Composite flora for specifically degrading antioxidant 168 as well as screening method and application of composite flora

Abstract

The invention discloses a composite flora capable of specifically degrading an antioxidant 168 as well as a screening method and application of the composite flora. The composite flora comprises serratia sp. LY-1 and bacillus pacificus, wherein the volume ratio of a single bacterium suspension of the serratia sp. LY-1 to a single bacterium suspension of the bacillus pacificus is 1: 1. The screening method comprises the following steps: respectively inoculating a sludge sample containing microorganisms into an inorganic salt culture medium containing an antioxidant 168 and bis (2, 4-di-tert-butylphenyl) hydrogen phosphate as a carbon source and an energy source, and carrying out enrichment culture; and gradually separating out the bacterial strain for degrading the antioxidant 168 and bis (2, 4-di-tert-butylphenyl) hydrogen phosphate by a gradient dilution method. The composite flora capable of specifically degrading the antioxidant 168 can be used for efficiently degrading the antioxidant 168.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composite bacterial community capable of specifically degrading antioxidant 168, a screening method and application thereof. Background Art

[0002] Antioxidant 168 is a widely used antioxidant that belongs to the family of organophosphites. This compound is widely used in the manufacture of various polymers, such as polyethylene, polypropylene, polyester, polyamide, and polyvinyl chloride. It is often used as a stabilizer in the production of plastics, synthetic rubber, and other easily oxidized materials. Antioxidant 168 is a commonly used organophosphite antioxidant in various industrial applications, such as plastics, rubbers, adhesives, and lubricants. Despite its usefulness, the widespread use of antioxidant 168 has led to its widespread presence in the environment, resulting in potentially significant negative impacts on human health and ecosystems. Antioxidant 168 is known to be persistent in the environment, can accumulate in soil, sediments, and water systems, and bioaccumulate in aquatic organisms, leading to potential toxicity.

[0003] Antioxidant 168 (tris(2,4-di-tert-butylphenyl) phosphite) is a common antioxidant widely used in the polyolefin industry to prevent polymer aging and oxidation during processing. However, Antioxidant 168 may degrade into several degradation products such as 2,4-di-tert-butylphenol, 1,3-di-tert-butylbenzene, tris(2,4-di-tert-butylphenyl) phosphate (oxidized form of Antioxidant 168).

[0004] In addition, antioxidant 168 can undergo photochemical degradation, resulting in the formation of reactive intermediates that can harm living organisms. However, the persistence and potential toxicity of antioxidant 168 in the environment have raised concerns about its impacts on human health and ecosystems. The potential negative impacts of antioxidant 168 on the environment have led to increased research interest in its fate, migration, and impacts on ecosystems. Several studies have reported the detection of antioxidant 168 in various environmental matrices, including soil, sediment, and water systems. For example, a study by Zhang et al. (2021) reported that antioxidant 168 was widely present in soil and sediment samples collected from the Xiangjiang River in China. Similarly, a study by Jani et al. (2019) reported that antioxidant 168 was detected in several fish tissues collected from the Mediterranean Sea. The widespread use of antioxidant 168 in various industrial applications has led to its widespread presence in the environment, which may have negative effects on human health and ecosystems. Therefore, how to degrade antioxidant 168 has become one of the issues that need to be urgently addressed. Summary of the invention

[0005] The purpose of the present invention is to provide a composite bacterial community that can specifically degrade antioxidant 168, and a screening method and application thereof, which can degrade antioxidant 168 and its metabolites.

[0006] In one aspect of the present invention, the present invention provides a composite bacterial community that specifically degrades antioxidant 168. According to an embodiment of the present invention, the composite bacterial community includes Serratia sp.LY-1 and Bacillus pacificus in a single bacterial suspension volume ratio of 1:1.

[0007] In another aspect of the present invention, the present invention provides a method for screening a composite bacterial community that specifically degrades antioxidant 168. According to an embodiment of the present invention, the method comprises the following steps:

[0008] The sludge samples containing microorganisms were inoculated in inorganic salt medium containing antioxidant 168 and di-2,4-di-tert-butylphenyl hydrogen phosphate for enrichment culture; then the strains for the purpose of degrading antioxidant 168 and di-2,4-di-tert-butylphenyl hydrogen phosphate were gradually separated by gradient dilution method, and these colonies were further characterized and identified based on their appearance and color. The streak plate method was used to separate and obtain pure cultures of bacteria from the mixed population. After cultivation, the growth of single bacterial colonies on the plate was observed, and then similar colonies were identified and separated. The strains that efficiently degrade antioxidant 168 and di-2,4-di-tert-butylphenyl hydrogen phosphate were compounded with a single bacterial suspension volume ratio of 1:1 to construct a synthetic bacterial community and used to degrade antioxidant 168.

[0009] In addition, the screening method for a composite bacterial community that specifically degrades antioxidant 168 according to the above embodiment of the present invention may also have the following additional technical features:

[0010] In some embodiments of the present invention, the sewage sample needs to be pretreated before inoculation, and the treatment method is as follows: PBS buffer is added to the sewage sample for mixing, oscillation and centrifugation are performed, and the supernatant is poured out. The process is repeated multiple times to completely remove organic matter in the sample.

[0011] In some embodiments of the present invention, the method for preparing an inorganic salt culture medium containing an antioxidant 16 is as follows: 2.5008 g / L KH2PO4, 1.5003 g / L K2HPO4, 1.0001 g / L NH4Cl, and 0.5002 g / L MgSO4·7H2O are dissolved in 1 liter of distilled water to prepare a mineral salt solution, and the pH value is adjusted to 7.0. After adjusting the pH value, trace elements and antioxidant 168 are added.

[0012] In some embodiments of the present invention, the antioxidant 168 is added in an amount of 10 mg / L.

[0013] In some embodiments of the invention, NaOH is used to adjust the pH.

[0014] In another aspect of the present invention, the present invention proposes an application of the composite bacterial community for degrading specific antioxidant 168 in degrading antioxidant 168 in sewage. According to an embodiment of the present invention, the composite bacterial community is added to sewage and stirred evenly.

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

[0016] Microbial degradation of antioxidant 168 has not been reported. The present invention uses a composite bacterial strain to degrade antioxidant 168, which can degrade 100% of antioxidant 168 within 72 hours, while a single bacterial strain that degrades antioxidant 168 can only degrade 60% of antioxidant 168. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the evolutionary tree of the 16SrRNA gene sequence of the best degradation bacteria (Serratia) of antioxidant 168 in Example 2 of the present invention;

[0018] Figure 2 is the mass spectrum of the antioxidant 168 after 7 days of inoculation with Serratia in Example 4 of the present invention;

[0019] Figure 3 is a schematic diagram of the biodegradation pathway of antioxidant 168 in Example 4 of the present invention;

[0020] Figure 4 is a graph showing the degradation rate of the antioxidant 168 degraded by the composite bacterial community in Example 6 of the present invention;

[0021] Figure 5 This is a graph showing the degradation rate of antioxidant 168 in actual wastewater degraded by the composite bacterial community in Example 7 of the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1

[0024] A method for screening a composite bacterial community that specifically degrades antioxidant 168 comprises the following steps:

[0025] (1) Sample collection

[0026] Sludge and wastewater were collected from the aerobic pool of the Dongzhi Chemical Industrial Park Wastewater Treatment Plant. Sludge and wastewater were stored in refrigerated containers and transported to the laboratory. The samples were taken out and frozen in a -80°C refrigerator and then placed in a 4°C refrigerator overnight. After all samples were thawed, an appropriate amount of PBS buffer was added to the thawed samples for mixing and vortexed at 3200 rpm. The mixed sample was placed on a vortex oscillator for 5 minutes and then centrifuged in a high-speed centrifuge (12000 rpm) for 5 minutes. Gently remove the sample and pour out the supernatant. This process was repeated several times to completely remove the organic matter in the sample. The treated sludge sample was resuspended in PBS buffer.

[0027] (2) Culture medium

[0028] Antioxidant 168-degrading bacteria were isolated using the selective culture enrichment technique in mineral salt medium (MSM). Sludge was enriched in a mineral salt medium (pH 7) containing antioxidant 168 as the sole carbon source. The mineral salt solution was prepared by dissolving the following salts in 1 liter of distilled water: KH2PO4 (2.5008 g / L), K2HPO4 (1.5003 g / L), NH4Cl (1.0001 g / L), MgSO4·7H2O (0.5002 g / L). After preparing the mineral salt medium, the pH was measured and adjusted to 7.0 with NaOH. After adjusting the pH, 100 ml of the mineral salt solution was poured into nine 250 ml conical flasks and 0.1 ml of trace elements were added to each conical flask. The amount of antioxidant 168 added was 10 mg / L.

[0029] (3) Strain isolation method

[0030] The treated sludge sample containing microorganisms (1) was inoculated in an inorganic salt medium. 1 mL of sludge was added to each conical flask, leaving one as a blank sample. The inorganic salt solution was then placed in a shaking incubator at 37°C and shaken at 120 rpm. After one week in the shaking incubator, repeated enrichment was performed and the enriched culture was placed in a shaking incubator.

[0031] After the first microbial enrichment was performed in an orbital shaker (120 rpm) at 37°C for 1 week, a 5 mL aliquot of the enrichment culture was inoculated into another 250 mL conical flask containing 100 mL of fresh MSM containing the same amount of antioxidant 168 for the second enrichment. After four consecutive enrichments, the culture with a series of concentration gradients was inoculated on MSM agar plates. The bacterial consortium was isolated and purified on nutrient agar plates by conventional spread plate technology. The plates were cultured at 37°C.

[0032] After enrichment, further cultivation of the microorganisms was performed to isolate bacteria capable of degrading antioxidant 168. Further cultivation was performed using the inverted plate technique, and pure colonies were obtained by repeated streaking. After a period of cultivation (48 hours), the culture plate was removed from the incubator. The agar surface was then checked for the presence of microbial colonies. These colonies were further characterized and identified based on their appearance and color. Pure cultures of bacteria were isolated and obtained from a mixed population using the streak plate method. After cultivation, the growth of individual bacterial colonies on the plate was observed. Similar colonies were then identified and isolated.

[0033] Example 2

[0034] The strains isolated from Example 1 were identified:

[0035] Using a sterile loop, carefully pick individual microbial colonies from the plate and transfer them to a small tube containing 2 ml of glycerol; they are then cryopreserved in glycerol. After cryopreservation, they are revived and shipped to the testing center for analysis and identification of the microorganisms (bacteria). The 16S rRNA gene was obtained by PCR amplification using universal bacterial primers 27F and 1492R. For PCR, the reaction was set at 95°C denaturation for 5 minutes, followed by 30 cycles of 95°C for 45 seconds, 55°C for 45 seconds, 72°C for 90 seconds, and a final step of 72°C for 5 minutes and extension at 72°C for 6 minutes. The resulting sequences were compared with the RNA sequences available in GenBank using the Basic Local Alignment Search Tool (BLAST).

[0036] Table 1 Sequencing of antioxidant 168-degrading bacteria Serratia sp.

[0037] bacteria Homology (%) GenBank Sequence ID Serratiasp.LY-1 99.65％ OR393043

[0038] Example 3

[0039] Determination of the degradation rate of antioxidant 168 by Serratia sp.LY-1 alone:

[0040] 1 ml of the activated Serratia sp.LY-1 bacterial suspension was inoculated into 100 mL of MSM medium with Irgafos 168 as the sole carbon source (concentration of 10 mg / L) and cultured at 37°C at 150 rpm for 7 days. A sample (1 mL) was taken out after 12 hours and the degradation rate was determined by high performance liquid chromatography (HPLC) within 7 days. The antioxidant 168 sample was centrifuged (10 minutes, 10,000 rpm) to separate the cell mass and the supernatant. The sample was extracted using an inorganic solvent (n-hexane) for analysis. Chromatographic separation was performed using an HPLC system with a mobile phase consisting of methanol at a flow rate of 1.5 mL / min, a Varian C-18 column (250 mm × 4.6 mm), a column temperature of 25°C, and a UV detector set to 210 nm.

[0041] Serratia sp.LY-1 could degrade antioxidant 168, with the degradation rates being 60.33% in 72 hours and 90.17% in 144 hours.

[0042] Table 2 Degradation rate of antioxidant 168 by Serratia

[0043] bacteria Degradation rate on the third day Degradation rate on the 7th day Serratiasp.LY-1 60.33％ 90.17％

[0044] Example 4

[0045] The metabolites obtained by degradation of antioxidant 168 were identified:

[0046] The samples were analyzed in a gas chromatography-mass spectrometer (GC-MS) (Agilent Technologies, USA) equipped with a HP-5MS capillary column (30.0 m x 250 m x0.25 m) in a mass spectrometer (Agilent 7890B ion trap MS) equipped with a manual injector containing a split / splitless capillary injection system and an array detector with a total scan of 30-500 nm. The column temperature was initially maintained at 80°C for 5 minutes, then raised to 200°C at 80°C for 5 minutes, and then raised to 260°C at 150°C for 5 minutes. The ionization energy was maintained at 70 eV, and the temperatures of the transmission line and ion trap were maintained at 280°C and 230°C, respectively. In the 1:50 split mode, the injection volume at 250°C was 1.0 μL of the n-hexane extract in Example 3. Helium was used as the carrier gas at a flow rate of 1.5 ml / min. During the degradation process, antioxidant 168 was decomposed into degradation products, which were then identified using gas chromatography-mass spectrometry.

[0047] like Figure 2 As shown in the ion chromatography, the main degradation products are di-2,4-di-tert-butylphenyl hydrogen phosphate and 2,4-di-tert-butylphenol. Figure 3 As shown, the degradation pathway of antioxidant 168 by Serratia sp. LY-1 is that Serratia sp. LY-1 strain decomposes antioxidant 168 into di-2,4-di-tert-butylphenyl hydrogen phosphate and 2,4-di-tert-butylphenol.

[0048] Example 5

[0049] The screening method for the degradation bacteria of the metabolite bis-2,4-di-tert-butylphenyl hydrogen phosphate is the same as that of Example 1, except that the same concentration of bis-2,4-di-tert-butylphenyl hydrogen phosphate is used instead of the antioxidant 168. The strains obtained by screening were identified, and the results are shown in Table 3.

[0050] Table 3 Sequencing of double 2,4-di-tert-butylphenyl hydrogen phosphate degrading bacteria Bacillus pacificus sp

[0051] bacteria Homology (%) GenBank Sequence ID Bacillus pacificus sp. 99.65％ CP041979

[0052] Degradation rate of the strain obtained in Example 5:

[0053] 1 ml of the activated Bacillus pacificus. bacterial suspension was inoculated into 100 mL of MSM medium with bis-2,4-di-tert-butylphenyl hydrogen phosphate as the sole carbon source (concentration of 10 mg / L) and cultured at 37°C with continuous shaking at 150 rpm for 7 days. Samples (1 mL) were taken out after 12 hours and the degradation rate was determined by high performance liquid chromatography (HPLC) within 7 days. The bis-2,4-di-tert-butylphenyl hydrogen phosphate sample was centrifuged (10 min, 10,000 rpm) to separate the cell mass and the supernatant. The sample was extracted with an inorganic solvent (acetonitrile) for analysis. Chromatographic separation was performed using an HPLC system with a mobile phase consisting of methanol and water (80:20; v / v) at a flow rate of 1.0 mL / min, a Varian C-18 column (250 mm × 4.6 mm), a column temperature of 25°C, and a UV detector set at 230 nm.

[0054] The strain was able to degrade bis-2,4-di-tert-butylphenyl hydrogen phosphate at a concentration of 10 ppm. The degradation percentage was calculated from the HPLC data. The calculated degradation percentage of bis-2,4-di-tert-butylphenyl hydrogen phosphate was the highest, and it was able to degrade bis-2,4-di-tert-butylphenyl hydrogen phosphate by 83% after 7 days. After 7 days of cultivation under the same experimental conditions, there was almost no change in the sterile control group.

[0055] Table 4 Degradation rate of Bacilluspacificus sp. on bis-2,4-di-tert-butylphenyl hydrogen phosphate

[0056] bacteria Degradation rate on the third day Degradation rate on the 7th day Bacillus pacificus sp. 55.68％ 83.24％

[0057] Example 6

[0058] The composite bacterial community includes Serratia sp.LY-1 and Bacillus pacificus in a single bacterial suspension volume ratio of 1:1, and 1 ml of the composite bacterial solution is inoculated into 100 mL of MSM medium with Irgafos 168 as the sole carbon source (concentration of 10 mg / L), and is continuously shaken at 150 rpm for 3 days at 37°C. After 24 hours, a sample (1 mL) is taken out and the degradation rate is determined by high performance liquid chromatography (HPLC) within 3 days. The extraction of the test sample and the high performance liquid chromatography conditions are consistent with those in Example 3.

[0059] like Figure 4 As shown, the composite bacterial community can efficiently degrade antioxidant 168, with a degradation rate of 75% in 48 hours and 100% in 72 hours.

[0060] Example 7

[0061] The composite bacterial consortium constructed in Example 6 was used to degrade Irgafos 168 as follows: the composite bacterial solution was inoculated into wastewater collected from a sewage treatment plant at a volume ratio of 2% to degrade Irgafos 168; the bacterial solution was transferred to 10 ml LB medium for overnight shake flask culture and cultured until the bacterial concentration was OD 600 =1; that is, in the wastewater collected from the sewage treatment plant, the bacterial solution was inoculated at a volume ratio of 2% and cultured for three days, and samples were taken at 0h, 24h, 48h, and 72h to determine the degradation performance of the strain on Irgafos 168; the concentration of Irgafos 168 in the wastewater was determined according to the detection method of Implementation Case 3.

[0062] like Figure 5 As shown, the strain has a good degradation effect on Irgafos 168 with a concentration of 7.2 mg / L in actual wastewater. It can remove 88% of Irgafos 168 within 48 hours and reach a degradation rate of 100% within 72 hours. This has important practical significance for the development of efficient microbial agents for degrading organic pollutants and their application in wastewater treatment.

[0063] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A composite bacterial community that specifically degrades antioxidant 168, characterized in that: The composite bacterial community comprises Serratia sp.LY-1 and Bacillus pacificus in a single bacterial suspension volume ratio of 1:

1.

2. A method for screening a composite bacterial community that specifically degrades antioxidant 168, characterized in that: The following steps are involved: The sludge samples containing microorganisms were inoculated into inorganic salt culture media containing antioxidant 168 and bis-2,4-di-tert-butylphenyl hydrogen phosphate for enrichment culture; then the strains for the purpose of degrading antioxidant 168 and bis-2,4-di-tert-butylphenyl hydrogen phosphate were gradually separated by the gradient dilution method, and these colonies were further characterized and identified based on their appearance and color. The streak plate method was used to separate and obtain pure cultures of bacteria from the mixed population. After culture, the growth of single bacterial colonies on the plate was observed, and then similar colonies were isolated and identified.

3. The method for screening a composite bacterial flora capable of specifically degrading antioxidant 168 according to claim 2, characterized in that: The sludge sample needs to be pretreated before inoculation. The treatment method is as follows: PBS buffer is added to the sludge sample for mixing, oscillation and centrifugation are performed, and the supernatant is poured out. This process is repeated multiple times to completely remove organic matter in the sample.

4. The method for screening a composite bacterial flora capable of specifically degrading antioxidant 168 according to claim 2, characterized in that: The preparation methods of inorganic salt culture media containing antioxidant 168 and bis-2,4-di-tert-butylphenyl hydrogen phosphate are as follows: 2.5008 g / L KH2PO4, 1.5003 g / L K2HPO4, 1.0001 g / L NH4Cl, and 0.5002 g / L MgSO4·7H2O are dissolved in 1 liter of distilled water to prepare a mineral salt solution, the pH value is adjusted to 7.0, and after adding trace elements, antioxidant 168 and bis-2,4-di-tert-butylphenyl hydrogen phosphate are added respectively.

5. The method for screening a composite bacterial flora capable of specifically degrading antioxidant 168 according to claim 4, characterized in that: The added amount of the antioxidant 168 is 10 mg / L.

6. The method for screening a composite bacterial flora capable of specifically degrading antioxidant 168 according to claim 4, characterized in that: The pH was adjusted using NaOH.

7. An application of the composite bacterial community for specific degradation of antioxidant 168 according to claim 1 in degradation of antioxidant 168 in sewage, characterized in that: Add the composite bacterial flora into the sewage and stir evenly.

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