Bacteria for degrading ethylene oxide and uses thereof

Abstract

The present disclosure relates to strains for degrading ethylene oxide and degradation agents comprising the same, wherein the strains are Acetobacter peroxydans EO-01 strain with Deposit number of CGMCC No. 18431; Lactobacillus fermentum EO-02 strain with Deposit number of CGMCC No. 18432; or Bacillus subtilis EO-03 strain with Deposit number of CGMCC No. 18433. The strains are capable of safely and efficiently degrading ethylene oxide. The present disclosure also provides a method for purifying and producing strains that can degrade ethylene oxide, and a method for biodegrading ethylene oxide.

Claims

1. A product which is a a bacterial strain Lactobacillus fermentum EO-02 with Deposit number of CGMCC No. 18432 capable of degrading ethylene oxide.

2. The product according to claim 1, wherein the bacterial strain has a concentration from 10.sup.10 cfu/mL to 10.sup.12 cfu/mL.

3. The product according to claim 1, wherein the Lactobacillus fermentum EO-02 with Deposit number of CGMCC No. 18432 is capable of degrading ethylene oxide in sewage, sludge, or exhaust gas.

4. The product according to claim 1, wherein the Lactobacillus fermentum EO-02 with Deposit number of CGMCC No. 18432 is capable of degrading ethylene oxide at a rate at least 10% greater relative to the degradation rate of ethylene oxide in the absence of Lactobacillus fermentum EO-02 with Deposit number of CGMCC No. 18432.

5. The product according to claim 1, wherein the Lactobacillus fermentum EO-02 with Deposit number of CGMCC No. 18432 strain bacterium is capable of using ethylene oxide as a carbon source and is capable of growing normally with ethylene oxide as the main carbon source in the culture.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIGS. 1A to 1C show bacterial colony growth of the EO-degrading potential bacteria in the second enrichment medium after growing for 48 hours at a constant temperature of 37° C., wherein the EO-degrading potential bacteria were, FIG. 1A, Acetobacter peroxydans EO-01 original strain, FIG. 1B, Lactobacillus fermentum EO-02 original strain, and FIG. 1C Bacillus subtilis EO-03 original strain obtained by the enrichment, purification and screening processes according to Example 1 of the present disclosure.

(2) FIGS. 2A to 2C show Gram staining results of the EO-degrading potential bacteria, wherein the EO-degrading potential bacteria are, FIG. 2A, Acetobacter peroxydans EO-01 original strain, FIG. 2B. Lactobacillus fermentum EO-02 original strain, and FIG. 2C Bacillus subtilis EO-03 original strain obtained by the enrichment, purification and screening processes according to Example 1 of the present disclosure.

(3) FIGS. 3A to 3C are the phylogenetic evolution diagrams of EO-degrading potential bacteria, wherein the EO-degrading potential bacteria were (FIG. 3A) Acetobacter peroxydans EO-01 original strain, (FIG. 3B) Lactobacillus fermentum EO-02 original strain, and (FIG. 3C) Bacillus subtilis EO-02 original strain obtained by the enrichment, purification and screening according to Example 1 of the present disclosure.

(4) FIGS. 4A and 4B show bacterial colony growth of Acetobacter peroxydans EO-01 strain (FIG. 4A) after the inducted acclimation of Example 2, and (FIG. 4B) before the inducted acclimation of Example 2 in a liquid Sabouraud induction medium with 800 mg/L ethylene oxide after growing at a constant temperature of 37° C. for 48 hours in a comparative experiment of ethylene oxide degradation in Example 3 of the present disclosure.

(5) FIGS. 5A and 5B show bacterial colony growth of Lactobacillus fermentum EO-02 strain (FIG. 5A) after the inducted acclimation of Example 2, and (FIG. 5B) before the inducted acclimation of Example 2 in a liquid Sabouraud induction medium with 800 mg/L ethylene oxide after growing at a constant temperature of 37° C. for 48 hours in a comparative experiment of ethylene oxide degradation in Example 3 of the present disclosure.

(6) FIGS. 6A and 6B show bacterial colony growth of Bacillus subtilis EO-03 strain (FIG. 6A) after the inducted acclimation of Example 2, and (FIG. 6B) before the inducted acclimation of Example 2 in a liquid Sabouraud induction medium with 800 mg/L ethylene oxide after growing at a constant temperature of 37° C. for 48 hours in a comparative experiment of ethylene oxide degradation in Example 3 of the present disclosure.

DETAILED DESCRIPTION OF EMBODIMENTS

(7) Detailed description will be given below with referral to the accompanying figures to facilitate understanding of the present disclosure. Preferred embodiments are shown in the figures. However, the present disclosure may be implemented in various ways, without being limited to the examples presented in the description. The purpose of these embodiments is merely for illustration and better comprehension of the present disclosure.

(8) Unless otherwise defined, all the technical and scientific terms herein shall be understood as the same meaning with those commonly accepted by a person skilled in the art. Such terms, as used herein, are for the purpose of describing specific embodiments of, without limiting, the present disclosure. The term “and/or” as used herein refers to any and all combinations of one or more items recited.

(9) Enrichment, Purification, Screening and Identification of Strains with Ethylene Oxide Degradation Ability

(10) Below is an example of enrichment, purification, screening, and identification of strains with ethylene oxide degradation ability.

Example 1

(11) I. Enrichment, Purification and Screening

(12) A sample of the sludge mixture was collected at the sewage outlet of a suburban sewage disposal plant in Guangzhou, Guangdong Province, and used for the enrichment, purification and screening experiments of this example.

(13) A first enrichment medium was prepared as follows: 40 g of glucose, 5 g of casein trypsin digest, and 5 g of animal tissue pepsin digest were mixed, adjusted to pH 5.4-5.8, filled to 1000 mL with distilled water and thoroughly mixed. Portions of 250 ml of the prepared medium were added to 500 mL Erlenmeyer flasks, sterilized at 121° C. for 20 min, and cooled to room temperature. Pure liquid ethylene oxide was cooled down in an ice bath before 28 μL was taken and injected into the sterilized medium by a sealed syringe, providing 100 mg/L ethylene oxide in the medium complying with the national emission standard, to obtain the first enrichment medium.

(14) The screening and purification medium was prepared as follows: 40 g of glucose, 5 g of casein trypsin digest, 5 g of animal tissue pepsin digest and 15 g of agar were mixed, adjusted to pH of 5.4, and filled to 1000 mL with distilled water and thoroughly mixing. Portions of 250 ml of the prepared medium were added into 500 mL Erlenmeyer flasks, sterilized at 121° C. for 20 minutes, and cooled to about 50-56° C. 28 μL of liquid ethylene oxide was injected into the sterilized medium by a sealed syringe to obtain the screening and purification medium.

(15) A second enrichment medium was prepared as follows: 40 g of glucose, 5 g of casein trypsin digest, and 5 g of animal tissue pepsin digest were mixed, adjusted to pH 5.4-5.8, filled to 1000 mL with distilled water and thoroughly mixed. Portions of 250 ml of the prepared medium were added into 500 mL Erlenmeyer flasks, sterilized at 121° C. for 20 minutes, and cooled to room temperature to obtain the second enrichment medium.

(16) 10.0 g of the sludge mixture sample was weighed, added with 100 mL of 0.03 mol/L phosphate buffer, well mixed, allowed to stand for 120 min for clarification, and filtered to remove large particles of sediment and obtain a suspension.

(17) 1 mL of the suspension was added into 10 mL of the first enrichment medium in each of four test tubes and placed in a shaker for oxygen-consuming enrichment culture for 24 to 48 hours (200 rpm, 37° C.).

(18) The predominant strains from the first enrichment medium were streaked on the screening and purification medium for isolation to obtain ethylene oxide-degrading predominant strains.

(19) The ethylene oxide-degrading predominant strains were incubated in the second enrichment medium for 24 hours to obtain three EO-degrading potential strains, designated as the EO-01 original strain, EO-02 original strain and EO-03 original strain, respectively. The three EO-degrading potential strains were preserved at −80° C. using the glycerin preservation method (medium: 50% glycerol=1:1).

(20) At 48 hours of incubation in the screening and purification medium, the colony morphology of the EO-01 original strain was milky white needle-like petites with gray or off-white cells, a smooth and moist surface, a round shape, regular edges, a colony diameter of about 1.0 mm, and no pigment. The colony morphology of the EO-02 original strain was milky white needle-like petites, colorless and transparent, with a smooth and moist surface, a round shape, regular edges, a colony diameter from 0.5 mm to 1.0 mm, and no pigment. The colony morphology of the EO-03 original strain was off-white or light yellow and opaque, with a rough surface, uneven edges, high viscosity, a colony diameter about 4.0-5.0 mm, and no pigment.

Example 2 Characterization and Identification of Ethylene Oxide-Degrading Bacteria Strains

(21) The following identification methods were used:

(22) Morphological characterization, including observation of colony morphology, microscopic morphology, culture characteristics and Gram staining;

(23) Physiological and biochemical characterization, including nutrition type, nitrogen and carbon source utilization capacity, and biochemical tests;

(24) Molecular biological characterization (16s rDNA sequencing), including the procedure of bacterial culture, bacterial DNA extraction, PCR amplification, 16s rDNA sequencing and sequence alignment analysis, wherein the primer pair for PCR amplification was as follows:

(25) Upstream primer 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, as shown in SEQ ID NO: 1; and

(26) Downstream primer 1492R: 5′-GGTTACCTTGTTACGACTT-3′, as shown in SEQ ID NO: 2.

(27) The above characterization and identification methods are well known to those skilled in the art.

(28) The colony morphologies of the EO-01 original strain, EO-02 original strain, and EO-03 original strain are shown in FIGS. 1A, 1B and 1C, their Gram staining results shown in FIGS. 2A, 2B and 2C, and their phylogenetic trees shown in FIGS. 3A, 3B and 3C, respectively. According to the characterization results of morphology, physiology, biochemistry, and molecular biology, the EO-01 original strain was Acetobacter peroxydans, belonging to the genus Acetobacter; the EO-02 original strain was Lactobacillus fermentum, belonging to the genus Lactobacillus; and the EO-03 original strain was Bacillus subtilis, belonging to the genus Bacillus. The characterization and identification results of the three EO-degrading potential strains are summarized in Table 1 below.

(29) TABLE-US-00001 TABLE 1 Characterization and identification results of EO-01 original strain, EO-02 original strain, and EO-03 original strain Strain EO-01 original strain EO-02 original strain EO-03 original strain Colony morphology milky white needle-like petites, with colorless and transparent, off-white or light yellow gray or off-white cells, no pigment, smooth and moist surface, and opaque, rough smooth and moist surface, a round round shape, regular edges, surface, uneven edges, shape, regular edges, colony colony diameter from high viscosity, colony diameter of about 1.0 mm, 0.5 mm to 1.0 mm, and no diameter from 4.0 mm pigment to 5.0 mm, no pigment. Microscopic morphology oval, or short straight rod shape long straight rod shape, oval shape, sporing. generally in the form of short-chain or binary fission. Physiological Gram staining Gram-negative bacteria Gram-positive bacteria Gram-positive bacteria and results (red) (purple) (purple) biochemical Culture strict aerobic bacteria, the most Facultative anaerobic Aerobic bacteria, characteristics characteristics suitable growth temperature ranging bacteria, the most suitable extreme resistance to from 20° C. to 35° C. growth temperature ranging stress; Fast growth, from 30° C. to 40° C., good relatively low resistance to acid, well nutritional requirements, growth under acidic capability of efficiently conditions . secreting many proteins and metabolites, and producing no toxins 16s rDNA sequencing and 16s rDNA is as listed in SEQ ID NO: 16s rDNA is as listed in 16s rDNA is as listed in sequence alignment results 3, and has 99% homology with SEQ ID NO: 4, and has SEQ ID NO: 5, and has Acetobacter peroxydans 16S rDNA. 99% homology with 99% homology with Lactobacillus fermentum Bacillus subtilis 16S 16S rDNA. rDNA. Strain identification results Acetobacter peroxydans, belonging Lactobacillus fermentum, Bacillus subtilis, to the genus Acetobacter belonging to the genus belonging to the genus Lactobacillus Bacillus

Example 3 Induced Acclimation of Ethylene Oxide-Degrading Potential Bacteria

(30) This is an example of induced acclimation of ethylene oxide-degrading potential bacteria strains, including induced acclimation of ethylene oxide tolerance and acclimation of ethylene oxide degradation ability.

(31) Phase I: Induced Acclimation of Ethylene Oxide Tolerance

(32) Four ethylene oxide-tolerance acclimation mediums containing different concentrations of ethylene oxide were prepared as follows: 10 g of peptone, 40 g of glucose, and 15 g of agar were dissolved in distilled water, and adjusted to pH 5.4-5.8, filling up to 1000 mL with distilled water and thoroughly mixed, dividing the prepared medium into portions of 250 mL and sterilized at 121° C. for 20 min; and, before use, heating the medium to melt, allowing to cool to about 50-56° C., and injecting 25 mg, 50 mg, 125 mg or 200 mg of ethylene oxide respectively with a sealed syringe to make ethylene oxide-tolerance acclimation medium plates with four different concentrations of ethylene oxide (100 mg/L, 200 mg/L, 500 mg/L or 800 mg/L), designated as ethylene oxide-tolerance acclimation medium plates A, B, C, and D, respectively.

(33) Using the method of plate streaking, the three ethylene oxide-degrading potential bacteria, namely EO-01 original strain, EO-02 original strain, and EO-03 original strain, were inoculated into the ethylene oxide-tolerance acclimation medium plate A and incubated at a constant temperature of 37° C. for 48 h. Then a first single colony with the largest radius on each plate A was picked respectively and subcultured into the ethylene oxide-tolerance acclimation medium plate B and incubated at 37° C. for 48 h. Again, a second single colony with the largest colony radius on each plate B was picked respectively and subcultured into the ethylene oxide-tolerance acclimation medium plate C and incubated at a constant temperature of 37° C. for 48 hours. A third single colony with the largest colony radius on each plate C was picked and subcultured onto the ethylene oxide-tolerance acclimation medium plate D and incubated at a constant temperature of 37° C. for 48 hours. Then a fourth single colony with the largest colony radius on the plate D was picked and incubated at a constant temperature of 37° C. for 48 hours, to further obtain ethylene oxide-tolerance EO-01 strain, ethylene oxide-tolerance EO-02 strain, and ethylene oxide-tolerance EO-03 strain, respectively.

(34) Phase II: Induced Acclimation of Ethylene Oxide Degradation Ability

(35) Four ethylene oxide-degradation acclimation mediums containing a carbon source in different contents were prepared as follows: 10 g of peptone, glucose (20 g, 12 g, 4 g, or 0 g), and 15 g of agar were dissolved in distilled water, adjusted to pH 7.0-7.5, filled up to 1000 mL with distilled water and thoroughly mixing; dividing the medium prepared into 250 ml portions and sterilized at 121° C. for 20 min; and, before use, heating the medium to melt, allowing to cool to about 50-56° C., and injecting 200 mg of ethylene oxide with a sealed syringe to make Four ethylene oxide-degradation acclimation medium plates containing a carbon source in different contents (20 g/L 12 g/L, 4 g/L, and 0 g/L), designated as ethylene oxide-degradation acclimation medium plates A, B, C, and D, respectively.

(36) Using the method of plate streaking, the ethylene oxide-tolerance EO-01 strain, ethylene oxide-tolerance EO-02 strain, and ethylene oxide-tolerance EO-03 strain were inoculated into the ethylene oxide-degradation acclimation medium plate A and incubated at a constant temperature of 37° C. for 48 hours, respectively. Then a first single colony with the largest radius on each plate A was picked respectively and subcultured into the ethylene oxide-degradation acclimation medium plate B and incubated at a constant temperature of 37° C. for 48 hours; a second single colony with the largest colony radius on each plate B was picked respectively and subcultured into the ethylene oxide-degradation acclimation medium plate C and incubated at a constant temperature of 37° C. for 48 hours; a third single colony with the largest colony radius on each plate C was picked respectively and subcultured into the ethylene oxide-degradation acclimation medium plate D and incubated at a constant temperature of 37° C. for 48 hours; a fourth single colony with the largest colony radius on each plate D was picked and stored on bevels made from agar medium containing nutrients corresponding to the ethylene oxide degradation acclimation medium plate D, so that strains with tolerance and degradation ability against ethylene oxide, namely Acetobacter peroxydans EO-01, Lactobacillus fermentum EO-02, and Bacillus subtilis EO-03, were obtained and deposited with Deposit numbers CGMCC No. 18431, CGMCC No. 18432, and CGMCC No. 18433 respectively.

(37) The results of induced acclimation of ethylene oxide tolerance and degradation ability are shown in Table 2.

(38) TABLE-US-00002 TABLE 2 Results of induced acclimation of ethylene oxide degradation ability Phase I Phase II Carbon source (%) 100 100 100 100 50 30 10 0 EO concentration 100 200 500 800 800 800 800 800 (mg/L) EO-01 growth + + + + + + + + EO-02 growth + + + + + + + + EO-03 growth + + + + + + + + Note: “+” represents bacterial growth.

(39) The carbon source of 100%, 50%, 30%, 10%, and 0% in above table 2 corresponds to the glucose concentrations of 40 g/L, 20 g/L, 12 g/L, 4 g/L, and 0 g/L in ethylene oxide-degradation acclimation mediums, respectively.

(40) The results in Table 2 show that the EO-01, EO-02, and EO-03 strains, after induced acclimation as described above, all grow well under the culture conditions with ethylene oxide as the only carbon source, and may use ethylene oxide as the carbon source.

(41) According to the identification method described in Example 1, by morphological characterization, physiological and biochemical characterization, and molecular biological characterization, the EO-01, EO-02, and EO-03 strains were identified as follows:

(42) The EO-01 strain after the inducted acclimation was Acetobacter peroxydans, belonging to the genus Acetobacter.

(43) The EO-02 strain after the inducted acclimation was Lactobacillus fermentum, belonging to the genus Lactobacillus.

(44) The EO-03 strain after the inducted acclimation was Bacillus subtilis, belonging to the genus Bacillus.

Example 4 Comparative Experiment of Ethylene Oxide Degradation

(45) In the example below, comparative experiments were conducted to test the ability of the Acetobacter peroxydans EO-01 strain, Lactobacillus fermentum EO-02 strain, and Bacillus subtilis EO-03 strain.

(46) 1. Experimental Method:

(47) Liquid Sabouraud medium was prepared as follows: weighing 40 g of glucose, 5 g of casein trypsin digest, and 5 g of animal tissue pepsin digest, mixing in distilled water, adjusting to pH 5.4-5.8, and filling up to 1000 mL with distilled water; dividing the medium into portions of 250 mL in 500 mL Erlenmeyer flasks, sterilizing at 121° C. for 20 min, and allowing to cool to room temperature

(48) Two liquid Sabouraud induction medium with different ethylene oxide concentrations were made as follows: weighing 10 g peptone, filling up to 1000 mL with distilled water, mixing thoroughly; dividing into 400 mL portions, sterilizing at 121° C. for 20 min, and allowing to cool to room temperature; injecting 160 mg or 320 mg of ethylene oxide with a sealed syringe to make two liquid Sabouraud induction mediums containing ethylene oxide in different concentrations (400 mg/L and 800 mg/L respectively).

(49) Culture and activation: ethylene oxide-degrading potential bacteria EO-01, EO-02, and EO-03 original strains, which were subjected to no induced acclimation according to Example 1, and EO-01, EO-02, and EO-03 strains after induced acclimation according to Example 2, were each inoculated at 10 μL into 100 mL of liquid Sabouraud medium, and incubated at 37° C. for 48 h with shaking at 200 rpm, so as to obtain activation solutions of EO-01, EO-02, and EO-03 original strains respectively and activation solutions of EO-01, EO-02, and EO-03 strains with a strain concentration from 10.sup.10 cfu/mL to 10.sup.12 cfu/mL in the each activation solutions, respectively.

(50) To conduct a comparative experiment of ethylene oxide degradation, the following treatment and control groups were incubated in a 37° C. incubator for 48 hours.

(51) Experimental group 1A (acclimated strains/800 mg/L ethylene oxide): 5 mL each of the activated solutions of the EO-01, EO-02, EO-03 strains after induced acclimation was inoculated in 400 mL of the liquid Sabouraud induction medium containing 800 mg/L ethylene oxide, with cell count in the medium being 10.sup.8-10.sup.10 cfu/mL;

(52) Experimental group 1B (unacclimated original strain/800 mg/L ethylene oxide): 5 mL each of the activated solutions of the EO-01, EO-02, EO-03 original strains before induced acclimation was inoculated in 400 mL of the liquid Sabouraud induction medium containing 800 mg/L ethylene oxide, with cell count in the medium being 10.sup.8-10.sup.10 cfu/mL;

(53) Control Group 1: Liquid Sabouraud induction medium containing 800 mg/L ethylene oxide without inoculation of any strain.

(54) Experimental group 2A (acclimated strains/400 mg/L ethylene oxide): 5 mL each of the activated solutions of the EO-01, EO-02, EO-03 strains after induced acclimation was inoculated in 400 mL of the liquid Sabouraud induction medium containing 400 mg/L ethylene oxide, with cell count in the medium being 10.sup.8-10.sup.10 cfu/mL;

(55) Experimental group 2B (unacclimated original strain/400 mg/L ethylene oxide): 5 mL each of the activated solutions of the EO-01, EO-03, EO-03 original strains before induced acclimation was inoculated in 400 mL of the liquid Sabouraud induction medium containing 400 mg/L ethylene oxide, with cell count in the medium being 10.sup.8-10.sup.10 cfu/mL;

(56) Control group 2: Liquid Sabouraud induction medium containing 400 mg/L ethylene oxide without inoculation of any strain.

(57) To calculate the concentrations of residual ethylene oxide and the degradation rates, samples were taken from the above Treatment groups and Control groups after the comparative test for gas chromatography analysis according to the methods described in “Sanitary Standards for Disposable Hygiene Products” (GB15979-2002) of China National Standards as follows:

(58) a certain volume of pure ethylene oxide gas was collected with a sealed syringe and dissolved in deionized water to make a series of ethylene oxide standards of 0-1000 mg/L concentrations;

(59) the subject samples to be analyzed were prepared by diluting samples from the treatment and control groups 5 times with deionized water;

(60) after the Gas chromatography (GC) instrument is stabilized and under the same conditions, 2 μL each of the ethylene oxide standards and the diluted samples to be analyzed were injected into the GC instrument, wherein each sample was measured twice in parallel;

(61) qualitive determination was conducted according to the retention time and quantitative calculation on each peak area was performed to take the average value;

(62) an ethylene oxide standard curve was plotted according to the measurement data of the ethylene oxide standards, and the concentrations of residual ethylene oxide within each sample from the control and treatment groups were found based on the peak area corresponding to ethylene oxide thereof; and

(63) the degradation rate of ethylene oxide for each sample was calculated according to the following formula: Degradation Rate (%)=(Control Group Concentration—Treatment Group Concentration)/Control Group Concentration×100; specifically, the degradation rates of Treatment groups 1 and 2 were calculated based on Control Group 1, while those of Treatment groups 3 and 4 calculated based on Control Group 2.

(64) Other details of the experiment include Column: Chromosorb 101HP60-80 mesh, glass column 2 m long, diameter 3 mm Column temperature: 120° C. Detector: 150° C., Gasifier: 150° C.; Carrier gas volume: Nitrogen: 35 ml/min, Hydrogen: 35 ml/min, Air: 350 ml/min, and the pre-column pressure is about 108 Kpa.

(65) Additionally, promotion in the degradation ability for ethylene oxide of the strain before and after acclimation was calculated according to the following formula:

(66) Promotion of degradation ability (%)=(Degradation Rate (%) of the strain after acclimation—Degradation Rate (%) of the strain before acclimation)/Degradation Rate (%) of the strain before acclimation.

(67) 2. Experimental Results

(68) The experimental results are shown in Table 3 and FIGS. 4A to 6B. As shown in Table 3, Acetobacter Peroxydans EO-01 original strain, Lactobacillus fermentum EO-02 original strain, and Bacillus subtilis EO-03 original strain were acclimated to obtain outstanding resistance and significant degradability against high concentration of ethylene oxide, capable of degrading high concentration of ethylene oxide with no or low carbon source. Specifically, for 400 mg/L ethylene oxide, the EO-01, EO-02, and EO-03 strains after acclimation have degradation rates of 63.82%, 83.93%, and 72.96%, respectively, which were higher than the original strains before acclimation by 290.81%, 327.56%, and 319.31%, respectively; and for 800 mg/L ethylene oxide, EO-01, EO-02, and EO-03 strains after acclimation have degradation rates of 51.28%, 52.54%, and 57.19%, respectively, which were higher than the original strains before acclimation by 758.96%, 762.73%, and 545.5%, respectively.

(69) TABLE-US-00003 TABLE 3 Comparative experiment results of ethylene oxide degradation by EO-01, EO-02 and EO-03 strains before and after induced acclimation EO concentration EO concentration after test (mg/L) Degradation rate (%) Promotion of before test Before After Control Before After degradation ability Strain (mg/L) acclimation acclimation group acclimation acclimation (%) EO-01 800 568.8 294.7 604.9  5.97% 51.28% 758.96% 400 192.2 83.1 229.7 16.33% 63.82% 290.81% EO-02 800 566.1 286.1 602.8  6.09% 52.54% 762.73% 400 187.1 37.4 232.8 19.63% 83.93% 327.56% EO-03 800 547.3 257.1 600.5  8.86% 57.19%  545.5% 400 190.3 62.3 230.4 17.40% 72.96% 319.31%

Example 5—Treatment of Ethylene Oxide Sterilization Waste Gas

(70) In general, ethylene oxide sterilization waste gas can be absorbed into water. The water containing the absorbed ethylene oxide can be contacted with a strain of the present invention in a method of biodegrading ethylene oxide. The water containing the absorbed ethylene oxide can be discharged or transferred to an anaerobic vessel, such as an anaerobic sewage tank. A strain of the present invention can then be added to the tank, thereby biodegrading the ethylene oxide.

(71) In particular, 1) After the ethylene oxide sterilizer has sterilized, the ethylene oxide sterilization exhaust gas generated is fed into a hydration system, which uses the internal circulating water to absorb the incoming ethylene oxide sterilization exhaust gas, and several cycles of absorption produce ethylene oxide wastewater containing ethylene oxide.

(72) (2) The waste water was passed into an anaerobic ethylene oxide treatment cell inoculated with EO-01, EO-02, and EO-03 strains, the strain concentration was 10.sup.10-10.sup.12 cfu/mL, the inoculation amount was 1%-2%, the strain(s) used the active sludge in the anaerobic ethylene oxide treatment cell as the culture, and ethylene oxide was used as the carbon source and energy for metabolism, growth and proliferation, thus achieving the purpose of ethylene oxide treatment.

(73) The wastewater was treated in an anaerobic biological ethylene oxide treatment cell inoculated with the strain mixture, the mixture in the treatment cell was continuously stirred, the temperature was controlled at 32° C.-42° C. and the treatment time was 48 hours. The residual concentration of ethylene oxide in the treated wastewater was 25.89 mg/L with a treatment efficiency of 85.64%.

(74) The above concentrations were detected by gas chromatography in accordance with GB 15979-2002 (Appendix D), which is explained above. The degradation rate was calculated according to the following formula: Degradation rate=(starting concentration−residual concentration)/starting concentration.

(75) As another practical application, activated sludge can be contacted with a strain of the present invention, thereby biodegrading ethylene oxide in the activated sludge.

(76) Comparative tests and applications may be carried out in other samples containing ethylene oxide, such as sewage, sludge, exhaust gas, or wastewater, such as industrial (including industries related to petroleum and derivative products), medical treatment (such as ethylene oxide sterilant) and other sewage, sludge, exhaust gas, or wastewater using strains of the invention

(77) In the above-described tests and applications, the degradation rate is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 1000%, 1100%, 1200%, 1300%, 1400%, or 1500% greater relative to the degradation rate of ethylene oxide in the absence of the Acetobacter peroxydans strain EO-01, the Lactobacillus fermentum strain EO-02; the Bacillus subtilis strain EO-03, the Alcaligenes faecalis strain EO-05 or Alcaligenes faecalis strain comprising the 16S rDNA sequence of SEQ ID NO: 6, the Acetobacter peroxydans strain with the 16S rDNA of SEQ ID NO: 3; the Lactobacillus fermentum strain with the 16S rDNA of SEQ ID NO: 4; or the Bacillus subtilis strain with the 16S rDNA of SEQ ID NO: 5.

(78) The detailed embodiments described herein are only for the purpose of illustrating the present disclosure and are not intended to limit the scope of the present disclosure in any way. It would be understood by a person skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present disclosure. Such changes and modifications are contemplated by the present disclosure, the scope of which should only be defined by the following claims.