Phenol high-efficiency degradation related bacterial flora and application thereof

Abstract

The application discloses phenol high-efficiency degradation related strains and application thereof, and discloses strains with phenol degradation capacity, namely, Citrobacter farmeri with a preservation number of CGMCC No. 25400, Acinetobacter lwoffi with a preservation number of CGMCC No. 25399, Acidovorax soli with a preservation number of CGMCC No. 25478 and Diaphorobacter nitroreducens with a preservation number of CGMCC No. 25438. A method for degrading phenol by using the above strains is disclosed. The strains and the degradation method have the advantages of high efficiency, low consumption, simple operation, various treatment forms, less influence on the environment and no secondary pollution, and lay a foundation for the biological degradation of phenol.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to strains related to the efficient degradation of phenol and their applications. Background Technology

[0002] Phenol is a colorless, needle-like crystal with a characteristic odor. It is miscible with chloroform, ethanol, glycerol, and ether, and possesses weak acidity and reducing properties. Classified as highly toxic, it is an important raw material in the chemical, pharmaceutical, and agricultural industries, and is used in the production of certain bactericides, preservatives, drugs (such as aspirin), and resins. Phenol was first isolated from coal tar by the German chemist Runge in 1834, hence it is also known as carbolic acid. With the increasing demand for phenol across various industries, my country's phenol imports and consumption have remained at high levels. Phenol has become one of the major pollutants in many industrial wastewaters, primarily originating from coke ovens, steelmaking, the chemical industry, pharmaceuticals, and resin production. During phenol use, problems such as excessive phenol usage, phenol leakage and seepage, and improper treatment of phenol-containing wastewater have led to phenol pollution. As a carcinogenic, teratogenic, and mutagenic organic compound, phenol can severely impact aquatic organisms even at extremely low concentrations, and this harm becomes increasingly pronounced with increasing concentration. Phenolic wastewater is highly toxic, and its direct discharge into open water bodies can cause serious environmental and ecological problems. Furthermore, long-term human exposure to phenol can lead to chronic phenol poisoning. Phenol poisoning can occur through skin absorption, inhalation, ingestion, and other routes, thus affecting health. Therefore, removing phenol from water bodies is crucial for both environmental protection and human health.

[0003] Phenol is a highly mobile pollutant that readily migrates from contaminated sites into groundwater, making it a critical concern in contaminated site remediation. With increasing attention being paid to groundwater pollution and in-depth research, groundwater remediation technologies have made significant progress. Currently, widely used remediation technologies can be categorized into in-situ and ex-situ remediation methods. Ex-situ remediation involves extracting and treating the groundwater, including extraction treatment technologies and multiphase extraction technologies. However, these technologies are expensive, waste water resources significantly, damage the original natural environment, and do not address the root cause of the pollution, while also having a limited treatment range. In-situ remediation technologies commonly include permeable reactive barrier technology and in-situ aeration technology, but these face risks such as high cost of reaction media, concerns about reactivity and physicochemical stability, limited lifespan, and secondary pollution. Furthermore, aeration remediation places high demands on the geological environment of the groundwater. Summary of the Invention

[0004] The technical problem this application aims to solve is how to efficiently degrade phenol, and in particular, to provide a bacterial community related to the efficient degradation of phenol.

[0005] To address the aforementioned issues, this application provides a microbial agent for degrading phenol.

[0006] The microbial agent is a 1+2+3+4 microbial agent, a 1+3+4 microbial agent, a 1+3 microbial agent, a 1+2+3 microbial agent, a 1+2+4 microbial agent, a 1+2 microbial agent, a 1+4 microbial agent, or a 1-microbial agent;

[0007] The active ingredients of the 1+2+3+4 bacterial agent are composed of Acinetobacter lwoffii BF1, Citrobacter farmeri BF2, Acidovorax soli BF3 and Diaphorobacter nitroreducens BF4.

[0008] The active ingredients of the 1+3+4 bacterial agent consist of Acinetobacter lwoffii BF1, Acidovorax soli BF3 and Diaphorobacter nitroreducens BF4;

[0009] The active ingredients of the 1+3 bacterial agent are composed of Acinetobacter lwoffii BF1 and Acidovorax soli;

[0010] The 1+2+3 bacterial agent is composed of Acinetobacter lwoffii BF1, Citrobacter farmeri BF2 and Acidovorax soli BF3.

[0011] The 1+2+4 bacterial agent is composed of Acinetobacter lwoffii BF1, Citrobacter farmeri BF2 and Diaphorobacter nitroreducens BF4;

[0012] The 1+2 bacterial agent consists of Acinetobacter lwoffii BF1 and Citrobacter farmeri BF2;

[0013] The active ingredients of the 1+4 bacterial agent consist of Acinetobacter lwoffii BF1 and Diaphorobacter nitroreducens BF4;

[0014] The active ingredient of the bacterial agent is Acinetobacter lwoffii BF1;

[0015] The Acinetobacter lwoffii BF1 strain has the accession number CGMCC No. 25399 from the China General Microbiological Culture Collection Center.

[0016] The Citrobacter farmeri BF2 strain has the accession number CGMCC No. 25400 from the China General Microbiological Culture Collection Center.

[0017] The accession number of Acidovorax soli BF3 at the China General Microbiological Culture Collection Center is CGMCC No. 25478;

[0018] The Diaphorobacter nitroreducens BF4 has the accession number CGMCC No. 25438 from the China General Microbiological Culture Collection Center.

[0019] In the above-mentioned 1+2+3+4 bacterial agent, the ratio of colony-forming units of Acinetobacter lwoffii BF1, Citrobacter farmeri BF2, Acidovorax soli BF3 and Diaphorobacter nitroreducens BF4 is 1:1:1:1.

[0020] In the above-mentioned 1+2+3+4 bacterial agent, Acinetobacter lwoffii BF1 contains 4 × 10 8 Colony-forming unit.

[0021] In the 1+3+4 bacterial agent, the colony-forming unit ratio of Acinetobacter lwoffii BF1, Acidovoraxsoli BF3, and Diaphorobacter nitroreducens BF4 was 5.3 × 10⁻⁶. 8 .

[0022] In the above-mentioned 1+3+4 bacterial agent, Acinetobacter lwoffii BF1 contains 5.3 × 10⁻⁶. 8 Colony-forming unit.

[0023] In the 1+3 bacterial agent, the ratio of Acinetobacter lwoffii BF1 to Acidovorax soli in colony-forming units is 1:1.

[0024] In the above-mentioned 1+3 bacterial agent, Acinetobacter lwoffii BF1 contains 8 × 10⁻⁶ 8 Colony-forming unit.

[0025] In the 1+2+3 bacterial agent, the colony-forming unit ratio of Acinetobacter lwoffii BF1, Citrobacter farmeri BF2, and Acidovorax soli BF3 is 1:1:1;

[0026] In the above-mentioned 1+2+3 bacterial agent, Acinetobacter lwoffii BF1 contains 5.3 × 10⁻⁶. 8 Colony-forming unit.

[0027] In the 1+2+4 bacterial agent, the colony-forming unit ratio of Acinetobacter lwoffii BF1, Citrobacter farmeri BF2, and Diaphorobacter nitroreducens BF4 is 1:1:1;

[0028] In the above-mentioned 1+2+4 bacterial agent, Acinetobacter lwoffii BF1 contains 5.3 × 10⁻⁶. 8 Colony-forming unit.

[0029] In the 1+2 bacterial agent, the colony-forming unit ratio of Acinetobacter lwoffii BF1 and Citrobacter farmeri BF2 is 1:1.

[0030] In the 1+4 bacterial agent, the colony-forming unit ratio of Acinetobacter lwoffii BF1 and Diaphorobacter nitroreducens BF4 is 1:1.

[0031] In the above-mentioned 1+4 bacterial agent, Acinetobacter lwoffii BF1 contains 8 × 10⁻⁶ mmol / L. 8 Colony-forming unit.

[0032] The bacterial agent mentioned above contains Acinetobacter lwoffii BF1.

[0033] In the above text, the bacterial agent contains 16 × 10⁻⁶ Acinetobacter lwoffii BF1. 8 Colony-forming unit.

[0034] To address the aforementioned issues, this application also provides Acinetobacter rumeni.

[0035] The Acinetobacter lwoffii mentioned is Acinetobacter lwoffii BF1, which has the accession number CGMCC No. 25399 at the China General Microbiological Culture Collection Center.

[0036] To address the aforementioned problems, this application also provides a microbial agent for degrading phenol.

[0037] The microbial agent is a 2+3+4 microbial agent, a 2+3 microbial agent, a 2+4 microbial agent, or a 2-microbial agent;

[0038] The active ingredients of the 2+3+4 bacterial agent consist of Citrobacter farmeri BF2, Acidovorax soli BF3 and Diaphorobacter nitroreducens BF4;

[0039] The active ingredients of the 2+3 bacterial agent consist of Citrobacter farmeri BF2 and Acidovorax soli BF3;

[0040] The active ingredients of the 2+4 bacterial agent consist of Citrobacter farmeri BF2 and Diaphorobacter nitroreducens BF4;

[0041] The active ingredient of the two bacterial agents is Citrobacter farmeri BF2;

[0042] The Acinetobacter lwoffii BF1 strain has the accession number CGMCC No. 25399 from the China General Microbiological Culture Collection Center.

[0043] The Citrobacter farmeri BF2 strain has the accession number CGMCC No. 25400 from the China General Microbiological Culture Collection Center.

[0044] The accession number of Acidovorax soli BF3 at the China General Microbiological Culture Collection Center is CGMCC No. 25478;

[0045] The Diaphorobacter nitroreducens BF4 has the accession number CGMCC No. 25438 from the China General Microbiological Culture Collection Center.

[0046] In the above-mentioned 2+3+4 bacterial agent, the ratio of colony-forming units of Citrobacter farmeri BF2, Acidovorax soli BF3 and Diaphorobacter nitroreducens BF4 is 1:1:1.

[0047] In the above-mentioned 2+3+4 bacterial agent, Citrobacter farmeri BF2 contains 5.3 × 10⁻⁶. 8 Colony-forming unit.

[0048] In the above-mentioned 2+3 bacterial agent, the colony-forming unit ratio of Citrobacter farmeri BF2 and Acidovorax soli BF3 is 1:1.

[0049] In the above-mentioned 2+3 bacterial agent, Citrobacter farmeri BF2 contains 8 × 10 8 Colony forming unit

[0050] In the above-mentioned 2+4 bacterial agent, the ratio of Citrobacter farmeri BF2 and Diaphorobacter nitroreducens BF4 to colony-forming units is 1:1.

[0051] In the above-mentioned 2+4 bacterial agent, Citrobacter farmeri BF2 contains 8 × 10 8 Colony forming unit

[0052] In the above text, among the two bacterial agents, Citrobacter farmeri BF2 contains 16 × 10⁻⁶.8 Colony-forming unit.

[0053] To address the aforementioned issues, this application also provides Citrobacter.

[0054] The citrobacter mentioned is Citrobacter farmeri BF2, which has the accession number CGMCC No. 25400 at the China General Microbiological Culture Collection Center.

[0055] To address the aforementioned problems, this application also provides a microbial agent for degrading phenol.

[0056] The microbial agent is a 3+4 microbial agent or a 3-microbial agent;

[0057] The active ingredients of the 3+4 microbial agent consist of Acidovorax soli BF3 and Diaphorobacternitroreducens BF4;

[0058] The active ingredient of the three bacterial agents is Acidovorax soliBF3;

[0059] The Acinetobacter lwoffii BF1 strain has the accession number CGMCC No. 25399 from the China General Microbiological Culture Collection Center.

[0060] The Citrobacter farmeri BF2 strain has the accession number CGMCC No. 25400 from the China General Microbiological Culture Collection Center.

[0061] The accession number of Acidovorax soli BF3 at the China General Microbiological Culture Collection Center is CGMCC No. 25478;

[0062] The Diaphorobacter nitroreducens BF4 has the accession number CGMCC No. 25438 from the China General Microbiological Culture Collection Center.

[0063] In the above text, the ratio of colony-forming units of Acidovorax soli BF3 and Diaphorobacternitroreducens BF4 in the 3+4 bacterial agent is 1:1.

[0064] In the above-mentioned 3+4 bacterial agent, Acidovorax soli BF3 contains 8×10 8Colony-forming unit.

[0065] In the above text, among the three bacterial agents, Acidovorax soli BF3 contains 16 × 10⁻⁶. 8 Colony-forming unit.

[0066] To address the aforementioned issues, this application also provides Acidovorax soli.

[0067] The accession number of Acidovorax soli at the China General Microbiological Culture Collection Center is CGMCC No. 25478.

[0068] To address the aforementioned problems, this application also provides a microbial agent for degrading phenol.

[0069] The bacterial agent is a 4-strain agent; the active ingredient of the 4-strain agent is Diaphorobacter nitroreducens BF4; the accession number of Diaphorobacter nitroreducens BF4 at the China General Microbiological Culture Collection Center is CGMCC No. 25438.

[0070] In the above text, among the four bacterial agents, Diaphorobacter nitroreducens BF4 contains 16 × 10⁻⁶. 8 Colony-forming unit.

[0071] To address the aforementioned issues, this application also provides Diaphorobacter nitroreducens.

[0072] The accession number of Diaphorobacter nitroreducens at the China General Microbiological Culture Collection Center is CGMCC No. 25438.

[0073] In addition to the active ingredients, the aforementioned microbial agents may also include excipients such as water, carbon sources, and / or ammonia sources. Carbon sources are nutrients for microbial growth; they are carbon-containing compounds, including sugars, oils, organic acids and esters, and small-molecule alcohols, which are both fast-acting and slow-acting carbon sources. Hydrogen sources refer to substances that provide the nitrogen element required for microbial nutrition, including peanut meal, soybean meal, yeast powder, peptone, ammonia, ammonium salts, and nitrates, which are both fast-acting and slow-acting hydrogen sources.

[0074] In addition to the active ingredients, the above-mentioned microbial agents may also contain a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, plant material, or a polymer compound; the mineral material may be at least one selected from clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one selected from corn flour, soybean flour, and starch; the polymer compound may be polyvinyl alcohol and / or polyethylene glycol. The liquid carrier may be vegetable oil, mineral oil, or water; the organic solvent may be decane and / or dodecane. In the microbial agent, the active ingredients may exist in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate. The dosage form of the microbial agent may be various, such as liquid, suspension, powder, granules, wettable powder, or water-dispersible granules.

[0075] The above-mentioned microbial agents, Acinetobacter rumeni, Citrobacter, Acidovorax soli, or Diaphorobacter nitroreducens are used in any of the following applications:

[0076] A1) Application of the above materials in the degradation of phenol;

[0077] A2) Application of the above materials in the preparation of degradable phenol products;

[0078] A3) Application of the aforementioned materials in biodegradable phenol

[0079] A4) Application of the above materials in the preparation of biodegradable phenol products;

[0080] A5) Application of the above materials in the degradation of phenol in natural organic matter;

[0081] A6) Application of the above materials in the preparation of phenol products from degraded natural organic matter.

[0082] To address the aforementioned problems, this application also provides a method for culturing the aforementioned Acinetobacter rumeni, Citrobacter, Acidovorax soli, or Diaphorobacter nitroreducens, characterized in that the method includes the step of culturing the strains in a culture medium.

[0083] Microorganisms that act as catalysts for living cells during fermentation include four main categories: bacteria, actinomycetes, yeasts, and molds.

[0084] In the above text, *Citrobacter faecium*, *Acinetobacter rumeni*, *Acidovorax soli*, and *Diaphorobacter nitroreducens* can be the cultured strains.

[0085] The culture step may involve placing the above-mentioned bacterial strain in LB liquid medium and culturing it at 150 r / min and 30°C with shaking until the OD600 is greater than 1.

[0086] The culture time mentioned above can be 38 hours.

[0087] In the above text, the inoculum size for the culture can be 1×10⁻⁶. 8 CFU / ml. The degradation of phenol described above can be carried out in the presence of natural organic matter (NOM). The concentration of NOM can be 0-10 mg C / L. NOM mainly originates from the decomposition residues of plants and animals and is widely present in water bodies, soil, atmosphere, and rock strata. It has significant ecological and environmental importance to the global carbon and nitrogen cycle. Previous studies have investigated the effects of NOM on bacterial communities, finding that NOM can increase the activity of enzymes within bacteria in a short period, and this promoting effect is related to the type and concentration of NOM. In this study, the degradation efficiency of phenol by the degrading bacteria is highly correlated with the relevant enzymes and their activities. Since the Suwannee River natural organic matter provided by the International Humic Acid Association is commonly used in studies related to natural organic matter...

[0088] Beneficial effects

[0089] This invention screened out four strains with the ability to degrade phenol: Citrobacter farmeri (CGMCC No. 25400), Acinetobacter rumenii (CGMCC No. 25399), Acidovorax soli (CGMCC No. 25478), and Diaphorobacter nitroreducens (CGMCC No. 25438).

[0090] Furthermore, the conditions for its degradation of phenol were optimized, and it was found that a high degradation capacity could be maintained at pH 7-8 and salt concentration <0.1%. Moreover, the natural organic matter SRNOM did not affect its degradation rate.

[0091] The strains screened and the methods provided in this application can convert pollutants into non-toxic and harmless carbon dioxide, water or other non-toxic and harmless substances. They are highly efficient and low-consumption, simple to operate, have diverse treatment methods, have minimal impact on the environment and basically do not produce secondary pollution.

[0092] Preservation Instructions

[0093] Bacterial species name: Acinetobacter ruvidii

[0094] Latin name: Acinetobacter lwoffii

[0095] Strain number: BF1

[0096] Depositary institution: China General Microbiological Culture Collection Center

[0097] Abbreviation of depositary institution: CGMCC

[0098] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0099] Deposit date: July 25, 2022

[0100] Registration number in the deposit center: CGMCC No.25399

[0101] Species name: Citrobacter farmeri

[0102] Latin name: Citrobacter farmeri

[0103] Strain number: BF2

[0104] Depositary institution: China General Microbiological Culture Collection Center

[0105] Abbreviation of depositary institution: CGMCC

[0106] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0107] Deposit date: July 25, 2022

[0108] Registration number in the deposit center: CGMCC No.25400

[0109] Species name: Acidovorax soli

[0110] Latin name: Acidovorax soli

[0111] Strain number: BF3

[0112] Depositary institution: China General Microbiological Culture Collection Center

[0113] Abbreviation of depositary institution: CGMCC

[0114] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0115] Deposit date: August 3, 2022

[0116] Registration number in the deposit center: CGMCC No.25478

[0117] The Chinese name of the Acidovorax soli bacterium is acid bacterium.

[0118] Strain name: Diaphorobacter nitroreducens

[0119] Latin name: Diaphorobacter nitroreducens

[0120] Strain number: BF4

[0121] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0122] Collection institution abbreviation: CGMCC

[0123] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0124] Date of preservation: August 3, 2022

[0125] Collection Center Registration Number: CGMCC No. 25438

[0126] The Chinese name for the bacteria is *Diaphorobacter nitroreducens*. Attached Figure Description

[0127] Figure 1 This is the phylogenetic tree of strain BF1.

[0128] Figure 2 This is the phylogenetic tree of strain BF2.

[0129] Figure 3 This is the phylogenetic tree of strain BF3.

[0130] Figure 4 This is the phylogenetic tree of strain BF4.

[0131] Figure 5 This is the standard curve for phenol.

[0132] Figure 6 The values ​​represent the degradation rate of a single bacterial species, with circles representing strain BF1, triangles representing strain BF2, interpolations representing strain BF3, and squares representing strain BF4.

[0133] Figure 7 The degradation rate changes between the two bacterial strains.

[0134] Figure 8 The variation in degradation rate of multi-strain combinations. Detailed Implementation

[0135] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0136] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0137] The following examples used SPSS 16 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0138] Example 1: Screening, isolation, sequencing, and identification of highly efficient phenol-degrading bacteria

[0139] Experimental materials

[0140] Inorganic salt medium (MSM medium): NaCl 1.0 g / L, K₂HPO₄·3H₂O 0.79 g / L, MgSO₄·7H₂O 0.2 g / L, NH₄NO₃ 1 g / L, trace elements 1 mL / L. Add 20 g / L agar when preparing the inorganic salt solid medium. Autoclave at 121℃ for 20 min, adding phenol as needed.

[0141] Experimental protocol

[0142] The inoculum was obtained from soil samples collected by the applicant, Wang Jie (jiewangcau@cau.edu.cn), from Luanhong Coking Plant in Tangshan on November 20, 2021. A 100mL glass culture bottle was used, with a 1cm layer of soil sample at the bottom of the bottle. 80mL of inorganic salt culture medium was added, and phenol solution was added at a concentration of 500mg / L, the sole carbon source. The culture was incubated at 20℃ and 150r / min with shaking for 3-4 days. 10mL of the bacterial suspension was transferred to a new culture medium and incubated at 20℃ and 150r / min with shaking for 3-4 days. This process was repeated 4-5 times.

[0143] Take a turbid solution containing bacteria in good growth condition, and after serial dilution, select 10... -4 10 -5Two dilutions of the bacterial culture were used, with 100 μL spread onto a solid medium containing 500 mg / L phenol. The cultures were incubated at 20°C for 15 days, during which time colony morphology and growth were observed. After colony growth was complete, streaking was performed until single strains were isolated. The strains were then preserved and identified.

[0144] PCR amplification was performed using forward primer 27F and reverse primer 1492R. The 27F primer sequence was AGAGTTTGATCMTGGCTCAG (where M is a or c), and the 1492R primer sequence was TACGGYTACCTTGTTACGACTT (where Y is t or c). The PCR reaction system consisted of: 5 μL of 10×Ex Tap buffer, 4 μL of 2.5 mM×dNTP Mix, 12 μL of 10p primer, 2 μL of 10p primer, 0.5 μL of 5 μL Ex Tap, 2 μL of Template, and 4.5 μL of ddH2O. The PCR reaction program was as follows: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s; annealing at 54 °C for 30 s; extension at 72 °C for 1 min 30 s, repeated 24 times, followed by a final extension at 72 °C for 10 min. The amplified sequences were compared for homology using NCBI to identify the strain.

[0145] Strain identification

[0146] To understand, explore, and apply strains, it is first necessary to identify their species, which allows for understanding their relationship with closely related genera and species. Contemporary strain identification techniques include classical classification and modern classification methods. Classical classification methods are time-consuming and experimentally complex, while modern classification methods, such as analytical biological methods like 16S rDNA identification, offer the advantage of rapid species analysis of location samples, making them more suitable for the research context of this paper. Therefore, this paper chooses 16S rDNA for strain identification.

[0147] Four phenol-degrading bacteria were obtained through screening and isolation of highly efficient phenol-degrading bacteria, named BF1, BF2, BF3, and BF4. The 16S rDNA was amplified using the aforementioned 1492R and 27F rDNA sequences. These amplified fragments were then sequenced by Beijing Ruienuo Biotechnology Co., Ltd. (results shown below). Homology alignment was performed using NCBI, and a phylogenetic tree was constructed to identify the species. The results showed that the 16S rDNA sequence of the tested strain BF1 had 100% similarity to *Acinetobacter* and was most closely related to *Acinetobacter lwoffii*. Figure 1The strain was identified as *Acinetobacter lwoffii*; the 16S rDNA sequence of BF2 showed 61% similarity to *Citrobacter*, and was most closely related to *Citrobacter farmeri*. Figure 2 The strain was identified as *Citrobacter* and named *Citrobacter sp. BF2*. The 16S rDNA sequence of BF3 showed 100% similarity to *Acidovorax* genus and was most closely related to *Acidovorax soli*. Figure 3 The strain was identified as *Acidovorax soli*; the 16S rDNA sequence of BF4 showed 100% similarity to bacteria of the genus *Acidovorax soli*, and was most closely related to *Diaphorobacter nitroreducens*. Figure 4 The strain was identified as a nitrifying bacterium (Diaphorobacter nitroreducens).

[0148] The 16S rDNA sequence of BF1 is as follows:

[0149]

[0150] The 16S rDNA sequence of BF2 is as follows:

[0151]

[0152] The 16S rDNA sequence of BF3 is as follows:

[0153]

[0154] The 16S rDNA sequence of BF4 is as follows:

[0155]

[0156] Based on morphological characteristics, culture characteristics, physiological and biochemical properties, analysis using the Biolog automated microbial analysis system, and 16S rDNA analysis, BF1 was identified as *Acinetobacter lwoffii*. *Acinetobacter lwoffii* BF1 was deposited on July 25, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with the CGMCC registration number CGMCC No. 25399. Hereinafter, it will be referred to as *Acinetobacter lwoffii* CGMCC No. 25399.

[0157] Based on morphological characteristics, culture characteristics, physiological and biochemical properties, analysis using the Biolog automated microbial analysis system, and 16S rDNA analysis, BF2 was identified as *Citrobacter farmeri*. *Citrobacter farmeri* BF2 was deposited on July 25, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with the CGMCC registration number CGMCC No. 25400. Hereinafter, it will be referred to as *Citrobacter farmeri* CGMCC No. 25400.

[0158] Based on morphological characteristics, culture characteristics, physiological and biochemical properties, analysis using the Biolog automated microbial analysis system, and 16S rDNA analysis, BF3 was identified as *Acidovorax soli*. *Acidovorax soli* BF3 was deposited on August 3, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with the CGMCC registration number CGMCC No. 25478. Hereinafter referred to as *Acidovorax soli* CGMCC No. 25478.

[0159] Based on morphological characteristics, culture characteristics, physiological and biochemical properties, analysis using the Biolog automated microbial analysis system, and 16S rDNA analysis, BF4 was identified as *Diaphorobacter nitroreducens*. *Diaphorobacter nitroreducens* BF4 was deposited on August 3, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with the CGMCC registration number CGMCC No. 25438. Hereinafter referred to as *Acidovorax soli* CGMCC No. 25438.

[0160] Example 2: Investigation of the degradation characteristics of highly efficient phenol-degrading bacteria

[0161] This study screened and isolated four phenol-degrading bacteria. Although the bacteria came from the same environmental sample, their degradation characteristics for phenol differed due to their different species. Therefore, it was necessary to investigate the degradation characteristics of phenol by the four single bacterial species. On the other hand, single bacterial strains generally have poor colonization ability in soil, while strain combinations have a strong adaptability to the soil environment. Therefore, this study set up experiments to degrade phenol with different bacterial combinations in order to find the optimal bacterial combination and achieve better practical applications for phenol degradation.

[0162] Experimental materials

[0163] Yeast peptone medium (LB medium): 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, the remainder being water, autoclaved at 121°C for 20 min.

[0164] Inorganic salt medium (MSM medium): NaCl 1.0 g / L, K2HPO4·3H2O 0.79 g / L, MgSO4·7H2O 0.2 g / L, NH4NO3 1 g / L, trace elements (ZnSO4·7H2O 0.1 g, MnCl2·4H2O 0.03 g, H3BO3 0.3 g, CoCl2·6H2O 0.2 g, CuCl2·2H2O 0.01 g, NiCl2·6H2O 0.02 g, Na2MoO4·2H2O 0.03 g, deionized water 1000 mL, pH 3.4) 1 mL / L, the remainder being water, autoclaved at 121℃ for 20 min.

[0165] Phenol, hydrochloric acid, sodium hydroxide, natural organic matter (using Suwannee River natural organic matter provided by the International Humic Acid Association to represent natural organic matter in the experiment), and sodium chloride were added as needed to set different culture conditions and explore the degradation characteristics of the degrading bacteria.

[0166] Phenol-inorganic salt medium (MSM medium): NaCl 1.0 g / L, K2HPO4·3H2O 0.79 g / L, MgSO4·7H2O 0.2 g / L, NH4NO3 1 g / L, trace elements (ZnSO4·7H2O 0.1 g, MnCl2·4H2O 0.03 g, H3BO3 0.3 g, CoCl2·6H2O 0.2 g, CuCl2·2H2O 0.01 g, NiCl2·6H2O 0.02 g, Na2MoO4·2H2O 0.03 g, deionized water 1000 mL, pH 3.4) 1 mL / L, the remainder being water, autoclaved at 121℃ for 20 min. Then, a phenol solution filtered through a 0.22-micron membrane was added to bring the final phenol concentration to 500 mg / L; the pH was adjusted using 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution to bring the culture medium pH to 7.0.

[0167] Experimental indicators

[0168] Determination of bacterial growth: Using a blank culture medium as a reference, the light absorbance of the bacterial solution was measured at a wavelength of 600 nm and recorded as OD600nm, which reflects the growth status of the strain in the liquid culture medium.

[0169] Phenol content determination: High performance liquid chromatography (HPLC) was performed using an Eclipse Plus C18 column with a 40 / 60 (v / v) methanol / water mobile phase. The detection wavelength was 270 nm, the flow rate was 1 mL / min, and the injection volume was 10 μL. Phenol degradation rate = (Phenol concentration in blank sample - Phenol concentration in sample) / Phenol concentration in blank sample × 100%.

[0170] Phenol standard curve:

[0171] A series of phenol standard solutions with varying concentrations were prepared and determined using high-performance liquid chromatography (HPLC). Based on the relationship between peak area and phenol concentration, a phenol standard curve was plotted with phenol concentration on the x-axis and peak area on the y-axis. Figure 5 The concentration showed a good linear relationship in the range of 0–50 mg / L, with the equation y = 10.759x - 2.2885 and a correlation index R0. 2 With a value of 0.999, the accuracy is relatively high, so it can be used as a reference for subsequent experiments.

[0172] 1. Effects of different microbial agents on phenol degradation

[0173] The isolated single strains BF1, BF2, BF3, or BF4 were inoculated into LB liquid medium for expansion culture. The culture was carried out with shaking at 150 rpm and 30°C for 38 h until the OD600nm value was greater than 1, indicating optimal bacterial growth. The culture was then centrifuged at 2500 rpm for 8 min, and the precipitate (bacterial cells) was collected. The cells were washed with MSM liquid medium and collected. The bacterial suspension was diluted with MSM liquid medium to an OD600nm value of 1.6 (using MSM liquid medium as a blank control) for further inoculation and investigation.

[0174] 1. Preparation of phenol-degrading bacterial agent

[0175] 1.1 Preparation of microbial agents

[0176] A single colony of *Acinetobacter rumenella* CGMCC No. 25399 (BF1) was picked and inoculated into LB liquid medium. The medium was incubated at 30°C with shaking for 38 hours to prepare a stock solution. This stock solution was then added to LB liquid medium at a ratio of 1% (v / v) and incubated at 30°C with shaking for 38 hours. The fermentation broth was collected, and this fermentation broth is the primary inoculum. The active ingredient of this primary inoculum is *Acinetobacter rumenella* CGMCC No. 25399, and the concentration of *Acinetobacter rumenella* CGMCC No. 25399 in this primary inoculum is 16 × 10⁻⁶. 8 cfu / mL. cfu stands for colony forming unit.

[0177] 1.2 Preparation of microbial agents

[0178] A single colony of *Citrobacter farnesiana* CGMCC No. 25400 (BF2) was picked and inoculated into LB broth. The culture was incubated at 30°C with shaking for 38 hours to prepare a stock solution. This stock solution was then added to LB broth at a ratio of 1% (v / v) and incubated at 30°C with shaking for 38 hours. The fermentation broth was collected; this fermentation broth is the two-strain inoculum. The active ingredient in this two-strain inoculum is *Citrobacter farnesiana* CGMCC No. 25400, and the concentration of *Citrobacter farnesiana* CGMCC No. 25400 in this two-strain inoculum is 16 × 10⁻⁶. 8 cfu / mL. cfu stands for colony forming unit.

[0179] 1.3 Preparation of microbial agents

[0180] A single colony of *Acidovorax soli* CGMCC No. 25478 (BF3) was inoculated into LB broth and cultured at 30°C with shaking for 38 hours to prepare a stock solution. This stock solution was then added to LB broth at a ratio of 1% (v / v) and cultured at 30°C with shaking for 38 hours. The fermentation broth was collected; this fermentation broth is the 3-strain inoculum. The active ingredient in this 3-strain inoculum is *Acidovorax soli* CGMCC No. 25478, and the concentration of *Acidovorax soli* CGMCC No. 25478 in this 3-strain inoculum is 16 × 10⁻⁶. 8 cfu / mL. cfu stands for colony forming unit.

[0181] 1.4 Preparation of bacterial agents

[0182] A single colony of *Diaphorobacter nitroreducens* CGMCC No. 25438 (BF4) was inoculated into LB broth and cultured at 30°C with shaking for 38 hours to prepare a stock solution. This stock solution was then added to LB broth at a ratio of 1% (v / v) and cultured at 30°C with shaking for 38 hours. The fermentation broth was collected; this fermentation broth is the 4-strain inoculum. The active ingredient in this 4-strain inoculum is *Acinetobacter nitroreducens* CGMCC No. 25438, and the concentration of *Diaphorobacter nitroreducens* CGMCC No. 25438 in this 4-strain inoculum is 16 × 10⁻⁶. 8 cfu / mL. cfu stands for colony forming unit.

[0183] Preparation of 1.5-1+2 microbial agent

[0184] The above-mentioned bacterial agent 1 and bacterial agent 2 are mixed to obtain a 1+2 bacterial agent. The active ingredients of this 1+2 bacterial agent are *Acinetobacter rumenella* CGMCC No. 25399 and *Citrobacter freundii* CGMCC No. 25400, and the content of *Acinetobacter rumenella* CGMCC No. 25399 in this 1+2 bacterial agent is 8 × 10⁻⁶. 8 The cfu / mL concentration of Citrobacter farnesianum CGMCC No. 25400 was 8 × 10⁻⁶. 8 cfu / mL.

[0185] 1.6 Preparation of 1+3 bacterial agent

[0186] The above-mentioned bacterial agent 1 and bacterial agent 3 are mixed to obtain a 1+3 bacterial agent. The active ingredients of this 1+3 bacterial agent are Acinetobacter rumenella CGMCC No. 25399 and Acidovorax soli CGMCC No. 25478, and the content of Acinetobacter rumenella CGMCC No. 25399 in this 1+3 bacterial agent is 8 × 10⁻⁶. 8 The concentration of CFU / mL and Acidovorax soli CGMCC No. 25478 was 8 × 10⁻⁶. 8 cfu / mL.

[0187] 1.7 Preparation of 1+4 microbial agent

[0188] The above-mentioned bacterial agent 1 and bacterial agent 4 were mixed to obtain a 1+4 bacterial agent. The active ingredients of this 1+4 bacterial agent are Acinetobacter rumenella CGMCC No. 25399 and Diaphorobacter nitroreducens CGMCC No. 25438, and the content of Acinetobacter rumenella CGMCC No. 25399 in this 1+4 bacterial agent is 8 × 10⁻⁶. 8 The concentration of cfu / mL and Diaphorobacter nitroreducens CGMCC No. 25438 was 8 × 10⁻⁶. 8 cfu / mL.

[0189] 1.8 Preparation of 2+3 bacterial agent

[0190] The above-mentioned bacterial agents 2 and 3 were mixed to obtain a 2+3 bacterial agent. The active ingredients of this 2+3 bacterial agent are *Citrobacter freundii* CGMCC No. 25400 and *Acidovorax soli* CGMCC No. 25478, and the content of *Citrobacter freundii* CGMCC No. 25400 in this 2+3 bacterial agent is 8 × 10⁻⁶. 8 The concentration of CFU / mL and Acidovorax soli CGMCC No. 25478 was 8 × 10⁻⁶. 8 cfu / mL.

[0191] 1.9 Preparation of 2+4 bacterial agent

[0192] The above-mentioned two bacterial agents and four bacterial agents were mixed to obtain a 2+4 bacterial agent. The active ingredients of this 2+4 bacterial agent are *Citrobacter freundii* CGMCC No. 25400 and *Diaphorobacter nitroreducens* CGMCC No. 25438. The content of *Citrobacter freundii* CGMCC No. 25400 in this 1+2 bacterial agent is 8 × 10⁻⁶. 8The concentration of cfu / mL, Diaphorobacternitroreducens CGMCC No. 25438 was 8 × 10⁻⁶. 8 cfu / mL.

[0193] 1.10 Preparation of 3+4 microbial inoculant

[0194] The above-mentioned 3-inoculant and 4-inoculant were mixed to obtain a 3+4 inoculant. The active ingredients of this 3+4 inoculant are Acidovorax soli CGMCC No. 25478 and Diaphorobacter nitroreducens CGMCC No. 25438. The content of Acidovorax soli CGMCC No. 25478 in this 1+2 inoculant is 8 × 10⁻⁶. 8 The concentration of cfu / mL, Diaphorobacternitroreducens CGMCC No. 25438 was 8 × 10⁻⁶. 8 cfu / mL.

[0195] 1.11 Preparation of 1+2+3 microbial inoculant

[0196] The above-mentioned bacterial agents 1, 2, and 3 were mixed to obtain a 1+2+3 bacterial agent. In this 1+2+3 agent, *Acinetobacter rumenella* CGMCC No. 25399, and the active ingredients are *Citrobacter freundii* CGMCC No. 25400 and *Acidovorax soli* CGMCC No. 25478. The content of *Acinetobacter rumenella* CGMCC No. 25399 in this 1+2+3 agent is 5.3 × 10⁻⁶. 8 The cfu / mL concentration of Citrobacter farnesianum CGMCC No. 25400 was 5.3 × 10⁻⁶. 8 The concentration of Acidovorax soli CGMCC No. 25478 was 5.3 × 10⁻⁶ CFU / mL. 8 cfu / mL.

[0197] 1.12 Preparation of 1+2+4 microbial agent

[0198] The above-mentioned bacterial agent 1, bacterial agent 2, and bacterial agent 4 were mixed to obtain a 1+2+4 bacterial agent. The active ingredients of this 1+2+4 bacterial agent are *Acinetobacter rumenella* CGMCC No. 25399, *Citrobacter freundii* CGMCC No. 25400, and *Diaphorobacter nitroreducens* CGMCC No. 25438. The content of *Acinetobacter rumenella* CGMCC No. 25399 in this 1+2+4 bacterial agent is 5.3 × 10⁻⁶. 8The cfu / mL concentration of Citrobacter farnesianum CGMCC No. 25400 was 5.3 × 10⁻⁶. 8 The concentration of Diaphorobacter nitroreducens CGMCC No. 25438 was 5.3 × 10⁻⁶ CFU / mL. 8 cfu / mL.

[0199] 1.13 Preparation of 1+3+4 microbial agent

[0200] The above-mentioned bacterial agent 1, bacterial agent 3, and bacterial agent 4 were mixed to obtain a 1+3+4 bacterial agent. The active ingredients of this 1+3+4 bacterial agent are *Acinetobacter rumenella* CGMCC No. 25399, *Acidovorax soli* CGMCC No. 25478, and *Diaphorobacter nitroreducens* CGMCC No. 25438. The content of *Acinetobacter rumenella* CGMCC No. 25399 in this 1+3+4 bacterial agent is 5.3 × 10⁻⁶. 8 The concentration of Acidovorax soli CGMCC No. 25478 was 5.3 × 10⁻⁶ CFU / mL. 8 The concentration of Diaphorobacter nitroreducens CGMCC No. 25438 was 5.3 × 10⁻⁶ CFU / mL. 8 cfu / mL.

[0201] 1.14 Preparation of 2+3+4 microbial inoculant

[0202] The above-mentioned bacterial agents 2, 3, and 4 were mixed to obtain a 2+3+4 bacterial agent. The active ingredients of this 2+3+4 bacterial agent are *Citrobacter freundii* CGMCC No. 25400, *Acidovorax soli* CGMCC No. 25478, and *Diaphorobacter nitroreducens* CGMCC No. 25438. The content of *Citrobacter freundii* CGMCC No. 25400 in this 2+3+4 bacterial agent is 5.3 × 10⁻⁶. 8 The concentration of Acidovorax soli CGMCC No. 25478 was 5.3 × 10⁻⁶ CFU / mL. 8 The concentration of Diaphorobacter nitroreducens CGMCC No. 25438 was 5.3 × 10⁻⁶ CFU / mL. 8 cfu / mL.

[0203] 1.15 Preparation of 1+2+3+4 microbial agent

[0204] The above-mentioned bacterial agents 1, 2, 3, and 4 are mixed to obtain a 1+2+3+4 bacterial agent. The active ingredients of this 1+2+3+4 bacterial agent are *Acinetobacter rumenella* CGMCC No. 25399, *Citrobacter freundii* CGMCC No. 25400, *Acidovorax soli* CGMCC No. 25478, and *Diaphorobacter nitroreducens* CGMCC No. 25438. The content of *Acinetobacter rumenella* CGMCC No. 25399 in this 2+3+4 bacterial agent is 4 × 10⁻⁶. 8 The cfu / mL concentration of Citrobacter farnesianum CGMCC No. 25400 was 4 × 10⁻⁶. 8 The concentration of CFU / mL and Acidovorax soli CGMCC No. 25478 was 4 × 10⁻⁶. 8 The concentration of cfu / mL, Diaphorobacter nitroreducens CGMCC No.25438 was 4 × 10⁻⁶. 8 cfu / mL.

[0205] 2. Degradation of phenol

[0206] Phenol-inorganic salt medium (MSM medium): NaCl 1.0 g / L, K2HPO4·3H2O 0.79 g / L, MgSO4·7H2O 0.2 g / L, NH4NO3 1 g / L, trace elements (ZnSO4·7H2O 0.1 g, MnCl2·4H2O 0.03 g, H3BO3 0.3 g, CoCl2·6H2O 0.2 g, CuCl2·2H2O 0.01 g, NiCl2·6H2O 0.02 g, Na2MoO4·2H2O 0.03 g, deionized water 1000 mL, pH 3.4) 1 mL / L, the remainder being water, autoclaved at 121℃ for 20 min. Then, a phenol solution filtered through a 0.22-micron membrane was added to bring the final phenol concentration to 500 mg / L; the pH was adjusted using 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution to bring the culture medium pH to 7.0.

[0207] The procedures described below are the same except for the different microbial agents used.

[0208] 2.1 Degradation of phenol using a microbial agent

[0209] The specific method is as follows:

[0210] The above-mentioned bacterial agent 1 was inoculated into a 500mL Erlenmeyer flask containing 100mL of culture medium, so that the content of bacterial agent 1 in the Erlenmeyer flask, calculated as the content of Acinetobacter rouxii CGMCC No. 25399, was 0.16×10⁻⁶.8 Fermentation broth was cultured at 30℃ and 150 rpm for 2, 4, 6, 8, and 10 days with shaking at 30℃ and 150 rpm. The broth was collected, centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC) to determine the phenol concentration. A blank control (no inoculum) was also prepared by incubating 500 mL Erlenmeyer flasks containing 100 mL of culture medium at 30℃ and 150 rpm for 2, 4, 6, 8, and 10 days with shaking at 30℃ and 150 rpm. The broth was collected, centrifuged, and the supernatant was analyzed by HPLC to determine the phenol concentration. The experiment was repeated three times, with 10 Erlenmeyer flasks at each time point in each repetition.

[0211] 2.2 Degradation of phenol using two microbial agents

[0212] The two bacterial agents were inoculated into a 500mL Erlenmeyer flask containing 100mL of culture medium, so that the concentration of the two bacterial agents in the Erlenmeyer flask, calculated as the concentration of Citrobacter fargesii CGMCC No. 25400, was 0.16 × 10⁻⁶. 8 Fermentation broth was cultured at 30℃ and 150 rpm for 2, 4, 6, 8, and 10 days with shaking at 30℃ and 150 rpm. The broth was collected, centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC) to determine the phenol concentration. A blank control (no inoculum) was also prepared by incubating 500 mL Erlenmeyer flasks containing 100 mL of culture medium at 30℃ and 150 rpm for 2, 4, 6, 8, and 10 days with shaking at 30℃ and 150 rpm. The broth was collected, centrifuged, and the supernatant was analyzed by HPLC to determine the phenol concentration. The experiment was repeated three times, with 10 Erlenmeyer flasks at each time point in each repetition.

[0213] 2.3 Degradation of phenol using three microbial agents

[0214] The above three bacterial agents were inoculated into a 500mL Erlenmeyer flask containing 100mL of culture medium, so that the content of one bacterial agent in the Erlenmeyer flask, calculated as the content of Acidovorax soli CGMCC No.25478, was 0.16×10⁻⁶. 8 Fermentation broth was cultured at 30℃ and 150 rpm for 2, 4, 6, 8, and 10 days with shaking at 30℃ and 150 rpm. The broth was collected, centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC) to determine the phenol concentration. A blank control (no inoculum) was also prepared by incubating 500 mL Erlenmeyer flasks containing 100 mL of culture medium at 30℃ and 150 rpm for 2, 4, 6, 8, and 10 days with shaking at 30℃ and 150 rpm. The broth was collected, centrifuged, and the supernatant was analyzed by HPLC to determine the phenol concentration. The experiment was repeated three times, with 10 Erlenmeyer flasks at each time point in each repetition.

[0215] 2.4 Degradation of phenol using four microbial agents

[0216] The above four bacterial agents were inoculated into a 500mL Erlenmeyer flask containing 100mL of culture medium, so that the concentration of the four bacterial agents in the Erlenmeyer flask, calculated as Diaphorobacter nitroreducens CGMCC No.25438, was 0.16×10⁻⁶. 8 Fermentation broth was cultured at 30℃ and 150 rpm for 2, 4, 6, 8, and 10 days with shaking at 30℃ and 150 rpm. The broth was collected, centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC) to determine the phenol concentration. A blank control (no inoculum) was also prepared by incubating 500 mL Erlenmeyer flasks containing 100 mL of culture medium at 30℃ and 150 rpm for 2, 4, 6, 8, and 10 days with shaking at 30℃ and 150 rpm. The broth was collected, centrifuged, and the supernatant was analyzed by HPLC to determine the phenol concentration. The experiment was repeated three times, with 10 Erlenmeyer flasks at each time point in each repetition.

[0217] 2.5 Degradation of phenol using two-component microbial agents

[0218] The specific method is as follows:

[0219] The above-mentioned 1+2, 1+3, 1+4, 2+3, 2+3, or 3+4 bacterial inoculants were inoculated into 500mL Erlenmeyer flasks containing 100mL of culture medium, respectively, to obtain fermentation flasks for phenol degradation using 1+2, 1+3, 1+4, 2+3, 2+3, and 3+4 bacterial inoculants, respectively. The bacterial load in each fermentation flask is shown in Table 5. The flasks were cultured at 30℃ and 150rpm for 2, 4, 6, 8, and 10 days, respectively. The fermentation broth was collected, centrifuged, and the supernatant was used for high-performance liquid chromatography (HPLC) analysis to determine the phenol content and obtain the phenol concentration of the sample. Simultaneously, a control (blank sample) without inoculation was set up. 500mL Erlenmeyer flasks containing 100mL of culture medium were incubated at 30℃ and 150rpm for 2, 4, 6, 8, and 10 days, respectively. The fermentation broth was collected, centrifuged, and the supernatant was analyzed for phenol content using the above-mentioned high-performance liquid chromatography (HPLC) to obtain the phenol concentration of the blank sample. The experiment was repeated three times, with 10 Erlenmeyer flasks at each time point in each repetition.

[0220] Table 5

[0221]

[0222] 2.6 Degradation of phenol using a three-component microbial agent

[0223] The above-mentioned 1+2+3, 1+2+4, 1+3+4, and 2+3+4 bacterial inoculants were inoculated into 500mL Erlenmeyer flasks containing 100mL of culture medium, respectively, to obtain fermentation flasks for phenol degradation using the 1+2+3, 1+2+4, 1+3+4, and 2+3+4 bacterial inoculants, respectively. The bacterial load in each fermentation flask is shown in Table 6. The flasks were cultured at 30℃ and 150rpm for 2, 4, 6, 8, and 10 days, respectively. The fermentation broth was collected, centrifuged, and the supernatant was used for high-performance liquid chromatography (HPLC) analysis to determine the phenol content and obtain the phenol concentration of the sample. Simultaneously, a control (blank sample) without inoculation was set up. 500mL Erlenmeyer flasks containing 100mL of culture medium were incubated at 30℃ and 150rpm for 2, 4, 6, 8, and 10 days, respectively. The fermentation broth was collected, centrifuged, and the supernatant was analyzed for phenol content using the above-mentioned high-performance liquid chromatography (HPLC) to obtain the phenol concentration of the blank sample. The experiment was repeated three times, with 10 Erlenmeyer flasks at each time point in each repetition.

[0224] Table 6

[0225]

[0226] 2.7 Degradation of phenol using a four-component microbial agent

[0227] The above-mentioned bacteria 1, 2, 3, and 4 were inoculated into a 500mL Erlenmeyer flask containing 100mL of culture medium, so that the content of bacteria 1 in the Erlenmeyer flask, calculated as the content of Acinetobacter farouterus CGMCC No. 25399, was 0.04×10⁻⁶. 8 The cfu / mL concentration of the two bacterial agents in the conical flask, calculated as the concentration of *Citrobacter freundii* CGMCC No. 25400, was 0.04 × 10⁻⁶. 8 The concentration of the three bacterial agents in the conical flask, calculated as Acidovorax soli CGMCC No. 25478, was 0.04 × 10⁻⁶ CFU / mL. 8 The concentrations of CFU / mL and the content of the four bacterial agents in the conical flask, calculated as Diaphorobacter nitroreducens CGMCC No. 25438, were 0.04 × 10⁻⁶. 8Fermentation broth was cultured at 30℃ and 150 rpm for 2, 4, 6, 8, and 10 days with shaking at 30℃ and 150 rpm. The broth was collected, centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC) to determine the phenol concentration. A blank control (no inoculum) was also prepared by incubating 500 mL Erlenmeyer flasks containing 100 mL of culture medium at 30℃ and 150 rpm for 2, 4, 6, 8, and 10 days with shaking at 30℃ and 150 rpm. The broth was collected, centrifuged, and the supernatant was analyzed by HPLC to determine the phenol concentration. The experiment was repeated three times, with 10 Erlenmeyer flasks at each time point in each repetition.

[0228] 3. The results are as follows.

[0229] The changes in phenol degradation rate in each group are as follows: Figure 6 , Figure 7 and Figure 8 As shown. The phenol degradation rate of different strains and mixed strains increased significantly in the first four days, and then fluctuated and increased over time, with the final degradation rate ranging from 77.94% to 94.56%. There were significant differences between the groups, and the degradation methods of different strains of phenol were different. They can be roughly divided into two situations: (1) A high phenol degradation rate was achieved in the early stage, and it increased in the later stage, but the efficiency increase was small. This type of bacteria is suitable for phenol degradation experiments with a long cycle, and the time scale is extended to explore its degradation characteristics; (2) The phenol degradation rate was low in the early stage, and it was greatly improved in the later stage. This type of bacteria is suitable for related research on short-term high-efficiency degradation of phenol.

[0230] Depend on Figure 6 It can be seen that BF1 maintained the highest degradation rate among the four strains throughout the entire degradation process, and its degradation rate steadily increased over time (2–6 days), making it the strain with the highest overall degradation performance (85.59%). BF2 initially exhibited the lowest phenol degradation rate, which significantly increased between 2 and 6 days, ultimately ranking second only to BF1. It is speculated that BF2 utilized and converted phenol degradation intermediates more rapidly during the degradation process, indicating higher o-phenyl dioxygenase activity, thus leading to a rapid increase in phenol degradation rate in the later stages. BF3 and BF4 showed similar changes in phenol degradation rates at each stage, both initially low and gradually increasing. The degradation rates of phenol by the four strains were not significantly different, ranging from 80.43% to 85.59%.

[0231] Compared to a single strain ( Figure 6 ), strain combination ( Figure 7This further amplified the differences between groups. In the experiment of degrading phenol with a combination of two bacterial species, BF1+4 showed the best initial degradation and its degradation efficiency remained relatively stable in the later stages. However, in the final stage, BF1+3's degradation efficiency surpassed that of BF1+4, and it was the best-performing bacterial species in both single-strain and mixed-strain experiments (89.56%) for phenol degradation. This is presumably because BF1+4 has a lower tolerance to intermediate products of phenol degradation, resulting in a slight decrease in its degradation efficiency in the later stages. BF1+2 showed significant fluctuations in degradation efficiency during the phenol degradation process, likely due to poor mutual adaptation between the two bacterial species, leading to inconsistent degradation capabilities. Groups BF2+3, BF2+4, and BF3+4 all showed poor initial degradation, with significant improvement from 2 to 4 days, followed by fluctuating increases in the later stages. In the investigation of degradation by mixed bacteria, the final degradation rates of each group ranged from 77.94% to 89.56%. The mutual promotion or inhibition between different bacteria amplified the differences in degradation characteristics between the bacterial species.

[0232] Multi-strain combination degradation of phenol ( Figure 8 At that time, the degradation rate of each group was 79.93%–89.75%, and the difference between groups was between that of a single bacterium ( Figure 6 ) and a combination of two strains ( Figure 7 The interaction between these microbial communities demonstrates the synergistic metabolic effects of mixed microbial groups. Generally, combinations of multiple microorganisms achieve better mutual benefit and dynamic equilibrium than combinations of two. This is similar to previous research, such as the degrading microbial community enriched by Sun Wenjing et al., which could degrade 72 mg / L of benzene under optimal conditions for 50 hours; and Zhang Lei et al.

[40] A group of degrading bacteria was isolated from contaminated soil. Under optimal conditions, this group could completely degrade 100 mg / L benzene within 6 hours. Zhang Yongmin et al. isolated a group of aerobic bacteria that degrade phenanthrene from river surface sediments. This group could degrade 95.78% of 100 mg / L phenanthrene within 60 hours, demonstrating strong environmental adaptability and stress resistance. Furthermore, previous studies have confirmed that the synergistic effect of multiple strains is more efficient and stable than that of a single degrading bacterium. In the initial stage of phenol degradation, the BF1+2+4 group showed the best degradation performance, exhibiting stable degradation with little change in degradation rate throughout the process. While the BF2+3+4 group showed a consistently low degradation rate throughout the process, its rate significantly increased between 2 and 4 days. The underlying mechanism warrants further investigation to better understand, regulate, and apply the degradation rate of the bacterial community. In subsequent experiments, considering the stability, applicability, and degradation efficiency throughout the entire degradation cycle, the BF1+BF2+BF3+BF4 combination was used.

[0233] Example 2: Effects of different environmental factors on phenol degradation

[0234] To investigate the effects of environmental conditions on the degradation rate of phenol-degrading bacteria, this study used MSM culture media with different pH values ​​(5, 6, 7, 8, 9), different salinities (0.5%, 1.0%, 2.0%), and different natural organic matter concentrations (2.5, 5.0, 10.0 mg C / L) to culture the bacteria. The phenol concentration was measured on day 10, and the effects of different conditions on the phenol degradation rate were analyzed.

[0235] 1. Effect of different pH values ​​on phenol degradation

[0236] pH has multifaceted effects on bacterial growth and metabolism. First, pH alters the electrical charges of biomolecules such as nucleic acids and proteins, thus affecting the biological activity of enzymes within the bacteria. Second, pH influences cell membrane charge, thereby changing the bacteria's ability to absorb external nutrients. Furthermore, pH can alter the availability of nutrients and the toxicity of harmful substances in the environment, ultimately impacting bacterial survival. Therefore, investigating the effect of pH on the efficiency of phenol degradation by degrading bacteria is essential.

[0237] 1. Preparation of phenol culture media with different pH values:

[0238] Preparation of pH 5 phenol medium:

[0239] Add 1.0g NaCl, 0.79g K2HPO4·3H2O, 0.2g MgSO4·7H2O, 1g NH4NO3, and 1mL trace elements to a container. Add ddH2O to bring the volume to 1000ml. Adjust the pH to 5 using 1mol / L HCl or 1mol / L NaOH solution. Autoclave at 121℃ for 20min to obtain a pH=5 culture medium. Add sterile membrane-filtered phenol to bring the final concentration to 500mg / L to obtain a pH=5 phenol culture medium. Repeat the above operation to obtain multiple pH=5 phenol culture media for subsequent experiments.

[0240] Preparation of pH=6 phenol medium: Repeat the above operation to obtain multiple pH=6 phenol mediums for subsequent experiments.

[0241] Preparation of pH=7 phenol medium: Repeat the above operation to obtain multiple pH=7 phenol mediums for subsequent experiments.

[0242] Preparation of pH=8 phenol medium: Add 1.0g NaCl, 0.79g K2HPO4·3H2O, 0.2g MgSO4·7H2O, 1g NH4NO3, and 1mL trace elements to a container. Add ddH2O to bring the volume to 1000ml. Adjust the pH to 8 using 1mol / L HCl solution or 1mol / L NaOH solution. Autoclave at 121℃ for 20min to obtain pH=8 medium. Add phenol filtered through a sterile membrane to bring the final concentration to 500mg / L to obtain pH=8 phenol medium. Repeat the above operation to obtain multiple pH=8 phenol mediums for subsequent experiments.

[0243] Preparation of pH=9 phenol medium: Repeat the above operation to obtain multiple pH=9 phenol mediums for subsequent experiments.

[0244] The above-mentioned strains 1, 2, 3, and 4 were respectively inoculated into 500 mL Erlenmeyer flasks containing 100 mL of the above-mentioned pH=5, pH=6, pH=7, pH=8, or pH=9 phenol medium, so that the content of strain 1 in the Erlenmeyer flask, calculated as the content of Acinetobacter farouterus CGMCC No. 25399, was 0.04 × 10⁻⁶. 8 The concentrations of two bacterial agents in the conical flask (CFU / mL, calculated as the concentration of *Citrobacter freundii* CGMCC No. 25400) and the concentrations of three bacterial agents in the conical flask (Acidovorax soli CGMCC No. 25478) were both 0.04 × 10⁻⁶. 8 The concentration of CFU / mL and the content of the four bacterial agents in the conical flask, calculated as Diaphorobacter nitroreducens CGMCC No. 25438, were 0.04 × 10⁻⁶. 8 Fermentation flasks containing four different bacterial cultures at varying pH values ​​were prepared for phenol degradation (see Table 7). These flasks were cultured at 30°C and 150 rpm for 10 days with shaking. The fermentation broth was collected, centrifuged, and the supernatant was analyzed using high-performance liquid chromatography (HPLC) to determine the phenol concentration. A blank control (no inoculum) was also prepared by incubating 500 mL Erlenmeyer flasks containing 100 mL of culture medium at 30°C and 150 rpm for 10 days. The fermentation broth was collected, centrifuged, and the supernatant was analyzed using HPLC to determine the phenol concentration. The experiment was repeated three times, with 10 Erlenmeyer flasks at each time point in each repetition.

[0245] Table 7

[0246]

[0247]

[0248] The results are shown in Table 2. The mixed bacterial community could not effectively degrade phenol in either highly acidic or highly alkaline environments. This is presumably because high concentrations of acid / alkali inhibited the activity of different enzymes within the bacteria, thus hindering their utilization of the carbon source phenol from the external environment and preventing them from obtaining sufficient energy for growth and reproduction. This cycle further reduced the phenol degradation efficiency. These results indicate that the optimal pH conditions for the bacterial community are 7-8.

[0249] Table 2

[0250]

[0251] Example 3: Effect of different salt concentrations on phenol degradation

[0252] Inorganic salts are essential elements for bacterial growth, playing a crucial role in maintaining cell membrane homeostasis, regulating osmotic pressure, and coordinating enzyme reactions during growth. To protect intracellular protoplasm, bacteria maintain normal physiological activities by regulating osmotic pressure within a certain salinity range. However, in high-salt environments, inorganic salts can damage the bacterial cell membrane and internal enzyme systems, thereby affecting their normal growth and reproduction. Therefore, environmental salinity has a significant impact on bacterial growth and metabolism. This study investigated the effects of salinity gradients of 0.5%, 1.0%, and 2.0% on phenol-degrading bacteria. The degradation results are shown in Table 3.

[0253] 1. Preparation of phenol culture media with different salt concentrations:

[0254] Preparation of 0.5% NaCl-phenol medium:

[0255] Add 5g NaCl, 0.79g K2HPO4·3H2O, 0.2g MgSO4·7H2O, 1g NH4NO3, and 1mL trace elements to a container. Add ddH2O to bring the volume to 1000ml. Adjust the pH to 7.0 using 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution. Autoclave at 121℃ for 20min to obtain 0.5% NaCl medium. Add sterile membrane-filtered phenol to bring the final concentration to 500mg / L, obtaining 0.5% NaCl-phenol medium. Repeat the above steps to obtain multiple 0.5% NaCl-phenol mediums for subsequent experiments.

[0256] Preparation of 1% NaCl phenol medium:

[0257] Add 10g NaCl, 0.79g K2HPO4·3H2O, 0.2g MgSO4·7H2O, 1g NH4NO3, and 1mL trace elements to a container. Add ddH2O to bring the volume to 1000ml. Adjust the pH to 7.0 using 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution. Autoclave at 121℃ for 20min to obtain 0.5% NaCl medium. Add sterile membrane-filtered phenol to bring the final concentration to 500mg / L, obtaining 1% NaCl-phenol medium. Repeat the above steps to obtain multiple 1% NaCl-phenol mediums for subsequent experiments.

[0258] Preparation of 2% NaCl phenol medium:

[0259] Add 20g NaCl, 0.79g K2HPO4·3H2O, 0.2g MgSO4·7H2O, 1g NH4NO3, and 1mL trace elements to a container. Add ddH2O to bring the volume to 1000ml. Adjust the pH to 7.0 using 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution. Autoclave at 121℃ for 20min to obtain 2% NaCl medium. Add sterile membrane-filtered phenol to bring the final concentration to 500mg / L, obtaining 2% NaCl-phenol medium. Repeat the above steps to obtain multiple 2% NaCl-phenol mediums for subsequent experiments.

[0260] The above-mentioned strains 1, 2, 3, and 4 were respectively inoculated into 500 mL Erlenmeyer flasks containing 100 mL of the above-mentioned 0.5% NaCl-phenol medium, 1% NaCl-phenol medium, or 2% NaCl-phenol medium, so that the content of strain 1 in the Erlenmeyer flask, calculated as the content of Acinetobacter farouterus CGMCC No. 25399, was 0.04 × 10⁻⁶. 8 The cfu / mL concentration of the two bacterial agents in the conical flask, calculated as the concentration of *Citrobacter freundii* CGMCC No. 25400, was 0.04 × 10⁻⁶. 8 The concentration of the three bacterial agents in the conical flask, calculated as Acidovorax soli CGMCC No. 25478, was 0.04 × 10⁻⁶ CFU / mL. 8 The concentrations of CFU / mL and the content of the four bacterial agents in the conical flask, calculated as Diaphorobacter nitroreducens CGMCC No. 25438, were 0.04 × 10⁻⁶. 8Fermentation flasks containing four different NaCl concentrations of bacterial culture were prepared to degrade phenol (as detailed in Table 8). The flasks were cultured at 30°C and 150 rpm for 10 days with shaking. The fermentation broth was collected, centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC) to determine the phenol concentration. A blank control (no inoculum) was also prepared by incubating 500 mL Erlenmeyer flasks containing 100 mL of culture medium at 30°C and 150 rpm for 10 days with shaking. The fermentation broth was collected, centrifuged, and the supernatant was analyzed by HPLC to determine the phenol concentration. The experiment was repeated three times, with 10 Erlenmeyer flasks at each time point in each repetition.

[0261] Table 8

[0262]

[0263]

[0264] Table 3

[0265] Table 3 shows that the phenol degradation rate of the phenol-degrading bacteria gradually decreased with increasing salinity, and at a salinity of 2.0%, it could only degrade 26.24% of 500 mg / L phenol in 10 days. However, at a salinity of 1%, the degradation effect of the bacteria on phenol did not decrease significantly, indicating that the surface bacteria could tolerate the effect of 1% salinity.

[0266]

[0267] 5. The Influence of Different Natural Organic Matter on Phenol Degradation

[0268] Natural organic matter (NOM) mainly originates from the decomposition residues of plants and animals and is widely found in water bodies, soil, atmosphere, and rock strata. It plays an important ecological and environmental role in the global carbon and nitrogen cycle. Previous studies have investigated the effects of NOM on bacterial communities, finding that NOM can increase the activity of enzymes within bacteria in a short period of time, and this promoting effect is related to the type and concentration of NOM. In this study, the degradation efficiency of phenol by degrading bacteria was highly correlated with the relevant enzymes and their activities. Since the Suwannee River natural organic matter provided by the International Humic Acid Association is commonly used in related studies of natural organic matter, this paper selected SRNOM (China Humic Acid Association, 2R101N) to represent natural organic matter for the experiment, and set concentration gradients of 2.5, 5.0, and 10.0 mg C / L.

[0269] 1. Preparation of phenol culture media with different natural organic matter contents:

[0270] Preparation of 0 mg C / L natural organic phenol culture medium:

[0271] Add 1.0 g NaCl, 0.79 g K2HPO4·3H2O, 0.2 g MgSO4·7H2O, 1 g NH4NO3, and 1 mL trace elements to a container. Add ddH2O to bring the volume to 1000 mL. Adjust the pH to 7.0 using 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution. Autoclave at 121°C for 20 min. Add phenol filtered through a sterile membrane to bring the final concentration to 500 mg / L, obtaining a 0 mg C / L natural organic phenol medium. Repeat the above steps to obtain multiple 0 mg C / L natural organic phenol mediums for subsequent experiments.

[0272] Preparation of 2.5 mg C / L natural organic phenol culture medium:

[0273] Add 4.5 mg of natural organic matter SRNOM, 1.0 g of NaCl, 0.79 g of K2HPO4·3H2O, 0.2 g of MgSO4·7H2O, 1 g of NH4NO3, and 1 mL of trace elements to a container. Add ddH2O to bring the volume to 1000 mL. Adjust the pH to 7.0 using 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution. Autoclave at 121 °C for 20 min. Add phenol filtered through a sterile membrane to bring the final concentration to 500 mg / L, obtaining a 2.5 mg C / L natural organic matter phenol medium. Repeat the above steps to obtain multiple 2.5 mg C / L natural organic matter phenol mediums for subsequent experiments.

[0274] Preparation of 5.0 mg C / L natural organic phenol culture medium:

[0275] Add 9.0 mg of natural organic matter SRNOM, 1.0 g of NaCl, 0.79 g of K2HPO4·3H2O, 0.2 g of MgSO4·7H2O, 1 g of NH4NO3, and 1 mL of trace elements to a container. Add ddH2O to bring the volume to 1000 mL. Adjust the pH to 7.0 using 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution. Autoclave at 121 °C for 20 min. Add phenol filtered through a sterile membrane to bring the final concentration to 500 mg / L, obtaining a 5.0 mg C / L natural organic matter phenol medium. Repeat the above steps to obtain multiple 5 mg C / L natural organic matter phenol mediums for subsequent experiments.

[0276] Preparation of 10 mg C / L natural organic phenol culture medium:

[0277] Add 18 mg of natural organic matter SRNOM, 1.0 g of NaCl, 0.79 g of K2HPO4·3H2O, 0.2 g of MgSO4·7H2O, 1 g of NH4NO3, and 1 mL of trace elements to a container. Add ddH2O to bring the volume to 1000 mL. Adjust the pH to 7.0 using 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution. Autoclave at 121°C for 20 min. Add phenol filtered through a sterile membrane to bring the final concentration to 500 mg / L, obtaining a 10 mg C / L natural organic matter phenol medium. Repeat the above steps to obtain multiple 10 mg C / L natural organic matter phenol mediums for subsequent experiments.

[0278] The above-mentioned strains 1, 2, 3, and 4 were respectively inoculated into 500 mL Erlenmeyer flasks containing 100 mL of the above-mentioned 0 mg C / L natural organic phenol medium, 2.5 mg C / L natural organic phenol medium, 5.0 mg C / L natural organic phenol medium, or 10 mg C / L natural organic phenol medium, so that the content of strain 1 in the Erlenmeyer flask, calculated as the content of Acinetobacter farouterus CGMCC No. 25399, was 0.04 × 10⁻⁶. 8 The cfu / mL concentration of the two bacterial agents in the conical flask, calculated as the concentration of *Citrobacter freundii* CGMCC No. 25400, was 0.04 × 10⁻⁶. 8 The concentration of the three bacterial agents in the conical flask, calculated as Acidovoraxsoli CGMCC No. 25478, was 0.04 × 10⁻⁶ CFU / mL. 8 The concentrations of CFU / mL and the content of the four bacterial agents in the conical flask, calculated as Diaphorobacter nitroreducens CGMCC No. 25438, were 0.04 × 10⁻⁶. 8 Fermentation flasks containing four different natural organic matter concentrations of CFU / mL were prepared for phenol degradation using a four-component bacterial agent (as detailed in Table 9). The flasks were cultured at 30°C and 150 rpm for 10 days with shaking. The fermentation broth was collected, centrifuged, and the supernatant was analyzed using high-performance liquid chromatography (HPLC) to determine the phenol concentration. A blank control (no inoculation) was also prepared by incubating 500 mL Erlenmeyer flasks containing 100 mL of culture medium at 30°C and 150 rpm for 10 days. The fermentation broth was collected, centrifuged, and the supernatant was analyzed using HPLC to determine the phenol concentration. The experiment was repeated three times, with 10 Erlenmeyer flasks at each time point in each repetition.

[0279] Table 9

[0280]

[0281]

[0282] The results, as shown in Table 4, indicate that the degradation effect of the mixed bacterial agent slightly increases with the increase of natural organic matter concentration, suggesting that the presence of natural organic matter in the water does not reduce the degradation of pollutants.

[0283] Table 4

[0284]

[0285] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A microbial agent for degrading phenol, wherein the microbial agent is a 1+2+3+4 microbial agent; The active ingredients of the 1+2+3+4 bacterial agent are composed of Acinetobacter lwoffii BF1, Citrobacter farmeri BF2, Acidovorax soli BF3 and Diaphorobacter nitroreducens BF4. The Acinetobacter lwoffii BF1 strain has the accession number CGMCC No. 25399 from the China General Microbiological Culture Collection Center. The Citrobacter farmeri BF2 strain has the accession number CGMCC No. 25400 from the China General Microbiological Culture Collection Center. The accession number of Acidovorax soli BF3 at the China General Microbiological Culture Collection Center is CGMCC No. 25478; The Diaphorobacter nitroreducens BF4 has the accession number CGMCC No. 25438 from the China General Microbiological Culture Collection Center.

2. The use of the microbial agent according to claim 1 in any of the following: A1) Application of the above materials in the degradation of phenol; A2) Application of the above materials in the preparation of degradable phenol products; A3) Application of the aforementioned materials in biodegradable phenol A4) Application of the above materials in the preparation of biodegradable phenol products; A5) Application of the above materials in the degradation of phenol in natural organic matter; A6) Application of the above materials in the preparation of phenol products from degraded natural organic matter.

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