Enterococcus faecalis and application thereof

Abstract

The application discloses a facultative anaerobic Enterococcus faecalis and application thereof, and the facultative anaerobic Enterococcus faecalis is Enterococcus faecalis (E. Enterococcus faecalis ) NPW, which is preserved in the China General Microbiological Culture Collection Center on July 30, 2025, and has a preservation number of CGMCC No. 35461. The bacteria exhibit excellent benzene series degradation capacity, and can be used for bioremediation of benzene series contaminated soil and underground water, and have the characteristics of rapid and efficient, safe and reliable, and environment-friendly. The strain fermentation process provided by the application can significantly improve the cell density of the facultative anaerobic Enterococcus faecalis NPW.

Description

Technical Field

[0001] This invention belongs to the fields of microbial technology and fermentation engineering, specifically relating to a facultative anaerobic Enterococcus faecalis ( Enterococcus faecalis ) and its applications. Background Technology

[0002] Benzene compounds generally refer to benzene and its homologues, mainly including benzene, toluene, ethylbenzene, and xylene. They are among the most basic and important chemical raw materials, widely used in petroleum refining and petrochemicals, coal coking, dyes, plastics, rubber, synthetic fibers, and pesticide manufacturing. Benzene compounds are commonly found in environmental media such as surface water, soil and sediments, groundwater, and the atmosphere. Due to their carcinogenic and mutagenic properties, even at very low concentrations, they can produce biotoxicity, posing increasingly serious threats to ecosystems and human health. Therefore, these compounds have become a focus of environmental concern. Benzene compounds mainly enter soil and groundwater through industrial emissions, leaks from oil tanks and pipelines, and other production activities, causing environmental pollution.

[0003] Currently, methods for remediating soil and groundwater pollution are mainly divided into three categories: physical, chemical, and biological.

[0004] Physical methods typically include soil filling and landfilling. These methods are simple and efficient, but the disposal costs are high. The high volatility of benzene compounds also poses a risk of secondary pollution during the disposal process, making these methods less suitable for the disposal of benzene compounds.

[0005] The main technology used in chemical methods is chemical oxidation, which requires oxidizing agents such as permanganate, persulfate, hydrogen peroxide, and ozone. While removing benzene series pollutants, this process also generates some ecotoxicous intermediate products, leading to secondary pollution. Therefore, although physical and chemical treatment technologies are relatively mature, they still face problems such as high treatment costs, high energy consumption, complex procedures, high pollutant residues, and serious secondary pollution. They can no longer meet my country's increasingly stringent environmental protection requirements, and their high treatment costs are not suitable for my country's national conditions. Unlike physical and chemical methods, bioremediation technologies, including phytoremediation and microbial remediation, are not only low-cost but also do not pose a risk of secondary pollution, and are considered the most promising remediation technologies.

[0006] However, traditional biological treatment technologies also suffer from problems such as low efficiency, poor applicability, and difficulty in industrial application. Summary of the Invention

[0007] In view of this, embodiments of the present invention provide a facultative anaerobic Enterococcus faecalis ( Enterococcus faecalis ) and its applications, benzene series pollutants can be degraded through bioaugmentation.

[0008] In a first aspect, embodiments of the present invention provide a facultative anaerobic Enterococcus faecalis, wherein the facultative anaerobic Enterococcus faecalis is Enterococcus faecalis (… Enterococcus faecalis NPW was deposited at the China General Microbiological Culture Collection Center on July 30, 2025, with accession number CGMCC No. 35461.

[0009] Secondly, embodiments of the present invention provide an application of facultative anaerobic Enterococcus faecalis in the degradation of benzene compounds.

[0010] Furthermore, the application process includes: Facultative anaerobic Enterococcus faecalis was inoculated into sterilized seed culture medium for fermentation culture; Seed culture solution was inoculated into degradation medium containing benzene series contaminated sample pairs for degradation.

[0011] Furthermore, the seed culture medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 5 mg / L FeSO4·7H2O, and pH=7.0.

[0012] Furthermore, the degradation culture medium has the following composition: NaNO3 6 g / L, KH2PO4 2 g / L, Na2HPO4·12H2O 2 g / L, KCl 1 g / L, NaCl 1 g / L, CaCl2 1 g / L, MgSO4 1 g / L, pH=6.60.

[0013] Furthermore, during the fermentation and degradation process, an anaerobic or facultative anaerobic environment is used, with a dissolved oxygen concentration not exceeding 30%.

[0014] Thirdly, embodiments of the present invention provide a microbial agent containing the aforementioned facultative anaerobic Enterococcus faecalis.

[0015] Fourthly, embodiments of the present invention provide a method for preparing a microbial agent, the method comprising: fermenting and culturing the above-mentioned facultative anaerobic Enterococcus faecalis.

[0016] Fifthly, embodiments of the present invention provide a method for remediating benzene-contaminated soil and / or groundwater, the method comprising injecting the aforementioned facultative anaerobic Enterococcus faecalis or the aforementioned bacterial agent into the benzene-contaminated soil and / or groundwater to be remediated.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a facultative anaerobic Enterococcus faecalis ( Enterococcus faecium NPW has the ability to degrade benzene compounds and can use benzene compounds as the sole carbon source for degradation. This invention utilizes this strain to biodegrade benzene-contaminated soil and / or groundwater, and is particularly suitable for low-oxygen or anaerobic environments. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] It should be noted that, unless otherwise specified, the features in the following embodiments and implementation methods can be combined with each other.

[0020] Diluted coating to obtain single colonies Take 2g of soil contaminated with petroleum hydrocarbons, place it in a bottle containing 40mL of sterile water, shake to disperse, and seal. Take 1mL of the upper layer of liquid and spread it on a plate. Incubate at 30℃ for 12h, and finally dilute 10... -5 Find suitable single colonies for research on plate. Inoculate the obtained single colonies into LB medium for expansion culture. The composition of LB liquid medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH=7.0.

[0021] Screening of benzene-degrading strains Strain screening was conducted using benzene compounds as the sole carbon source. 3 mL of the enriched strain solution was added to 100 mL of degradation medium containing 50 mg / L each of benzene, toluene, ethylbenzene, and xylene (total benzene concentration 200 mg / L), and cultured anaerobic or facultative anaerobic conditions in a shaker at 30°C. Strains surviving under these conditions were selected. The degradation medium consisted of: NaNO3 6 g / L, KH2PO4 2 g / L, Na2HPO4·12H2O 2 g / L, KCl 1 g / L, NaCl 1 g / L, CaCl2 1 g / L, MgSO4 1 g / L, pH 6.60.

[0022] Determination of OD of bacterial culture in inorganic salt culture medium 600 Values ​​were measured at 24h, 48h, and 72h for the bacterial culture in the inorganic salt medium. 600 The values ​​were 0.072, 0.367, and 0.691, respectively, indicating that the bacteria can degrade benzene compounds in an inorganic salt culture medium containing only benzene compounds as the sole carbon source.

[0023] Taxonomic identification of strains The screened degrading strains were identified using molecular biology methods. Their 16S rRNA sequences were determined to be the gene sequence shown in SEQ ID No. 1. Sequence alignment with the GenBank nucleic acid database confirmed them as *Enterococcus faecalis*. Enterococcus faecalis ), named Enterococcus faecalis ( Enterococcus faecalis The NPW was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 30, 2025, with accession number CGMCC No. 35461. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0024] The obtained bacteria were cultured in LB medium. To further improve the cell concentration during fermentation, the effects of ferrous ions and dissolved oxygen (DO) on cell concentration were investigated. The basal medium consisted of 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and pH 7.0.

[0025] Table 1

[0026] As shown in Table 1 above, 2-5 mg / L FeSO4·7H2O and 10-30% dissolved oxygen are more conducive to the growth of the strain. Under these conditions, the bacterial concentration can reach 0.698, which is 6.4 times higher than that without FeSO4·7H2O, thus promoting efficient and rapid degradation.

[0027] The obtained bacteria were OD in LB medium. 600 When the concentration of benzene was 0.69, it was inoculated into inorganic salt media with benzene, toluene, ethylbenzene, and xylene (200 mg / L) as the sole carbon source, respectively, and incubated in a constant temperature shaker at 30℃ for 3 days. The residual benzene series compounds in the media were then analyzed by GC-MS (gas chromatography-mass spectrometry) to show the degradation effect. Standards were 200 mg / L and 100 mg / L GC standard grade benzene, toluene, ethylbenzene, and xylene. The degradation medium consisted of: NaNO3 6 g / L, KH2PO4 2 g / L, Na2HPO4·12H2O 2 g / L, KCl 1 g / L, NaCl 1 g / L, CaCl2 1 g / L, MgSO4 1 g / L, pH=6.60.

[0028] The calculations showed that after 3 days of anaerobic culture, the degradation rates of benzene compounds by the *Enterococcus faecalis* were: benzene 77.66%, toluene 82.92%, ethylbenzene 91.31%, and xylene 78.19%.

[0029] The obtained bacteria were OD in LB medium. 600When the concentration of xylene (200 mg / L) was 0.69, the culture medium was inoculated into an inorganic salt medium with xylene (200 mg / L) as the sole carbon source. 2 g / L yeast extract, 2 g / L glucose, 100 mg / L Tween 80, 100 mg / L rhamnolipid, and 100 mg / L sophorolipid were added to the medium, respectively. The medium was then incubated at 30°C in a shaker for 3 days. The residual benzene compounds in the medium were analyzed by GC-MS (gas chromatography-mass spectrometry) to show the degradation effect. Standards were 200 mg / L and 100 mg / L GC-grade xylene. The degradation medium consisted of: NaNO3 6 g / L, KH2PO4 2 g / L, Na2HPO4·12H2O 2 g / L, KCl 1 g / L, NaCl 1 g / L, CaCl2 1 g / L, MgSO4 1 g / L, pH 6.60.

[0030] Calculations showed that the degradation rates of benzene compounds by *Enterococcus faecalis* after 3 days of anaerobic culture were as follows: 83.63% with yeast extract, 84.12% with glucose, 85.25% with Tween, 80.04% with rhamnolipid, and 86.27% with sophorolipid. This demonstrates that adding specific nutrients can enhance the degradation activity of *Enterococcus faecalis* NPW, laying a solid foundation for its practical engineering applications.

[0031] The above description is merely a preferred example of the present invention and is not intended to limit the scope of protection of the present invention. In addition to the above embodiments, the present invention may have other implementations. All technical solutions formed by equivalent substitutions or equivalent changes fall within the scope of protection claimed by the present invention.

Claims

1. A facultative anaerobic Enterococcus faecalis, characterized in that, The facultative anaerobic Enterococcus faecalis is Enterococcus faecalis ( Enterococcus faecalis NPW was deposited at the China General Microbiological Culture Collection Center on July 30, 2025, with accession number CGMCC No. 35461. The gene sequence of the 16S rRNA of the facultative anaerobic Enterococcus faecalis is shown in SEQ ID No.

1.

2. The use of the facultative anaerobic Enterococcus faecalis according to claim 1 in the degradation of benzene, toluene, ethylbenzene and / or xylene.

3. The application according to claim 2, characterized in that, The application process includes: Facultative anaerobic Enterococcus faecalis was inoculated into sterilized seed culture medium for fermentation culture; Seed culture solution was inoculated into degradation medium containing benzene series contaminated sample pairs for degradation.

4. The application according to claim 3, characterized in that, The seed culture medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 2-5 mg / L FeSO4·7H2O, and pH=7.

0.

5. The application according to claim 3, characterized in that, The degradation culture medium consists of: NaNO3 6 g / L, KH2PO4 2 g / L, Na2HPO4·12H2O 2 g / L, KCl 1 g / L, NaCl 1 g / L, CaCl2 1 g / L, MgSO4 1 g / L, pH=6.

60.

6. The application according to claim 3, characterized in that, During fermentation and degradation, an anaerobic or facultative anaerobic environment is used, with dissolved oxygen concentration not exceeding 30%.

7. A microbial agent, characterized in that, The bacterial agent contains the facultative anaerobic Enterococcus faecalis as described in claim 1.

8. A method for preparing a microbial agent, characterized in that, The method includes: fermenting and culturing the facultative anaerobic Enterococcus faecalis as described in claim 1.

9. A method for remediating benzene-contaminated soil and / or groundwater, characterized in that, The method includes injecting the facultative anaerobic Enterococcus faecalis of claim 1 or the bacterial agent of claim 7 into the soil and / or groundwater contaminated with benzene, toluene, ethylbenzene and / or xylene to be remediated.

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