Application of MR-1 Shewanella oneidensis EPS in degradation of plasticizers

Abstract

The application provides application of MR-1 Shewanella oneidensis in degradation of plasticizers and belongs to the technical field of biological treatment of environmental organic pollutants. EPS in a culture solution of MR-1 Shewanella oneidensis is extracted and added into polluted water for degrading plasticizers, the EPS is any one of total EPS, S-EPS, LB-EPS and TB-EPS, and the plasticizer is DBP. The EPS extracted from the culture solution of MR-1 Shewanella oneidensis has high DBP degradation efficiency and long duration and is environment-friendly and non-polluting.

Description

Technical Field

[0001] This invention belongs to the field of biological treatment technology for environmental organic pollutants, specifically involving the application of MR-1 Oneda Shewanella EPS in degrading plasticizers. Background Technology

[0002] In recent years, an increasing number of organic pollutants have been discharged into water and soil, causing serious pollution. Among these pollutants is dibutyl phthalate (DBP). DBP is an organic compound used as a plasticizer in polyvinyl acetate, alkyd resins, nitrocellulose, ethyl cellulose, and chloroprene and nitrile rubbers. It is primarily used in the manufacture of paints, printing inks, safety glass, pesticides, and fabric lubricants. It is not only highly toxic to aquatic organisms but can also accumulate in the human body through the food chain, posing a threat to human health. Therefore, how to efficiently and safely remove DBP from the environment is a question worthy of discussion.

[0003] Currently, plasticizers such as dibutyl phthalate (DBP), benzoic acid esters, and benzoic acid esters exhibit very slow hydrolysis and photodegradation rates in the natural environment, classifying them as recalcitrant substances. Artificial degradation methods for DBP include physical, chemical, and biological methods. Physical methods primarily utilize humic acid or activated carbon adsorption, relying on the strong pore structure and adsorption capacity of the adsorbents to remove DBP from water. Chemical methods mainly employ photocatalytic degradation, using ultraviolet light to photolyze and remove DBP from water. While both of these methods are effective in removing DBP from water, they have significant drawbacks, such as the unresolved fate of DBP attached to the adsorbent, high costs for adsorbent regeneration and replacement, slow photocatalytic degradation rates, and the need for exogenous catalysts. Biological methods have become the main pathway for the degradation of plasticizers such as DBP. However, biological methods generally result in slow degradation, especially due to the inconsistent degradation levels among different microbial species. Developing a biological method that achieves high DBP degradation rates and maintains degradation effectiveness over a long period is a pressing technical challenge. Summary of the Invention

[0004] This invention provides the application of MR-1 Shewanella onychomycosis EPS in degrading plasticizers. The EPS extracted from the MR-1 Shewanella onychomycosis culture broth has a significant and long-lasting ability to degrade DBP.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] This invention provides the application of MR-1 Oneda Shewanella EPS in degrading plasticizers.

[0007] Preferably, the EPS is any one of total EPS, S-EPS, LB-EPS, and TB-EPS.

[0008] Preferably, the plasticizer is DBP.

[0009] Preferably, the extraction method of MR-1 Oneida Shewanella EPS includes the following steps: MR-1 Oneida Shewanella is placed in a liquid culture medium for culture, and the bacterial solution is centrifuged to obtain MR-1 Oneida Shewanella EPS.

[0010] This invention provides a product of a degradable plasticizer containing MR-1 Oneda Shewanella EPS.

[0011] This invention provides a method for degrading DBP, comprising the following steps: extracting EPS from MR-1 Shewanella onychomycosis culture medium and adding EPS to polluted water for treatment.

[0012] Preferably, the EPS is any one of total EPS, S-EPS, LB-EPS, and TB-EPS.

[0013] Preferably, the mass ratio of the added EPS to the mass of DPB in the polluted water is 1 to 700:1.

[0014] Preferably, the treatment temperature is 15–25°C and the pH value is 7.0–7.5.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention is the first to utilize *Shewanella onychomycosis* MR-1 for the degradation of plasticizers. The EPS secreted by *Shewanella onychomycosis* MR-1 contains polysaccharides, proteins, and nucleic acids. The presence of proteins and polysaccharides allows EPS to adsorb organic pollutants in water or soil. Furthermore, because the EPS secreted by *Shewanella onychomycosis* MR-1 also contains cytochrome C, flavin, and enzymes, it greatly enhances the electron transfer between pollutants and EPS, thus enabling rapid and efficient degradation of pollutants by EPS. This invention adds EPS extracted from the culture broth of *Shewanella onychomycosis* MR-1 to water contaminated with DBP. After a certain period, analysis using gas chromatography-mass spectrometry (GC-MS) shows that most of the DBP has been degraded, with a degradation rate exceeding 70%. Moreover, EPS is a special type of soluble organic matter, making its introduction into the environment safe and environmentally friendly, without harming the ecological environment. Detailed Implementation

[0017] This invention provides the application of MR-1 *Shewanella onychomycosis* EPS in degrading plasticizers. The EPS described in this invention is further classified into soluble EPS (S-EPS), loosely bound EPS (LB-EPS), and tightly bound EPS (TB-EPS) based on its degree of binding with cells. The EPS described in this invention is a bioflocculant, mainly composed of polysaccharides, proteins, nucleic acids, etc. The presence of proteins and polysaccharides allows EPS to adsorb organic pollutants in water. Furthermore, the presence of cytochrome C, flavin, and enzymes in EPS greatly enhances the electron transfer between pollutants and EPS, thereby enabling rapid and efficient degradation of pollutants by EPS. The EPS described in this invention is any one of S-EPS, LB-EPS, and TB-EPS. The plasticizer described in this invention is DBP. The EPS described in this invention can also be a culture medium containing MR-1 *Shewanella onychomycosis* EPS and total EPS. The MR-1 *Shewanella onychomycosis* strain described in this invention was purchased from the American Type Culture Collection (ATCC), with accession number ATCC700550.

[0018] This invention provides a degradable plasticizer product containing MR-1 Onedashewanella EPS. The product may also include other substances that do not affect the activity of MR-1 Onedashewanella EPS.

[0019] This invention provides a method for degrading DBP in wastewater, comprising the following steps: extracting EPS from MR-1 Shewanella onenei culture medium, and adding the EPS to the polluted water for treatment. The EPS mentioned in this invention is any one of total EPS, S-EPS, LB-EPS, and TB-EPS. The total EPS, S-EPS, LB-EPS, and TB-EPS mentioned in this invention are extracted from MR-1 Shewanella onenei culture medium using different methods. This invention can either add different types of EPS separately to pollutants to degrade DBP, or extract total EPS and then use it for the degradation of DBP in pollutants.

[0020] In this invention, during the treatment of the pollutants, the mass ratio of EPS added to DPB in the polluted water is 1 to 700:1; the treatment temperature is 15 to 25°C, preferably 20°C; the pH value is 7.0 to 7.5, preferably 7.3; and the DPB concentration in the wastewater needs to be ≤15ppm, preferably 0.01 to 10ppm. When the EPS mentioned in this invention is total EPS, the mass ratio of the total EPS added to DPB in the polluted water is 200-300:1, preferably 230-250:1; when the EPS is S-EPS, the mass ratio of the S-EPS added to DPB in the polluted water is 550-750:1, preferably 600-700:1; when the EPS is LB-EPS, the mass ratio of the LB-EPS added to DPB in the polluted water is 10-70:1, preferably 20-50:1; when the EPS is TB-EPS, the mass ratio of the TB-EPS added to DPB in the polluted water is 1-15:1, preferably 4-10:1.

[0021] The present invention also provides a method for extracting EPS of MR-1 Oneydashewanella, comprising the following steps: MR-1 Oneydashewanella is placed in a liquid culture medium and cultured for 18-24 hours, followed by expansion culture to obtain a liquid culture medium containing secretions of MR-1 Oneydashewanella, which is then centrifuged to obtain MR-1 Oneydashewanella EPS.

[0022] In this invention, the liquid culture medium formula is: 4g yeast extract, 10g malt extract, 4g glucose, and 1.0L distilled water. The purchased MR-1 *Shewanella onenei* strain is inoculated into the liquid culture medium at a 20% volume ratio for cultivation. The cultivation temperature is 26–30°C, preferably 28°C, and the cultivation time is preferably 24 hours. Before inoculating the purchased *Shewanella onenei* strain into the liquid culture medium, it needs to be activated by inoculating it into a solid culture medium. The solid culture medium formula is: 5g malt extract, 2g yeast, 2g glucose, 7.5g agar, and pH adjusted to 7.3. The cultivation temperature of the MR-1 *Shewanella onenei* strain in the solid culture medium is 25–30°C, and the cultivation time is 36–60 hours.

[0023] In this invention, the method of scaling up the culture includes: dispensing the cultured bacterial solution into Erlenmeyer flasks at a volume of 80-120 ml in a clean bench, adding 8-12 ml of bacterial solution to each Erlenmeyer flask, and incubating on a shaker at 28°C and 160 rpm for 18-36 hours. This operation is repeated 2-4 times for scaling up the culture. The purpose of scaling up the culture in this invention is to obtain a larger quantity of EPS.

[0024] In this invention, as one possible implementation method, the liquid culture medium containing the secretions of MR-1 Shewanella onnae is dispensed into centrifuge tubes in a clean bench, sonicated at 10W for 2 minutes, heated in a water bath at 60°C for 30 minutes, centrifuged at 11000×g for 30 minutes, and the supernatant is taken as the total EPS.

[0025] In another possible implementation of this invention, the liquid culture medium containing MR-1 Shewanella onychomycosis secretions is dispensed into centrifuge tubes in a clean bench, centrifuged at 5999×g for 10 min, the supernatant is collected, and the precipitate is retained, which is S-EPS; 0.05% sodium chloride solution is added to the precipitate, mixed with a mixer for 30 s, sonicated at 10W for 2 min, centrifuged at 7995×g for 10 min, the supernatant is collected, and the precipitate is retained, which is LB-EPS; 0.05% sodium chloride solution is added to the precipitate again, mixed with a mixer for 30 s, sonicated at 10W for 2 min, heated in a 60℃ water bath for 30 min, centrifuged at 11000×g for 30 min, and the supernatant is collected, which is TB-EPS.

[0026] In this invention, the MR-1 Oneda Sheva strain was purchased from the American Center for Type Culture Collection (ATCC), and the accession number of this strain is ATCC 700550.

[0027] In this invention, unless otherwise specified, all components are commercially available products well known to those skilled in the art.

[0028] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0029] Example 1

[0030] 1. Culture of MR-1 Shewanella onedina

[0031] (1) The MR-1 Oneda Shiva strain (purchased from the American Type Culture Collection Library ATCC, accession number ATCC700550) was inoculated into a solid culture medium and placed in an incubator at 28°C for 48 hours.

[0032] (2) Use an inoculation loop to inoculate the colonies in the solid culture medium into the sterilized liquid culture medium, and place the liquid culture medium in a shaker (28℃, 160rpm) for 24 hours until the bacteria reach the stationary phase.

[0033] (3) 10 ml of the above-cultured MR-1 Oneda Shewanella culture was inoculated onto 100 ml of liquid culture medium on a clean bench and placed in a shaker (28℃, 160 rpm) for 24 hours; this operation was repeated for three subcultures; after 24 hours of the third culture, the bacterial growth reached the stationary phase and the OD value of the culture medium reached 0.976, and subsequent experiments were carried out.

[0034] The solid culture medium was prepared and processed as follows: 5g of malt extract, 2g of yeast, 2g of glucose, and 7.5g of agar were added and the pH was adjusted to 7.3. The mixture was then dispensed into petri dishes and solidified before being placed in an autoclave for sterilization (121℃, 30min).

[0035] Preparation of liquid culture medium: 10g malt extract, 4g yeast, and 4g glucose were dissolved in 1L of ultrapure water and the pH was adjusted to 7.3. The solution was dispensed into Erlenmeyer flasks and sterilized in an autoclave (121℃, 30min).

[0036] 2. Extraction of EPS from MR-1 Shewanella onychomycosis

[0037] (1) Dispense the cultured liquid culture medium into centrifuge tubes in a clean bench, sonicate at 10W for 2 min, heat in a 60℃ water bath for 30 min, centrifuge at 11000×g for 30 min, and the supernatant is the total EPS.

[0038] (2) Dispense the cultured liquid culture medium into centrifuge tubes in a clean bench, centrifuge at 5999×g for 10 min, and the supernatant is S-EPS. Add 0.05% sodium chloride solution to the precipitate, mix with a mixer for 30 s, sonicate at 10W for 2 min, centrifuge at 7995×g for 10 min, and the supernatant is LB-EPS. Add another 0.05% sodium chloride solution to the precipitate, mix with a mixer for 30 s, sonicate at 10W for 2 min, heat in a 60℃ water bath for 30 min, centrifuge at 11000×g for 30 min, and the supernatant is TB-EPS.

[0039] 3. Validation of DBP degradation by EPS in MR-1 Shewanella Oneda

[0040] DBP degradation experiments were conducted using EPS culture medium containing MR-1 Shewanella onychomycosis and the total EPS, S-EPS, LB-EPS, and TB-EPS extracted above, respectively. The steps are as follows:

[0041] Experiment 1: Seven centrifuge tubes were prepared, each containing 10 ml of MR-1 Shewanella onychomycosis EPS culture medium (EPS concentration of 4032 ppm, Bacteria with EPS). Three control groups were set up for each tube. 0.1 ml of 700 ppm DBP was added to each tube, making the DBP concentration in the reaction system 6.93 ppm. Hexane was added to each tube at 0, 2, 4, 6, 10, 15, and 20 hours of reaction for centrifugation extraction. Since DBP is soluble in hexane, the DBP that could be extracted by hexane was DBP that had not been degraded by the MR-1 Shewanella onychomycosis EPS culture medium. The residual amount of DBP in the extracted solution could be determined by GC-MS analysis, thus determining the amount of DBP degradation caused by the EPS-containing bacterial culture.

[0042] Experiment 2: Seven centrifuge tubes containing 10 ml of total EPS (1710 ppm) were prepared, with three control groups set up in each tube. 0.1 ml of DBP at a concentration of 700 ppm was added to each tube, bringing the DBP concentration in the reaction system to 6.93 ppm. Hexane was added to each tube at 0, 2, 4, 6, 10, 15, and 20 hours of reaction for centrifugation extraction. Since DBP is soluble in hexane, the DBP extracted by hexane was the undegraded DBP by S-EPS. GC-MS analysis of the extracted solution revealed the residual amount of DBP in the solution, thus determining the amount of DBP degradation caused by total EPS.

[0043] Table 1. Degradation results of DBP by EPS culture medium containing MR-1 Shewanella onychomycosis and total EPS.

[0044]

[0045] Experiment 3: Seven centrifuge tubes containing 10 ml of S-EPS (1632 ppm) were prepared, with three control groups set up for each tube. 0.1 ml of DBP at a concentration of 250 ppm was added to each tube, bringing the total DBP concentration in the reaction system to 2.48 ppm. Hexane was added to each tube at 0, 2, 4, 6, 10, 15, and 20 hours of reaction for centrifugation extraction. Since DBP is soluble in hexane, the DBP extracted by hexane was the undegraded DBP by S-EPS. GC-MS analysis of the extracted solution revealed the residual amount of DBP in the solution, thus determining the amount of DBP degraded by S-EPS.

[0046] Experiment 4: Seven centrifuge tubes containing 10 ml of LB-EPS (75 ppm) were prepared, with three control groups set up for each tube. 0.1 ml of DBP at a concentration of 250 ppm was added to each tube, bringing the total DBP concentration in the reaction system to 2.48 ppm. Hexane was added to each tube at 0, 2, 4, 6, 10, 15, and 20 hours of reaction for centrifugation extraction. Since DBP is soluble in hexane, the DBP extracted by hexane was the undegraded DBP by LB-EPS. GC-MS analysis of the extracted solution revealed the residual amount of DBP in the solution, thus determining the amount of DBP degraded by LB-EPS.

[0047] Experiment 5: Seven centrifuge tubes containing 10 ml of TB-EPS (16 ppm) were prepared, with three control groups set up for each tube. 0.1 ml of DBP at a concentration of 250 ppm was added to each tube, bringing the total DBP concentration in the reaction system to 2.48 ppm. Hexane was added to each tube at 0, 2, 4, 6, 10, 15, and 20 hours of reaction for centrifugation extraction. Since DBP is soluble in hexane, the DBP extracted by hexane was the undegraded DBP by TB-EPS. GC-MS analysis of the extracted solution revealed the residual amount of DBP in the solution, thus determining the amount of DBP degraded by TB-EPS.

[0048] Table 2 Degradation results of DBP by S-EPS, LB-EPS, and TB-EPS

[0049]

[0050] The experimental results are shown in Tables 1-2. All EPS (total EPS), S-EPS, LB-EPS, and TB-EPS exhibited highly efficient degradation of DBP within 20 hours, with degradation rates exceeding 50%. TB-EPS even showed a degradation rate exceeding 80%. Furthermore, Table 1 shows that the culture medium obtained from MR-1 Shewanella onychomycosis showed a high degradation rate of DBP. However, because the presence of MR-1 Shewanella onychomycosis may disrupt the aquatic ecological balance, culture media containing MR-1 Shewanella onychomycosis are not recommended.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of MR-1 *Shewanella onychomycosis* EPS in degrading plasticizers, characterized in that, The EPS can be any one of total EPS, S-EPS, LB-EPS, and TB-EPS; The extraction method of EPS from MR-1 Shewanella onione includes the following steps: MR-1 Shewanella onychomycosis was cultured in liquid culture medium. The cultured liquid culture medium was dispensed into centrifuge tubes in a clean bench, sonicated at 10W for 2 min, heated in a 60℃ water bath for 30 min, and centrifuged at 11000×g for 30 min. The supernatant was the total EPS. The cultured liquid culture medium was dispensed into centrifuge tubes in a clean bench and centrifuged at 5999×g for 10 min. The supernatant was S-EPS. 0.05% sodium chloride solution was added to the precipitate, and the mixture was mixed for 30 s with a mixer. The mixture was then sonicated at 10W for 2 min and centrifuged at 7995×g for 10 min. The supernatant was LB-EPS. 0.05% sodium chloride solution was added to the precipitate again, and the mixture was mixed for 30 s with a mixer. The mixture was then sonicated at 10W for 2 min, heated in a 60℃ water bath for 30 min, and centrifuged at 11000×g for 30 min. The supernatant was TB-EPS. The plasticizer is DBP.

2. A method for degrading DBP in the application of MR-1 Shewanella onychomycosis EPS according to claim 1 in degrading plasticizers, characterized in that, Includes the following steps: EPS was extracted from the MR-1 Shewanella onychomycosis culture medium and then added to polluted water bodies containing DBP for treatment.

3. The method according to claim 2, characterized in that, The ratio of the amount of EPS added to the mass of DPB in the polluted water is 1 to 700:

1.

4. The method according to claim 2, characterized in that, The treatment temperature is 15–25°C, and the pH value is 7.0–7.5.

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