across a wide salinity range of 1.0% to 5.0%, highlighting its excellent adaptability to seawater environments. The accelerated storage experiment in test example 3 demonstrates that the viable bacteria survival rate of the complete formulation bacterial agent after 6 months of storage at 37°C far exceeds conventional requirements and unstable formulations, highlighting its excellent storage stability. The degradation experiment in a real seawater environment in test example 4 further confirms its practical application effectiveness, with a degradation efficiency significantly superior to the comparative bacterial agent. Test example 5 demonstrates that the bacterial agent also has effective degradation ability for more difficult-to-degrade polypropylene microplastics, showcasing its broad-spectrum degradation potential. All test results collectively indicate that the composite microbial agent provided by this invention integrates synergistic degradation, environmental adaptability, long-term stability, and broad-spectrum action, exhibiting superior comprehensive performance and possessing potential for industrial application.

Claims

1. A composite microbial agent for the degradation of microplastics in seawater, characterized in that, It consists of effective live bacteria and auxiliary components; the effective live bacteria are a compound of marine-derived Halomonas, Acinetobacter, Bacillus and Pseudomonas, with a live bacteria ratio of (1-3):(1-3):(2-4):(1-2); the auxiliary components include a protectant and a nutrient, wherein the protectant is composed of trehalose, skim milk, glycerol and sucrose, and the nutrient is composed of glucose and yeast extract.

2. The compound microbial agent according to claim 1, characterized in that, The ratio of viable bacteria to live bacteria is 2:2:3:

1.

3. The compound microbial agent according to claim 1, characterized in that, The halomonas were selected from Halomonas var. longata, the Acinetobacter were selected from Acinetobacter venetianus, the Bacillus were selected from Bacillus subtilis, and the Pseudomonas were selected from Pseudomonas putida.

4. The compound microbial agent according to claim 1, characterized in that, Based on the total weight of the microbial agent, the amount of trehalose added is 5%-10%, the amount of skim milk added is 3%-8%, the amount of glycerol added is 1%-3%, the amount of sucrose added is 2%-5%, the amount of glucose added is 1%-3%, and the amount of yeast extract added is 0.5%-2%.

5. The compound microbial agent according to claim 1, characterized in that, The microbial agent is in the form of a powder obtained by freeze-drying or spray drying, and its total viable count is not less than 1.0 × 10^10 CFU / g.

6. The compound microbial agent according to claim 5, characterized in that, After the powder is sealed and stored at 25°C for 6 months, the survival rate of live bacteria is not less than 70%.

7. A method for preparing the composite microbial agent as described in any one of claims 1-6, characterized in that, Includes the following steps: S1: Separate fermentation: Halomonas, Acinetobacter, Bacillus and Pseudomonas were fermented at high density in suitable culture media and cultured to the late logarithmic growth stage to obtain four single bacterial cultures; S2: Centrifugation and concentration: The four single bacterial suspensions obtained in step S1 are centrifuged respectively, the bacterial sludge is collected, and the suspension is resuspended with sterile physiological saline to obtain a high concentration of single bacterial suspension. S3: Combination and protection: According to the proportions described in claim 2, measure the four high-concentration bacterial suspensions obtained in step S2 and mix them. Then, add the protectant and nutrient to the mixed bacterial solution and mix thoroughly to obtain a compound bacterial agent mixture. S4: Drying and shaping: The compound microbial agent mixture obtained in step S3 is dried to obtain the powdered microbial agent.

8. The application of the composite microbial agent as described in any one of claims 1-6 in the degradation of polyethylene (PE) or polypropylene (PP) microplastics.

9. The application of the composite microbial agent as described in any one of claims 1-6 in the degradation of microplastics in nearshore beaches or seawater, characterized in that, The bacterial agent is mixed with an inert carrier to form a solid bacterial agent, which is then spread on the intertidal zone of a beach polluted by microplastics. The tidal action allows the bacterial agent to come into full contact with the microplastics and degrade them.

10. The application of the composite microbial agent as described in any one of claims 1-6 in a microplastic treatment system for ship wastewater, characterized in that, The bacterial agent is immobilized on the biological packing material of a bioreactor for treating microplastics in ship ballast water or domestic sewage; the bioreactor is used in conjunction with an ultraviolet photocatalytic unit to form a synergistic treatment system of ultraviolet pretreatment-biodegradation.

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