Aeromicrobe margaritae JP4 and application thereof

Abstract

The invention discloses Aeromicrobium phocebe JP4 and application of the Aeromicrobium phocebe JP4, the strain is separated from a soil sample collected from a garbage treatment plant and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 2026184. The marsulfamicrobe JP4 is a newly separated strain and can be used for commercialized polyurethane sponge degradation treatment, the weight loss of the polyurethane sponge treated for 28 days based on the marsulfamicrobe JP4 can reach 32.44%, the degradation efficiency is high, and a novel strain resource is provided for green treatment and environment restoration of polyurethane products.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Marseille aerobic bacteria JP4 and its applications. Background Technology

[0002] Polyurethane (PU) is a multifunctional polymer synthesized from polyols and isocyanates. It comes in various forms and is widely used in construction, furniture, automotive, electronics, and footwear due to its excellent thermal insulation, mechanical properties, and durability. As one of the most commonly used plastics globally, its market continues to grow. However, PU materials are designed to have high environmental stability, making them extremely difficult to degrade in the natural environment after disposal. They can persist for thousands of years, resulting in serious resource waste and environmental pollution. Its waste, especially microplastics, not only disrupts the ecological balance of soil and water bodies but may also adsorb and accumulate toxic pollutants, posing a threat to biodiversity and human health.

[0003] Currently, conventional methods for treating PU waste mainly include landfill, incineration, physical recycling, and chemical recycling, but all have significant limitations. Landfilling, while low-cost, occupies large amounts of land, degrades extremely slowly, and poses a fire risk. Incineration can achieve volume reduction and energy recovery, but incomplete combustion of polyurethane easily produces toxic gases such as hydrogen cyanide and dioxins, causing secondary pollution. Physical recycling is simple to operate, but the mechanical properties of recycled products are usually poor, limiting their economic value. Chemical recycling can depolymerize PU into raw materials such as polyols, achieving a closed-loop cycle, but faces industrialization challenges such as harsh reaction conditions, high costs, and difficulties in product separation and purification.

[0004] In contrast, biodegradation using microorganisms or enzymes is considered a potential green and environmentally friendly solution. In recent years, research has identified various microorganisms capable of degrading PU, but most of these studies are based on laboratory scales and often use water-soluble PU models for screening, resulting in generally low degradation efficiency for commercially available PU products that are actually insoluble in water. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a novel type of Marseille aerobic microorganism JP4 that can effectively degrade commercial polyurethane products; the second purpose is to provide applications of this Marseille aerobic microorganism JP4.

[0006] Technical solution: The Marseille aerobic bacteria ( ) described in this invention Aeromicrobium phoceense JP4, with accession number CCTCC NO: M 2026184.

[0007] Preservation Notes: The *Aeromonas masei* JP4 of this invention is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026184, deposited on January 22, 2026. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430064, China.

[0008] The microbial preparation of the present invention contains Aeromonas JP4.

[0009] Preferably, the microbial preparation is any one of a culture medium containing live *Aeromonas bougainvillea* JP4, freeze-dried powder of live *Aeromonas bougainvillea* JP4, or immobilized live *Aeromonas bougainvillea* JP4.

[0010] The preparation method of the microbial preparation of the present invention comprises the following steps: after activation of Marseille aerobic bacteria JP4, inoculation into LB medium, and shaking culture at 28~32℃ for 45~50 h to obtain the microbial preparation.

[0011] Preferably, the LB medium contains 9-11 g / L peptone, 4-6 g / L sodium chloride, 4-6 g / L yeast extract, and the remainder is water.

[0012] The application of the Marseille aerobic bacteria JP4 or microbial preparations described in this invention in the degradation of polyurethane products.

[0013] Preferably, the polyurethane product is any one or more of the following: polyurethane sponge, foam, plastic, film, sheet, coating, adhesive, and fiber.

[0014] Preferably, the application process involves inoculating the aforementioned Marseille aerobic bacteria JP4 or the aforementioned microbial preparation into an inorganic salt culture medium containing polyurethane products, followed by shaking culture for degradation.

[0015] Preferably, the OD of the inorganic salt culture medium after inoculation... 600 It ranges from 1.3 to 1.7.

[0016] Preferably, the inorganic salt culture medium contains 1.9~2.1 g / L (NH4)2SO4, 0.4~0.6 g / L KH2PO4, 1.4~1.6 g / L K2HPO4, 0.4~0.6 g / L NaCl, 0.1~0.3 g / L MgSO4·7H2O, with the remainder being water.

[0017] Preferably, the culture temperature is 28~32℃.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The Marseille aerobic bacteria JP4 is a newly isolated strain that can be used for the degradation treatment of commercial polyurethane sponges. Based on the fact that the polyurethane sponges treated with this bacteria for 28 days can lose up to 32.44% of their weight, the degradation efficiency is high, providing a new strain resource for the green treatment and environmental remediation of polyurethane products. Attached Figure Description

[0019] Figure 1 For Marseille gas micro JP4 in Impranil ® Colony morphology on DLN plates; Figure 2 A schematic diagram of the Marseille gas microsphere JP4 in the phylogenetic tree; Figure 3 A statistical chart of weight loss after Marseille gas micro-JP4 degrades commercial polyurethane foam. Figure 4 Scanning electron microscope image of commercial polyurethane foam after degradation by Marseille gas micro-JP4; Figure 5 Infrared spectrum of commercial polyurethane foam after degradation by Marseille gas micro-JP4. Detailed Implementation

[0020] The technical solution of the present invention will be further described below.

[0021] Example 1: Screening and identification of Marseille aerobic bacteria JP4 1. Screening of Marseille Aerobicans JP4 Soil samples were collected from a waste treatment plant in Nanjing, Jiangsu Province. 1 g of soil was added to 100 mL of sterile water and incubated with shaking at 180 rpm for 2 h, followed by standing for 1 h. The supernatant was then diluted to 10 mL. -3 10 -4 10 -5 10 -6 Four concentration gradients were applied to Impranil at 0.2 mL dilutions. ® DLN selection plates were incubated at 30°C for 5 days, during which Impranil... ® DLN screening plates contain 1% (w / v) Impranil ® DLN (purchased from Covestro Polymers Ltd., product number Impranil) ® The ingredients are DLN-SD, 10 g / L peptone, 5 g / L sodium chloride, 5 g / L yeast extract, 15 g / L agar powder, and the remainder is water.

[0022] After culturing, select colonies with clear hydrolysis zones and inoculate them into fresh Impranil. ®DLN screening plates were used for secondary screening, and the cells were incubated at 30°C for 5 days. Colonies that still showed a clear hydrolysis zone after secondary screening were streaked for purification, ultimately yielding degradable Impranil. ® The DLN strain, named JP4, has the following colony morphology: Figure 1 As shown, this strain in Impranil ® The DLN screening plate is round, golden yellow, with smooth edges, and semi-transparent.

[0023] 2. Identification of Aerobicum JP4 in Marseilles Genomic DNA was extracted from strain JP4 using the Novizan FastPure Bacteria DNA Isolation Mini Kit (catalog number DC103) as a template, and the universal bacterial primers 27F / 1492R were used. The sequence of primer 27F is 5'-AGAGTTTGATCCTGGCTCAG-3', and the sequence of primer 1492R is 5'-TACGGCTACCTTGTTACGACTT-3'.

[0024] The 16S rRNA gene sequence of strain JP4 was amplified using the Phanta Max Super-Fidelity DNA Polymerase kit (catalog number P505) and sequenced by Beijing Qingke Biotechnology Co., Ltd.

[0025] Sequencing results were compared and analyzed using EzBioCloud (https: / / www.ezbiocloud.net / ), and the results showed that strain JP4 was similar to the type strain. Aeromicrobium phoceense The Marseille-Q0843 sequence similarity reached 99.93%. A phylogenetic tree was constructed by comparing strain JP4 with strains that showed high similarity.

[0026] The results are as follows Figure 2 As shown, strain JP4 and the type strain Aeromicrobium phoceense Marseille-Q0843 is in the same branch.

[0027] The whole genome of strain JP4 was sequenced by Wuhan Bena Technology Co., Ltd., and the relationship between strain JP4 and... Aeromicrobium phoceense The mean nucleic acid identity (ANI) and DNA-DNA hybridization (dDDH) values ​​between the two genomes of Marseille-Q0843 were found to be 97.47% and 78.70%, respectively, both significantly higher than the thresholds for same species (ANI threshold of 95% and dDDH threshold of 70%). Based on these results, strain JP4 was identified as *Aeromonas marseille*. Aeromicrobium phoceenseIt was named Marseilles aerobic bacteria JP4.

[0028] Example 2: Marseille aerobic bacteria JP4 used for the degradation of polyurethane foam Polyurethane (PU) sponges (purchased from Nantong Dagong Sponge Co., Ltd., white polyurethane filler) were cut into 1×1×1cm pieces, dried in an oven (approximately 0.1g per piece after drying), and then sterilized together in an inorganic salt culture medium. 0.3g of PU sponge was added to 100mL of the culture medium. The inorganic salt culture medium contained 2.0 g / L (NH4)2SO4, 0.5 g / L KH2PO4, 1.5 g / L K2HPO4, 0.5 g / L NaCl, 0.2 g / L MgSO4·7H2O, with the remainder being water.

[0029] *Aeromonas marsemata* JP4 was inoculated into LB medium (containing 10 g / L peptone, 5 g / L sodium chloride, 5 g / L yeast extract, and the remainder water), and cultured at 30°C and 180 rpm with shaking for 48 h. Afterward, it was inoculated into the aforementioned inorganic salt medium containing commercial PU sponge to achieve the final OD... 600 =1.5, and then placed in a shaking culture at 30℃ and 180 rpm; at the same time, an inorganic salt medium containing commercial PU sponge with an equal volume of LB medium was used as a control; each treatment was set up in 3 replicates.

[0030] 1. Statistics on weight loss of PU sponge after treatment At 14 and 28 days, respectively, PU sponges treated with Marseille aerobic bacteria JP4 and control group PU sponges were taken out. After being washed 5 times with ultrapure water, they were soaked in 0.4% (v / v) sodium hypochlorite solution for 24 h, ultrasonically treated for 10 min to remove residual bacteria on the sponges, washed 5 times with ultrapure water, and dried in an oven to constant weight. The weight loss of sponges before and after Marseille aerobic bacteria JP4 treatment was counted.

[0031] The results are as follows Figure 3 As shown, after 28 days of incubation, treatment with Marseille aerobic bacteria JP4 resulted in a weight loss of 32.44% in PU sponges.

[0032] 2. Morphological characterization of the treated PU sponge We commissioned Nanjing Jushang Testing Technology Co., Ltd. to acquire scanning electron microscopy images of PU sponges treated with Marseille aerobic bacteria JP4 for 28 days.

[0033] The results are as follows Figure 4 As shown, the control group PU sponge had an intact skeleton structure, a smooth surface, and a tightly connected mesh structure; the PU sponge treated with Aeromonas margaritifera JP4 showed obvious erosion marks on the surface, became very rough, had some structure destroyed, and the skeleton structure became thinner.

[0034] 3. Infrared spectroscopy determination of treated PU sponge The cleaned and dried PU sponges treated with Marseille aerobic bacteria JP4 and the control group PU sponges were subjected to Fourier transform infrared spectroscopy for data acquisition, with a scanning range of 500–4000 cm⁻¹. -1 The resolution is 4 cm. -1 The number of scans was 32.

[0035] The results are as follows Figure 5 As shown, compared with the control group, the characteristic peak (1725 cm⁻¹) of carbonyl (C=O) in PU sponges treated with Marseille aerobic bacteria JP4 after 28 days was significantly higher. -1 The significant decrease in strength indicates that the ester bonds in the sponge broke after treatment, and the sponge structure was damaged.

Claims

1. A type of Marseille aerobic bacteria ( Aeromicrobium phoceense JP4, with accession number CCTCC NO: M2026184.

2. A microbial preparation, characterized in that, The microbial preparation contains *Aeromonas masae* JP4.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation is any one of the following: a culture medium containing live *Aeromonas bougainvillea* JP4, freeze-dried powder of live *Aeromonas bougainvillea* JP4, or immobilized live *Aeromonas bougainvillea* JP4.

4. A method for preparing the microbial preparation according to claim 2, characterized in that, The steps are as follows: After activation of Marseille aerobic bacteria JP4, the bacteria are inoculated into LB medium and cultured at 28-32℃ with shaking for 45-50 h to obtain the microbial preparation.

5. The use of the Marseille aerobic bacteria JP4 as described in claim 1 or the microbial preparation as described in any one of claims 2 to 3 in the degradation of polyurethane products.

6. The application according to claim 5, characterized in that, The polyurethane product is any one or more of the following: polyurethane sponge, foam, plastic, film, sheet, coating, adhesive, and fiber.

7. The application according to claim 5 or 6, characterized in that, The application steps are as follows: inoculate the Marseille aerobic bacteria JP4 as described in claim 1 or the microbial preparation as described in any one of claims 2 to 3 into an inorganic salt culture medium containing polyurethane products, and then degrade it by shaking culture.

8. The application according to claim 7, characterized in that, The OD of the inorganic salt culture medium after inoculation 600 It ranges from 1.3 to 1.

7.

9. The application according to claim 7, characterized in that, The inorganic salt culture medium contains 1.9~2.1 g / L (NH4)2SO4, 0.4~0.6 g / L KH2PO4, 1.4~1.6 g / L K2HPO4, 0.4~0.6 g / L NaCl, 0.1~0.3 g / L MgSO4·7H2O, with the remainder being water.

10. The application according to claim 7, characterized in that, The culture temperature is 28~32℃.

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