Application of gordonibacter westensis eps in degradation of plasticizers

Abstract

The application provides application of EPS of Gordonia westlakei in degradation of plasticizers and belongs to the technical field of biological treatment of environmental organic pollutants. The EPS of Gordonia westlakei is extracted and added into polluted water bodies for degrading plasticizers, the EPS is any one of total EPS, S-EPS, LB-EPS or TB-EPS, and the plasticizer is DBP. The EPS secreted by Gordonia westlakei is used for degrading DBP in water bodies, and the method is fast and efficient, and does not easily cause harm to the environment.

Description

Technical Field

[0001] This invention belongs to the field of biological treatment technology for environmental organic pollutants, and in particular relates to the application of *Gordonella xiwahuensis* EPS in degrading plasticizers. Background Technology

[0002] Plasticizers are widely used in the processing of various products and can easily migrate from various plastic products and additives into environmental media such as the atmosphere, water, and soil, causing pollution and posing serious threats to human health and the ecological environment. Dibutyl phthalate (DBP) is one such plasticizer, mainly 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. Currently, DBP has been listed as a priority pollutant for control; therefore, how to efficiently and safely remove DBP from the environment is one of the key problems to be solved in this field.

[0003] Plasticizers can be degraded in various ways, including natural photolysis, chemical catalytic oxidation, and biodegradation. Among these, microbial degradation of plasticizers offers advantages such as high efficiency, stability, environmental friendliness, and economic feasibility. Current research shows that different strains of bacteria capable of degrading plasticizers exist in the environment or on plants, which can alleviate the stress of plasticizers on plants, reduce plasticizer residues in the environment, and decrease the accumulation of plasticizers by plants. *Goldenella* is a degrading bacterium; existing technologies have reported that *Goldenella isopreneophile* can grow in soil using dibutyl phthalate (DBP) as a carbon source, thereby reducing the DBP content in the soil. *Goldenella alkaline-eating* YC-RL2 shows broad application prospects in the bioremediation of plasticizers, indicating that the *Goldenella* genus has untapped potential in plasticizer degradation. However, there are currently no existing technologies reporting whether the secretions of *Goldenella xiwahuensis* have a plasticizer-degrading effect. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of *Gordonella asiatica* EPS in degrading plasticizers, which can rapidly and efficiently adsorb and degrade DBP in polluted water bodies, while not easily causing harm to the environment.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of *Gordonella spp.* EPS in degradable plasticizers.

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

[0008] This invention provides the combined application of *Gordonella spp.* EPS and biochar in degrading plasticizers.

[0009] Preferably, the plasticizer is DBP.

[0010] This invention provides a product of a degradable plasticizer containing *Gordonella spp.* EPS and biochar.

[0011] This invention provides a method for degrading DBP in wastewater, comprising the following steps: extracting EPS from the culture medium of *Goldenella oryzae* from Xiwa Lake, and adding the EPS to the wastewater for treatment.

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

[0013] Preferably, the mass ratio of EPS added to DBP in the wastewater is 1 to 1000:1; the treatment temperature is 18 to 25°C, and the pH value is 7.0 to 7.3.

[0014] Preferably, the concentration of DBP in the wastewater is 2 to 10 ppm.

[0015] Preferably, biochar is added to the wastewater at the same time, and the amount of biochar added is 0.001 to 0.003 g / mL.

[0016] The beneficial effects of this invention are:

[0017] This invention is the first to utilize *Goldenella xiwahuensis* EPS for the degradation of plasticizers. The EPS secreted by *Goldenella xiwahuensis* contains polysaccharides, proteins, and nucleic acids, enabling it to adsorb DBP from water or soil. Simultaneously, the enzymes, cytochrome C, and flavins present in the EPS significantly enhance the enzymatic catalytic interaction between DBP and EPS, resulting in rapid and efficient degradation of DBP by the EPS. Furthermore, EPS is a special type of soluble organic matter, making it safe and environmentally friendly when introduced into the environment without harming the ecosystem. Detailed Implementation

[0018] This invention provides the application of *Gordonella spp.* EPS in degrading plasticizers. The EPS described in this invention is any one of total EPS, S-EPS, LB-EPS, or TB-EPS; the S-EPS is a soluble EPS, the LB-EPS is a loosely bound EPS, and the TB-EPS is a tightly bound EPS; the plasticizer described in this invention is preferably dibutyl phthalate (DBP).

[0019] EPS secreted by *Gordonella oryzae* in Xiwa Lake contains polysaccharides, proteins, and nucleic acids, enabling it to adsorb DBP from the water. Simultaneously, the enzymes, cytochrome C, and flavin present in EPS significantly enhance the enzymatic catalytic reaction between DBP and EPS, resulting in rapid and efficient degradation of DBP by EPS. Furthermore, EPS is a high-molecular-weight complex secreted by microorganisms, acting as a binder connecting cells and extracellular polysaccharides and proteins. As dissolved organic matter of microbial origin in soil, EPS accounts for 50-90% of soil organic matter content, making its introduction into the environment safe and environmentally friendly, without harming the ecological environment.

[0020] This invention provides the combined application of *Goldenella spp.* EPS and biochar in the degradation of plasticizers. The biochar is preferably prepared from rice straw. As an optional embodiment, the biochar is prepared by pyrolyzing crushed rice straw in a high-temperature muffle furnace. This invention, by combining *Goldenella spp.* EPS with biochar, demonstrates a synergistic degradation effect, effectively improving the degradation rate of DBP in water.

[0021] This invention provides a biodegradable plasticizer product containing both *Goldenella spp.* EPS and biochar. The ratio of *Goldenella spp.* EPS to biochar in the product is 8–12 mL: 0.001–0.003 g.

[0022] This invention also provides a method for degrading DBP in wastewater, comprising the following steps: extracting EPS from *Goldenella spp.* culture medium from Xiwa Lake, and adding the EPS to the wastewater for treatment. The EPS described in this invention can be any one of total EPS, S-EPS, LB-EPS, or TB-EPS. Different types of EPS require different extraction conditions. This invention can extract different types of EPS separately and then add them individually to the wastewater to degrade DBP. Alternatively, total EPS can be extracted and used for the degradation of DBP in the wastewater.

[0023] As an optional implementation method, the present invention cultured *Gordonella xiwahuensis* in liquid culture medium, and after 18-24 hours of culture, expanded culture was carried out to extract EPS secreted by *Gordonella xiwahuensis*.

[0024] The method for extracting the total EPS can be as follows: the cultured liquid culture medium is subjected to ultrasound and water bath heating, followed by centrifugation, and the supernatant obtained is the total EPS. The ultrasound conditions are 10W for 2 minutes; the water bath conditions are heating at 60℃ for 30 minutes; and the centrifugation conditions are centrifugation at 11000×g for 30 minutes.

[0025] The extraction method for S-EPS can be as follows: centrifuge the cultured liquid culture medium to obtain the supernatant, which is S-EPS. The centrifugation conditions are 5999×g for 10 min.

[0026] The extraction method for LB-EPS can be as follows: add 0.05% sodium chloride solution to the precipitate after S-EPS extraction, mix and sonicate, centrifuge, and the supernatant is LB-EPS. The mixing is performed using a homogenizer for 30 seconds; the sonication conditions are 10W for 2 minutes; and the centrifugation conditions are 7995×g for 10 minutes.

[0027] The extraction method for TB-EPS can be as follows: add 0.05% sodium chloride solution to the precipitate after LB-EPS extraction, mix thoroughly, sonicate, heat in a water bath, and then centrifuge. The supernatant is TB-EPS. The mixing is performed using a homogenizer for 30 seconds; the sonication conditions are 10W for 2 minutes; the water bath conditions are heating at 60℃ for 30 minutes; and the centrifugation conditions are centrifugation at 11000×g for 30 minutes.

[0028] In the wastewater treatment process of this invention, the mass ratio of added EPS to DBP in the wastewater is 1–1000:1, preferably 2–800:1; more preferably, when the EPS is total EPS, the mass ratio of added EPS to DBP in the wastewater is 100–200:1; when the EPS is S-EPS, the mass ratio of added EPS to DBP in the wastewater is 700–800:1; when the EPS is LB-EPS, the mass ratio of added EPS to DBP in the wastewater is 30–40:1; when the EPS is TB-EPS, the mass ratio of added EPS to DBP in the wastewater is 2–3:1. The treatment temperature is 18–25℃, preferably 20–22℃; the pH value is 7.0–7.3, preferably 7.1–7.2; and the DBP concentration in the wastewater is 2–10 ppm, preferably 5–8 ppm.

[0029] The present invention adds biochar during the wastewater treatment process, wherein the amount of biochar added is 0.1-0.3 g / mL, preferably 0.2 g / mL.

[0030] 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.

[0031] In a specific embodiment of the present invention, *Gordonella xiwahuensis* was purchased from the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 4.2184. The liquid culture medium for *Gordonella xiwahuensis* consisted of: 4g yeast extract, 10g malt extract, 4g glucose, 1.0L distilled water, and pH adjusted to 7.3.

[0032] In a specific embodiment of the present invention, the method for preparing biochar is as follows: dried rice straw is pulverized and placed in a high-temperature muffle furnace, with a heating rate set at 20°C / min. -1 The mixture was pyrolyzed at a constant temperature of 550℃ for 1 hour, during which nitrogen (N2) was introduced to limit oxygen flow at a flow rate of 8 mL / min. -1 After the process is completed, the mixture is cooled to room temperature and removed. The prepared powder is then ground in a mortar and pestle and passed through a 120-mesh sieve for storage.

[0033] Unless otherwise specified, the following embodiments are all conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] Example 1

[0036] The *Gordonella xiwahuensis* strain was activated on solid culture medium: 500 ml of solid culture medium (5 g malt extract, 2 g yeast, 2 g glucose, 7.5 g agar) was prepared, pH adjusted to 7.3, and dispensed into Petri dishes. After solidification, *Gordonella xiwahuensis* was inoculated and cultured in an incubator (28℃) for 48 hours to activate the strain. Using an inoculation loop, the *Gordonella xiwahuensis* strain from the solid culture medium was transferred to sterilized liquid culture medium. The liquid culture medium was then incubated on a shaker (28℃, 160 rpm) for 36–48 hours until the bacteria reached the stationary phase. Subculturing was continued in a clean bench by adding 20 mL of bacterial culture medium to 100 mL of liquid culture medium.

[0037] EPS secreted by *Gordonella xiwahuensis* was extracted from the culture medium (OD600 value of 0.924).

[0038] (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 obtain the supernatant, which is the total EPS.

[0039] (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.

[0040] Experiments were conducted using the total EPS, S-EPS, LB-EPS, and TB-EPS extracted above:

[0041] Experiment 1: Seven centrifuge tubes containing 10 ml of S-EPS solution (concentration 1900 ppm) were prepared, with three control groups set up for each tube. 0.1 ml of DBP (concentration 250 ppm) was added to each tube, bringing the DBP concentration in the reaction system to 2.48 ppm. Hexane was added and centrifuged at 0, 2, 4, 6, 10, 15, and 20 hours after the reaction. Since DBP is soluble in hexane, the DBP extracted by hexane was the undegraded DBP by S-EPS. The extracted solution was analyzed by GC-MS to determine the residual amount of DBP in the solution, thus determining the amount of DBP degraded by S-EPS.

[0042] Experiment 2: Seven centrifuge tubes containing 10 ml of LB-EPS solution (concentration 98 ppm) were prepared, with three control groups set up for each tube. 0.1 ml of 250 ppm DBP was added to each tube, bringing the DBP concentration in the reaction system to 2.48 ppm. Hexane was added and centrifuged at 0, 2, 4, 6, 10, 15, and 20 hours after the reaction. Since DBP is soluble in hexane, the DBP extracted by hexane was the undegraded DBP by LB-EPS. The extracted solution was analyzed by GC-MS to determine the residual amount of DBP in the solution, thus determining the amount of DBP degraded by LB-EPS.

[0043] Experiment 3: Seven centrifuge tubes containing 10 ml of TB-EPS solution (7 ppm concentration) were prepared, with three control groups set up in each tube. 0.1 ml of 250 ppm DBP was added to each tube, bringing the 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. The extracted solution was analyzed by GC-MS to determine the residual amount of DBP in the solution, thus determining the amount of DBP degraded by TB-EPS.

[0044] The results of experiments 1-3 are shown in Table 1.

[0045] Table 1 Comparison of the degradation effects of three different EPS on DBP

[0046]

[0047] The results showed that S-EPS, LB-EPS, and TB-EPS all exhibited highly efficient degradation of DBP within 20 hours, with a degradation rate of about 50%, while TB-EPS showed a degradation rate of 93%.

[0048] Experiment 4: Seven centrifuge tubes containing 10 ml of total EPS solution (concentration 1980 ppm) were prepared, with three control groups set up for each tube. Additionally, 0.1 ml of DBP (concentration 1000 ppm) was added to each tube, bringing the DBP concentration in the reaction system to 9.90 ppm. Hexane was added and centrifuged at 0, 2, 4, 6, 10, 15, and 20 hours of reaction. Since DBP is soluble in hexane, the DBP extracted by hexane represents the undegraded DBP from the total EPS. The extracted solutions were analyzed by GC-MS to determine the residual DBP content, thus determining the amount of DBP degraded by the total EPS. The results are shown in Table 2. The degradation of DBP by EPS tended to stabilize after approximately 15 hours.

[0049] Table 2. Degradation effect of total EPS on DBP

[0050]

[0051] Experiment 5: Two sets of experiments were set up for comparison. 10 mL of S-EPS solution (concentration 1900 ppm) was added to 40 mL centrifuge tubes. One set added 0.02 g of biochar, and the other did not add biochar (Experiment 1). Simultaneously, 0.1 mL of DBP contaminant at a concentration of 250 ppm was added to both sets of solutions, making the DBP concentration in the reaction system 2.48 ppm. Degradation products were extracted at 0 h, 2 h, 4 h, 6 h, 10 h, 15 h, and 20 h for high-performance liquid chromatography (HPLC) analysis. Extraction method: First, 20 mL of cyclohexane was added and mixed well, then sonicated for 10 minutes to break the emulsion. After extraction, another 10 mL of cyclohexane was added, and the above operation was repeated. After extraction, nitrogen purging was performed, followed by the addition of 0.5 mL of cyclohexane to dissolve the product. The solution was filtered through a 0.45 μm filter membrane, and GC-MS was used for analysis.

[0052] Experiment 6: Two sets of experiments were set up for comparison. 10 mL of LB-EPS solution (concentration 98 ppm) was added to 40 mL centrifuge tubes. One set added 0.02 g of biochar, and the other did not add biochar (Experiment 2). Simultaneously, 0.1 mL of DBP contaminant at a concentration of 250 ppm was added to both sets of solutions, making the DBP concentration in the reaction system 2.48 ppm. Degradation products were extracted at 0 h, 2 h, 4 h, 6 h, 10 h, 15 h, and 20 h for high-performance liquid chromatography (HPLC) analysis. Extraction method: First, 20 mL of cyclohexane was added and mixed well, then sonicated for 10 minutes to break the emulsion. After extraction, another 10 mL of cyclohexane was added, and the above operation was repeated. After extraction, nitrogen purging was performed, followed by the addition of 0.5 mL of cyclohexane to dissolve the product. The solution was filtered through a 0.45 μm filter membrane, and GC-MS was used for analysis.

[0053] Experiment 7: Two sets of experiments were set up for comparison. 10 mL of TB-EPS solution (7 ppm concentration) was added to 40 mL centrifuge tubes. One set added 0.02 g of biochar, and the other did not add biochar (Experiment 3). Simultaneously, 0.1 mL of DBP contaminant at a concentration of 250 ppm was added to both sets of solutions, making the DBP concentration in the reaction system 2.48 ppm. Degradation products were extracted at 0 h, 2 h, 4 h, 6 h, 10 h, 15 h, and 20 h for high-performance liquid chromatography (HPLC) analysis. Extraction method: First, 20 mL of cyclohexane was added and mixed well, then sonicated for 10 minutes to break the emulsion. After extraction, another 10 mL of cyclohexane was added, and the above operation was repeated. After extraction, nitrogen purging was performed, followed by the addition of 0.5 mL of cyclohexane to dissolve the product. The solution was filtered through a 0.45 μm filter membrane, and GC-MS was used for analysis.

[0054] The results of experiments 5-7 are shown in Table 3.

[0055] Table 3 Comparison of the degradation effects of biochar mixed with different types of EPS on DBP

[0056]

[0057] The results showed that by combining the application of *Gordonella asiatica* EPS from Xiwa Lake with biochar, the two exhibited a synergistic degradation effect, which could effectively improve the degradation rate of DBP in the water.

[0058] Experiment 8: Two sets of experiments were set up for comparison. 10 mL of total EPS solution (concentration 1980 ppm) was added to 40 mL centrifuge tubes. One set added 0.02 g of biochar, and the other did not add biochar (Experiment 4). Simultaneously, 0.1 mL of DBP contaminant at a concentration of 1000 ppm was added to both sets of solutions, making the DBP concentration in the reaction system 9.90 ppm. Degradation products were extracted at 0 h, 2 h, 4 h, 6 h, 10 h, 15 h, and 20 h for high-performance liquid chromatography (HPLC) analysis. Extraction method: 20 mL of cyclohexane was added and mixed, then sonicated for 10 minutes to break the emulsion. After extraction, another 10 mL of cyclohexane was added, and the above operation was repeated. After extraction, nitrogen purging was performed, followed by the addition of 0.5 mL of cyclohexane to dissolve the product. The solution was filtered through a 0.45 μm filter membrane and analyzed by GC-MS. The results of Experiment 8 are shown in Table 4.

[0059] Table 4 Comparison of the degradation effects of biochar and total EPS on DBP

[0060]

[0061] The results showed that by combining total EPS from *Gordonella oryzae* in Xiwa Lake with biochar, the two exhibited a synergistic degradation effect, which could effectively improve the degradation rate of DBP in the water.

[0062] 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 combined application of *Gordonella asiatica* EPS and biochar in degrading plasticizers, characterized in that... EPS is any one of total EPS, S-EPS, LB-EPS or TB-EPS, and the plasticizer is DBP; The ratio of *Gordonella asiatica* EPS to biochar in the Xiwa Lake strain was 8-12 mL: 0.001-0.003 g. The method for extracting total EPS includes: after sonicating and heating the cultured *Gordonella xiwahuensis* liquid culture medium in a water bath, centrifuging it to obtain the supernatant as total EPS; The extraction method of S-EPS includes: centrifuging the cultured *Gordonella xiwahuensis* liquid culture medium to obtain the supernatant as S-EPS; The extraction method of LB-EPS includes: adding 0.05% sodium chloride solution to the precipitate after extracting S-EPS, mixing and sonicating, centrifuging, and the supernatant is LB-EPS; The extraction method of TB-EPS includes: adding 0.05% sodium chloride solution to the precipitate after LB-EPS extraction, mixing and sonicating, heating in a water bath, and then centrifuging, with the supernatant being TB-EPS.

2. A method for degrading DBP in wastewater using the combined application according to claim 1, characterized in that, Includes the following steps: EPS was extracted from the culture medium of *Gordonella argyrophylla* in Xiwa Lake and then added to the wastewater for treatment. Biochar is added to the wastewater at the same time.

3. The method according to claim 2, characterized in that, The mass ratio of EPS added to DBP in the wastewater is 1~1000:1; the treatment temperature is 18~25℃, and the pH value is 7.0~7.

3.

4. The method according to claim 2, characterized in that, The concentration of DBP in the wastewater is 2-10 ppm.

5. The method according to claim 2, characterized in that, The amount of biochar added is 0.001~0.003 g / mL.

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