Fusarium oxysporum and its application, pva degrading bacterial agent

Abstract

The application discloses a fusarium oxysporum and application thereof and a PVA degrading microbial agent, and belongs to a kind of fungal microorganism degradation substance, compared with existing PVA natural degradation, the degradation efficiency of PVA is greatly improved, and the damage of oxygen enrichment, ecological environment and biological chain in water body and soil is avoided.In addition, the fusarium oxysporum and the PVA degrading microbial agent of the application will not produce secondary pollution after degrading PVA, and the cost is low, economic and environmental protection, which is a kind of green and efficient PVA degrading microbial agent, and has high economic value.The application solves the problem of slow natural biological degradation rate of existing PVA.

Description

Technical Field

[0001] This invention relates to the technical field, specifically to a Fusarium oxysporum and its application, and a PVA-degrading bacterial agent. Background Technology

[0002] Polyvinyl alcohol (PVA) is a water-soluble polymer with excellent properties and is one of the few biodegradable polymers in the world. Due to its good water solubility, adhesion, abrasion resistance, and toughness, PVA is widely used in agricultural mulch films, textile sizing, and food packaging. However, PVA has high surface activity, resulting in a slow biodegradation rate in nature. Large quantities of slowly degrading PVA are widespread in nature, causing oxygen depletion in water and soil, ecological damage, and disruption of the food chain.

[0003] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, we provide Fusarium oxysporum and its applications, as well as a PVA-degrading bacterial agent, to solve the problem of the slow biodegradation rate of PVA in nature.

[0005] To achieve the above objectives, a Fusarium oxysporum strain is provided, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40272.

[0006] This invention provides an application of the above-mentioned Fusarium oxysporum, using Fusarium oxysporum for the degradation of PVA.

[0007] This invention provides a PVA-degrading bacterial agent, comprising a culture medium, a bacterial suspension of *Fusarium oxysporum* as described above, and a synergist, wherein the synergist comprises K + Mg 2+ Fe 3+ Ca 2+ .

[0008] Furthermore, the synergist includes CaCl2, MgSO4, K2HPO4, and FeSO4.

[0009] Furthermore, the culture medium is PDB medium.

[0010] Furthermore, based on the volume of the culture medium, the PVA-degrading bacterial agent comprises 5% to 25% of the bacterial solution.

[0011] Furthermore, the PVA-degrading microbial agent includes 8% to 12% of a synergist.

[0012] Furthermore, based on the volume percentage of the synergist, the synergist comprises 5% to 25% CaCl2, 5% to 25% MgSO4, 5% to 25% K2HPO4, and 5% to 25% FeSO4.

[0013] The beneficial effects of this invention are that the *Fusarium oxysporum* and PVA-degrading bacterial agent of this invention are fungal microbial degradation substances. Compared with existing natural PVA degradation methods, they significantly improve the degradation efficiency of PVA, avoiding oxygen enrichment in water and soil, ecological environmental damage, and disruption of the food chain. Furthermore, the *Fusarium oxysporum* and PVA-degrading bacterial agent of this invention do not produce secondary pollution after degrading PVA, are low-cost, economical, and environmentally friendly, and are a green and efficient PVA-degrading bacterial agent with high economic value. Attached Figure Description

[0014] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0015] Figure 1 This is a macroscopic morphological diagram of Fusarium oxysporum according to an embodiment of the present invention.

[0016] Figure 2 This is a microscopic morphological diagram of Fusarium oxysporum according to an embodiment of the present invention.

[0017] Figure 3 Phylogenetic tree of ITS rDNA sequences of Fusarium oxysporum and related species in embodiments of the present invention.

[0018] Figure 4 This is a phylogenetic tree of the EF1-α gene sequences of Fusarium oxysporum and related species in an embodiment of the present invention.

[0019] Figure 5 This is a line graph showing the degradation rate of Fusarium oxysporum at different temperatures according to an embodiment of the present invention.

[0020] Figure 6 This is a line graph showing the degradation rate of Fusarium oxysporum under different inoculum amounts according to an embodiment of the present invention. Detailed Implementation

[0021] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0022] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0023] Reference Figures 1 to 4 As shown, the present invention provides a Fusarium oxysporum, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40272, deposited on August 12, 2022, and located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0024] A preferred application of the *Fusarium oxysporum* strain of this invention is its use in degrading PVA. Compared to natural biodegradation, the *Fusarium oxysporum* strain of this invention can degrade PVA more efficiently.

[0025] Specifically, the application of Fusarium oxysporum in this invention includes the following steps:

[0026] S1: The isolated and purified Fusarium oxysporum was cultured in PDB (Potato Dextrose Broth) medium until the logarithmic growth phase.

[0027] S2: Prepare the degradation bacterial solution.

[0028] Take 1 mL of the logarithmic growth phase bacterial culture and add it to fresh PDB medium, then dilute to the bacterial culture OD value. 600 The degradation bacterial solution was obtained between 0.8 and 1.

[0029] S3: Add the substance to be degraded (polyvinyl alcohol PVA) to the sterilized culture medium (carbon-free solid culture medium) and pour it into plates.

[0030] S4: Drop the degrading bacterial solution onto the plate to degrade the substance to be degraded.

[0031] Specifically, the degrading bacterial solution was added to a plate, packaged in a light-proof newspaper, and incubated at a constant temperature of 24℃~28℃ for 7d~21d.

[0032] The present invention also provides a PVA-degrading bacterial agent comprising: a culture medium, a bacterial solution containing the aforementioned Fusarium oxysporum, and a synergist.

[0033] The culture medium is PDB medium.

[0034] The synergist contains K + Mg 2+ Fe 3+ Ca 2+ .

[0035] As a preferred embodiment, the PVA-degrading bacterial agent comprises 5% to 25% bacterial solution and 8% to 12% synergist, based on the volume of the culture medium.

[0036] Preferably, the PVA-degrading bacterial agent comprises 25% bacterial solution and 10% synergist, based on the volume of the culture medium. The synergist, based on its volume, comprises 5%–25% CaCl2, 5%–25% MgSO4, 5%–25% K2HPO4, and 5%–25% FeSO4.

[0037] The *Fusarium oxysporum* strain of this invention was isolated from the corroded and degraded plastic lid of a water bath in the laboratory of the Jiangsu Yangtze River Agricultural Research Institute. Specifically, the strain screening and identification process included:

[0038] a. Strain screening: Under aseptic conditions, the degraded material was picked up from the corroded plastic cap using a sterile inoculation loop and transferred to 50 mL PDB medium. The medium was incubated at 28°C with shaking (150 r / min) for 30 days, with three replicates. After 30 days, the mixed bacterial suspension with the best growth was selected.

[0039] b. Selection and acclimatization of bacterial strains: The mixed bacterial solution was streaked onto PDA (Potato Dextrose Agar) medium using an inoculation loop and incubated at 30°C for 10 days. After 10 days, white, yellow, and brownish-green colonies appeared. These unknown colonies were then inoculated onto PDB medium and incubated at 28°C with shaking (150 rpm) for 30 days. Different colony mixtures were then streaked onto PDA medium and incubated for 10 days. This streaking process was repeated on PDA medium to isolate and purify the strains until a single dominant colony grew on the plate.

[0040] c. Identification of strains

[0041] The obtained single strain was inoculated into PDA medium and cultured at 30℃ for 10 days. Colony morphology was observed, and the microscopic morphology of the strain was observed under a microscope. Molecular identification of the strain was performed according to the "FMIC-QO01-003 Method for Identification and Detection of Multiphasic Fungi" and the "QO-03-02 Operating Procedure for Molecular Biological Identification of Microbial Strains", namely, strain DNA extraction, PCR amplification, electrophoresis detection, 16S rDNA sequencing, and homology comparison.

[0042] Suspected bacterial strains were isolated from the corroded plastic cap and cultured for 7 days. The results were as follows: Figure 1 As shown, the macroscopic morphology of this strain is characterized by colonies with a diameter of 65mm to 68mm, white in the center and reddish-brown around the periphery; velvety texture; purplish-brown in the center and yellowish-brown around the periphery on the reverse side; no exudate is produced and no soluble pigment is produced.

[0043] like Figure 2 As shown, microscopic observation revealed that the hyphae were intertwined and branched, transparent and smooth, with septa, and 1.5μm to 3.5μm in diameter; phialids occurred on aerial hyphae, solitary, and 10μm to 35μm in size; microspores were fusiform or club-shaped, slightly curved, (4.5μm to 11μm) × (1.5μm to 2.8μm); megaspores were curved and sickle-shaped, with 1-3 transverse septa, (15μm to 25μm) × (2.8μm to 3.8μm).

[0044] The strain was identified, and its phylogenetic tree is shown below. Figure 3 and Figure 4 As shown, the results indicate that it is Fusarium oxysporum, named PDBF01.

[0045] Figure 3 The image shows the phylogenetic tree of the ITS rDNA sequence of strain "PDBF01" and related species, displayed using MEGA 5.0 software (MEGA is a toolkit for sequence analysis and comparative statistics, used to construct phylogenetic trees). The neighbor-joining method was used to display the phylogenetic tree. 1000 similarity replicates were performed. The phylogenetic tree nodes in the figure only show values ​​with Bootstrap values ​​greater than 50%. The superscript "T" represents the type strain.

[0046] Figure 4 The image shows a phylogenetic tree of the EF1-α gene sequence of strain "PDBF01" and related species, displayed using MEGA 5.0 software and the neighbor-joining method. 1000 similarity replication calculations were performed. The phylogenetic tree nodes in the figure only show values ​​with Bootstrap values ​​greater than 70%, and the superscript "T" indicates the type strain.

[0047] The degradation effect of strain PDBF01 on experimental PVA was measured under different temperature settings as follows: Figure 5 As shown, the results indicate that strain PDBF01 degrades PVA at 28℃ faster than at other temperature conditions. Below 28℃, the degradation rate increases with increasing temperature, but as the temperature continues to rise, the degradation rate shows a decreasing trend and then remains unchanged. The optimal degradation treatment temperature for strain PDBF01 is 28℃.

[0048] At different concentrations of PDBF01 bacterial suspensions, the degradation rate of the tested PVA by strain PDBF01 was as follows: Figure 6As shown, the results indicate that before the inoculum volume reaches 25% of the PVA-containing liquid volume (the volume ratio of culture medium to PVA solution is 1:1), the degradation effect of the target strain on PVA significantly increases with increasing inoculum volume. When the inoculum volume exceeds 25%, the degradation effect shows a slow increasing trend, but no significant difference. Furthermore, excessive inoculum leads to waste of the strain and problems with subsequent bacterial solution treatment. The optimal inoculum volume for strain PDBF01 is 25% of the volume of the PVA solution or culture medium.

[0049] Table 1 shows the degradation efficiency of strain PDBF01 on experimental PVA under different degradation times, optimal treatment temperatures, and optimal inoculum amounts.

[0050] Table 1. Degradation rate of PVA by strain PDBF01 at different treatment durations

[0051] 7 28.00 14 43.01 21 50.07 28 50.09 35 50.14

[0052] Table 1 shows the PVA-degrading agents used, including: culture medium and bacterial suspension containing the aforementioned *Fusarium oxysporum*, wherein the volume of the bacterial suspension was 25% of the culture medium volume. When degrading PVA at 28°C, the volume ratio of the culture medium to the PVA solution to be degraded was 1:1. The PVA degradation rate of strain PDBF01 increased with time before 21 days of cultivation; after 21 days, the rate of increase in PVA degradation rate tended to level off, and the increase in degradation rate showed no significant difference.

[0053] Based on the PVA-degrading bacterial agent used in Table 1, a synergist was added. The synergist comprised 10% of the culture medium volume. Calculated as 100% by volume, the synergist included 5%–25% CaCl2, 5%–25% MgSO4, 5%–25% K2HPO4, and 5%–25% FeSO4. Experiments showed that the PVA-degrading bacterial agent with added synergist achieved a degradation rate of 58.83% in the PVA solution to be degraded.

[0054] Specifically, the methods for determining PVA content can refer to the total chlorine content method in GB / T 39506-2020 Determination of Polyvinyl Chloride (PVC) Content in Rigid Polyvinyl Chloride (PVC-U) Pipes and Fittings, the starch-cadmium iodide method for determining PP degradation rate in Yu Bo et al.'s "A Review of Methods for Determining the Degradation Rate of Polyacrylamide in Wastewater" in Chemical Engineer (2016, 30(09)), the determination of PLA degradation rate by measuring lactic acid content in the degradation products in the study of bio-cyclic degradation of polylactic acid (PLA) by Zhan Yue et al. in Northeast Normal University (2013), and the iodine-potassium iodide-boric acid method for determining PVA degradation rate in the study of Cui Shuangke, Wang Zhigang, Qiang Xueni, Guo Yani et al.'s "An Improved Spectrophotometric Method for Determining PVA Content in Desizing Wastewater" in the Journal of Xi'an University of Technology (2014, 28(01): 68-71).

[0055] This invention provides an application method using a PVA-degrading microbial agent, comprising the following steps:

[0056] S11: Provides PVA-degrading bacterial agents.

[0057] S22: Pour the PVA degrading bacteria into the substance to be degraded (polyvinyl alcohol PVA) for degradation.

[0058] The volume ratio of PVA-degrading microbial agent to the substance to be degraded (polyvinyl alcohol PVA) is 1:1. The polyvinyl alcohol PVA to be degraded is mixed with 1g of water as a solvent in a container equipped with a stirrer. The stirring speed is controlled at 30-50 rpm. The mixture is soaked for 10 minutes, and the temperature is slowly raised to 85℃~99℃. After the PVA is completely dissolved, heating is stopped, and the mixture is cooled to obtain the desired substance to be degraded.

[0059] After the PVA-degrading bacterial agent is poured into the substance to be degraded and mixed evenly, it is incubated in a dark room at a constant temperature of 24℃~28℃ for 7d~21d.

[0060] The preferred degradation temperature is 28℃ and the degradation time is 21 days.

[0061] The volume of the PVA-degrading bacterial agent is 5% to 25% of the volume of the degrading material (polyvinyl alcohol PVA and PDB culture medium), and preferably, the inoculation amount is 25%.

[0062] The *Fusarium oxysporum* and PVA-degrading microbial agent of this invention are fungal microbial degradation substances. Compared with existing natural PVA degradation methods, they significantly improve the degradation efficiency of PVA, avoiding oxygen enrichment in water and soil, ecological damage, and disruption of the food chain. Furthermore, the *Fusarium oxysporum* and PVA-degrading microbial agent of this invention do not produce secondary pollution after degrading PVA, are low-cost, economical, and environmentally friendly, making them a green and highly efficient PVA-degrading microbial agent with high economic value.

[0063] The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A type of Fusarium oxysporum ( Fusarium oxysporum ), characterized in that, Fusarium oxysporum is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 40272.

2. An application of *Fusarium oxysporum* as described in claim 1, characterized in that, Fusarium oxysporum was used for the degradation of PVA.

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