<i>Alcaligenes faecalis </i>for degrading ethylene oxide

Abstract

The present disclosure provides an Alcaligenes faecalis strain capable of degrading ethylene oxide and uses thereof. The deposit number of the strain is CGMCC No. 18435. This strain can be used in pollution treatment, for example, to treat industrial sewage or wastewater containing ethylene oxide, which greatly improves the decontamination ability of ethylene oxide in manufacturing industries. The present disclosure also provides a degradation agent for degrading ethylene oxide and a method for biodegrading ethylene oxide.

Claims

1. A product which is a biologically pure culture of Alcaligenes faecalis strain EO-05 with the Deposit Number of CGMCC No. 18435 capable of biodegrading ethylene oxide at a higher rate than a naturally occurring Alcaligenes faecalis.

2. The product according to claim 1, wherein the product is prepared by culturing the Alcaligenes faecalis strain EO-05.

3. The product according to claim 1, wherein the Alcaligenes faecalis strain EO-05 has a concentration of at least 10.sup.8 cfu/mL.

4. A method for biodegrading ethylene oxide or decreasing the amount of ethylene oxide in a material comprising adding to the material comprising ethylene oxide an amount of the product of claim 1.

5. The method according to claim 4, wherein the method has a degradation rate of at least 10% greater relative to the degradation rate of ethylene oxide in the absence of the product.

6. The method according to claim 4, wherein the material comprising ethylene oxide comprises sewage or wastewater.

7. The method according to claim 6, wherein the wastewater is selected from the group consisting of wastewater from a petrochemical process and wastewater from a medical process.

8. A method for biodegrading ethylene oxide or decreasing the amount of ethylene oxide in material, wherein the method comprises: inoculating the product of claim 1 into tryptone soy broth culture medium for 24-48 hours of incubation at 37° C. using a shaker at 200 rpm to obtain an Alcaligenes faecalis EO-05 culture; and adding to the material comprising ethylene oxide an amount of the Alcaligenes faecalis EO-05 culture.

9. The method according to claim 4, wherein the product 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) FIG. 1 shows a photo of bacterial colony growth of the EO-degrading potential bacteria in enrichment medium without ethylene oxide at a constant temperature of 37° C. for 48 hours, wherein the EO-degrading potential bacteria was obtained by the screening and purification processes according to Example 1;

(2) FIG. 2 shows the Gram staining result of the EO-degrading potential bacteria in the identification tests according to Example 2, wherein the EO-degrading potential bacteria was obtained by the screening and purification processes according to Example 1;

(3) FIG. 3 shows the phylogenetic evolution diagram of the EO-degrading potential bacteria in the identification tests according to Example 2, wherein the EO-degrading potential bacteria was obtained by the screening and purification processes according to Example 1;

(4) FIG. 4 shows a photo of bacterial colony growth of the EO-05 strain in a carbon-free medium with 800 mg/L of ethylene oxide at a constant temperature of 37° C. for 48 hours in the comparative EO degradation test according to Example 4, wherein the EO-05 strain was obtained by the inductive acclimation processes according to Example 3; and

(5) FIG. 5 shows a photo of bacterial colony growth of the EO-degrading potential bacteria (i.e., the original strain before acclimation) in a carbon-free medium with 800 mg/L of ethylene oxide at a constant temperature of 37° C. for 48 hours in the comparative EO degradation test according to Example 4, wherein the EO-degrading potential bacteria was obtained according to Example 1.

DETAILED DESCRIPTION OF EMBODIMENTS

(6) Detailed description will be given below with referral to the accompanying figures to facilitate understanding of the present application. Preferred embodiments are shown in the figures. However, the present application 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.

(7) 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 application. The term “and/or” as used herein refers to any and all combinations of one or more items recited.

Example 1

Screening and Purification of Bacteria Strains with EO-Degrading Ability

(8) This is an example of the screening and purification of bacteria strains with EO-degrading ability.

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

(10) 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.

(11) Liquid enrichment medium (also known as TSB medium) was prepared as follows: 17 g of tryptone, 3 g of soy peptone, 5 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate and 2.5 g of glucose were mixed, adjusted to a pH of 7.4, and added to 1000 mL of distilled water, thoroughly mixed. Portions of 250 ml of the prepared medium was added to 500 mL Erlenmeyer flasks, sterilized at 121° C. for 20 min, and cooled to room temperature. Pure ethylene oxide liquid was placed on an ice box and 28 μL of ethylene oxide liquid was taken and injected into the sterilized medium by a sealed syringe, providing 100 mg/L of ethylene oxide in the medium complying with the national emission standard.

(12) 1 mL of the suspension was added to each of 4 test tubes containing 10 mL of liquid enrichment medium and placed in a shaker for oxygen-consuming enrichment culture for 24-48 h (200 rpm, 37° C.).

(13) Selection medium and selection culture plates were prepared as follows: 17 g of tryptone, 3 g of soy peptone, 5 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose and 15 g of agar were mixed, adjusted to a pH of 7.4, and added to 1000 mL of distilled water, thoroughly mixed. Portions of 250 ml of the prepared selection medium was added into 500 mL Erlenmeyer flasks, sterilized at 121° C. for 20 minutes, and cooled to about 50-56° C. 28 μL of ethylene oxide liquid was injected into the sterilized medium by a sealed syringe to make a selection culture plate.

(14) The dominant strains in the liquid enrichment medium were streaked on the selection culture plate for separation of the EO-degrading potential bacteria. As shown in FIG. 1, at 48 hours of culture, the colony was characterized by gray-white color, uneven edges, spreading growth, and with a colony diameter of 4.0-6.0 mm and blue-green fluorescent pigments.

(15) Single colonies were selected and cultured in the liquid enrichment medium without ethylene oxide for 24 hours to obtain EO-degrading potential bacteria, which were preserved at −80° C. using the glycerin preservation method (culture medium: 50% glycerol=1:

Example 2

Characterization and Identification of EO-Degrading Bacteria Strains

(16) This is an example of characterization and identification of EO-degrading bacteria strains, using the following identification methods:

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

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

(19) Molecular biological characterization (The DNA in the genome that produces the ribosomal RNA is called the “rRNA gene” or simply “rDNA.” 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:

(20) TABLE-US-00001 Upstream primer 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, as shown in  SEQ ID NO: 1; and Downstream primer 1492R: 5′-GGTTACCTTGTTACGACTT-3′, as shown in SEQ ID NO: 2.

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

(22) The characterization and identification results are as follows:

(23) Morphological characteristics: gray-white colonies, uneven edges, spreading growth, colony diameter 4.0-6.0 mm, with blue-green fluorescent pigment. Under microscope, the bacterial cells were short rods or spherical-shaped, separately arranged and without spores. The Gram stain gave a negative result as shown in FIG. 2;

(24) Molecular biological characteristics: the sequencing result of 16s rDNA is shown in SEQ ID NO: 3; the 16S rDNA sequence was subjected to nucleotide sequence alignment analysis by BLAST and showed a 99% sequence homology with Alcaligenes faecalis. The phylogenetic tree of this strain is shown in FIG. 3.

(25) According to the characterization results of morphology, physiology, biochemistry, and molecular biology, EO-degrading potential bacteria strain obtained by screening and purification according to Example 1 was Alcaligenes faecalis.

Example 3

Inductive Acclimation of EO-Degrading Potential Bacteria Strains

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

(27) Phase I: Inductive Acclimation of Ethylene Oxide Tolerance

(28) The tolerance acclimation medium and culture plates were prepared as follows: 10 g of peptone, 40 g of glucose and 15 g of agar were mixed, adjusted to a pH of 5.4-5.8, and added to 1000 mL of distilled water, thoroughly mixed. The prepared culture medium was divided into portions of 250 mL and sterilized at 121° C. for 20 min. Before use, the medium was heated to melt, allowed to cool to about 50-56° C., and added with 25 mg, 50 mg, 125 mg or 200 mg of ethylene oxide respectively by a sealed syringe to make medium plates with four different concentrations of ethylene oxide (100 mg/L, 200 mg/L, 500 mg/L or 800 mg/L).

(29) Using the method of plate streaking, the EO-degrading potential bacteria obtained from Example 1 was inoculated onto the tolerance acclimation medium with 100 mg/L ethylene oxide and incubated at a constant temperature of 37° C. for 48 h. Then the single colony with the largest radius was selected and subcultured onto the tolerance acclimation medium with 200 mg/L ethylene oxide and incubated at 37° C. for 48 h. Again, the single colony with the largest colony radius on the plate was selected and subcultured onto the tolerance acclimation medium with 500 mg/L ethylene oxide and incubated at a constant temperature of 37° C. for 48 h. The single colony with the largest colony radius on the plate was selected and subcultured onto the tolerance acclimation medium with 800 mg/L ethylene oxide and incubated at a constant temperature of 37° C. for 48 h. Then the single colony with the largest colony radius on the plate was selected as a strain with tolerance against high concentration of ethylene oxide.

(30) Phase II: Inductive Acclimation of Ethylene Oxide Degradation Ability

(31) The degradation acclimation medium and culture plates were prepared as follows: 10 g of peptone, glucose (20 g, 12 g, 4 g, or 0 g) and 15 g of agar were mixed, adjusted to a pH of 5.4-5.8, and added to 1000 mL of distilled water, thoroughly mixed to make mediums of four different carbon contents (50%, 30%, 10%, or 0%). The medium prepared as above were divided into 250 ml portions and sterilized at 121° C. for 20 min. Before use, the medium was heated to melt, allowed to cool to about 50-56° C., and added with 200 mg of ethylene oxide by a sealed syringe to make four types of culture plates with different amounts of carbon source (50%, 30%, 10%, or 0%) and 800 mg/L of ethylene oxide.

(32) Using the method of plate streaking, the strain with tolerance against high concentration of ethylene oxide obtained from Phase I was inoculated onto the degradation acclimation medium with 800 mg/L ethylene oxide and 50% carbon source, and incubated at a constant temperature of 37° C. for 48 h. Then the single colony with the largest radius was selected and subcultured onto the degradation acclimation medium with 800 mg/L ethylene oxide and 30% carbon source and incubated at 37° C. for 48 h. Again, the single colony with the largest colony radius on the plate was selected and subcultured onto the degradation acclimation medium with 800 mg/L ethylene oxide and 10% carbon source and incubated at a constant temperature of 37° C. for 48 h. The single colony with the largest colony radius on the plate was selected and subcultured onto the degradation acclimation medium with 800 mg/L ethylene oxide and 0% carbon source and incubated at a constant temperature of 37° C. for 48 h. The single colony with the largest colony radius on the plate was selected as a strain with strong tolerance and degradation ability against high concentration of ethylene oxide, which was then designated as the EO-05 strain.

(33) The EO-05 strain was preserved on agar medium slope with corresponding nutrients at −80° C. using the glycerin preservation method (culture medium: 50% glycerol=1:1).

(34) The results of Phase I and Phase II inductive acclimation of the bacteria strains were summarized in Table 1. It shows that the strain EO-05 was obtained with strong tolerance and degradation ability against high concentration of ethylene oxide by gradual control of the cultivation conditions of the dominant strains against ethylene oxide. The EO-05 strain was able to use ethylene oxide as a carbon source and grow normally with ethylene oxide being the sole carbon source in the culture.

(35) TABLE-US-00002 TABLE 1 Experiment results of induced acclimation of EO tolerance (Phase I) and EO degradation ability (Phase II) 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) Colony growth + + + + + + + + Note: “+” represents bacterial growth.

(36) The 16s rDNA sequence of the strain EO-05 acclimated in Example 3 was sequenced and the sequencing result is shown in SEQ ID NO: 4.

(37) The Alcaligenes faecalis EO-05 strain was deposited on Aug. 29, 2019 at China General Microbiological Culture Collection Center, with the deposit number being CGMCC No. 18435 and the deposit address being Institute of Microbiology of Chinese Academy of Sciences, NO. 1 West Beichen Road, Beijing 100101, China.

Example 4

Comparative Test

(38) This is an example testing the ability of the EO-05 strain to degrade ethylene oxide.

(39) I. Experimental Method:

(40) 1. Culture and activation: the EO-05 strain and the EO-degrading potential bacteria (i.e., the original strain before acclimation) were taken out from −80° C. refrigerator and 10 μL of each was inoculated in 100 mL tryptone soy broth (TSB) medium, respectively, and cultivate for 48 h (37° C., 200 rpm). The number of cells in the culture liquid is 10.sup.10-10.sup.12 cfu/mL.

(41) 2. Nutrient broth liquid culture medium was made as follows: 10 g peptone and 5 g sodium chloride were added to 1000 mL of distilled water, divided into 400 mL portions, sterilized at 121° C. for 20 min, and cool to room temperature for storage. To make liquid medium containing no carbon source but 400 mg/L or 800 mg/L ethylene oxide, 160 mg or 320 mg of ethylene oxide, respectively, were injected to the medium with a closed syringe before use.

(42) 3. Comparative Test of Ethylene Oxide Degradation

(43) 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.

(44) Treatment Group 1 (EO-05 strain/800 mg/L ethylene oxide): 5 mL of the bacterial culture of the pure cultured strain EO-05 obtained according to Example 3, with a live cell count of 10.sup.10-10.sup.12 cfu/mL, was inoculated into 400 mL of nutrient broth liquid medium containing no carbon source but 800 mg/L ethylene oxide as the inducer, with a live bacteria cell count in the medium being 10.sup.8-10.sup.10 cfu/mL. The cell culture result after 48 hours is shown in FIG. 4.

(45) Treatment Group 2 (original strain before acclimation/800 mg/L ethylene oxide): 5 mL of the bacterial culture of the EO-degrading potential bacteria (i.e., the original strain before acclimation) obtained according to Example 1, with a live cell count of 10.sup.10-10.sup.12 cfu/mL, was inoculated into 400 mL of nutrient broth liquid medium containing no carbon source but 800 mg/L ethylene oxide as the inducer, with a live bacteria cell count in the medium being 10.sup.8-10.sup.10 cfu/mL. The cell culture result after 48 hours is shown in FIG. 5.

(46) Control group 1 (No inoculation/800 mg/L ethylene oxide): Nutrient broth liquid medium containing no carbon source but 800 mg/L ethylene oxide without inoculation of the EO-05 strain or EO-degrading potential bacteria.

(47) Treatment Group 3 (EO-05 strain/400 mg/L ethylene oxide): 5 mL of the bacterial culture of the pure cultured strain EO-05 obtained according to Example 3, with a live cell count of 10.sup.10-10.sup.12 cfu/mL, was inoculated into 400 mL of nutrient broth liquid medium containing no carbon source but 400 mg/L ethylene oxide as the inducer, with a live bacteria cell count in the medium being 10.sup.8-10.sup.10 cfu/mL;

(48) Treatment Group 4 (original strain before acclimation/400 mg/L ethylene oxide): 5 mL of the bacterial culture of the EO-degrading potential bacteria (i.e., the original strain before acclimation) obtained according to Example 1, with a live cell count of 10.sup.10-10.sup.12 cfu/mL, was inoculated into 400 mL of nutrient broth liquid medium containing no carbon source but 400 mg/L ethylene oxide as the inducer, with a live bacteria cell count in the medium being 10.sup.8-10.sup.10 cfu/mL; and

(49) Control Group 2 (No inoculation/400 mg/L ethylene oxide): Nutrient broth liquid medium containing no carbon source but 400 mg/L ethylene oxide without inoculation of the EO-05 strain or EO-degrading potential bacteria.

(50) 4. Gas Chromatography (GC) Analysis

(51) To calculate the concentrations of residual ethylene oxide and the degradation rates, samples were taken from the above Treatment groups 1-4 and Control groups 1-2 after the comparative test and sent to the Shaanxi Provincial Center for Disease Control and Prevention for gas chromatography analysis according to the methods described in “Sanitary Standards for Disposable Hygiene Products” (GB15979-2002) of China National Standards as follows: a series of ethylene oxide standards of 0-200 mg/L concentrations were made by taking a certain volume of pure ethylene oxide gas with a sealed syringe for dissolving in deionized water; the subject samples to be analyzed were prepared by diluting samples from the treatment and control groups 5 times with deionized water; after the GC instrument with hydrogen flame ionization detector (FID) 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; qualitive determination was conducted according to the retention time and quantitative calculation on each peak area was performed to take the average value; 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 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.

(52) Additionally, the percentage of increase in the ethylene oxide degradation ability of the strain before and after acclimation was calculated according to the following formula:

(53) Percentage of increase in degradation ability (%)=(Degradation Rate (%) of the strain after acclimation−Degradation Rate (%) of the strain before acclimation)/Degradation Rate (%) of the strain before acclimation).

(54) 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 Kpaa.

(55) II. Experimental Results

(56) The experimental results are summarized in Table 2 below. As shown in Table 2, the EO-05 strain was capable of degrading ethylene oxide with concentrations as high as 400 mg/L and 800 mg/L with no carbon source, while the degradation rates were greatly improved compared to the original strain without acclimation. Specifically, the degradation rate of EO-05 strain of 400 mg/L ethylene oxide was as high as 92.90%, which was 350.97% higher than that of the original strain without acclimation. For 800 mg/L ethylene oxide, the EO-05 strain demonstrated a degradation rate of 68.65% and an increase of 585.12% compared to the original strain without acclimation.

(57) TABLE-US-00003 TABLE 2 Comparative ethylene oxide degradation experiment (no carbon source) of Alcaligenes faecalis EO-05 strain and the original Alcaligenes faecalis strain before acclimation EO concentration EO concentration Degradation Test Test before test after test Rate Group strain (mg/L) (mg/L) (%) Treatment EO-05 800 160.5 68.65% Group 1 strain Treatment Original 800 460.6 10.02% Group 2 strain Control N/A 800 511.9 / Group 1 Treatment EO-05 400 16.4 92.90% Group 3 strain Treatment Original 400 183.5 20.60% Group 4 strain Control N/A 400 231.1 / Group 2

(58) Comparative tests 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.

(59) An Alcaligenes faecalis strain of the invention comprising the 16S rDNA sequence of SEQ ID NO: 4 can also be used in comparative tests.

Example 5

Treatment of Ethylene Oxide Sterilization Waste Gas

(60) In general, ethylene oxide sterilization waste gas can be absorbed into water. The water containing the absorbed ethylene oxide can be contacted with an Alcaligenes faecalis 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. An Alcaligenes faecalis strain of the present invention can then be added to the tank, thereby biodegrading the ethylene oxide.

(61) 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 about 243.15 mg/L of ethylene oxide.

(62) (2) The ethylene oxide wastewater with the concentration of about 243.15 mg/L of ethylene oxide was passed into an aerobic bio-ethylene oxide treatment cell inoculated with the EO-05 strain, the strain concentration was 10.sup.10-10.sup.12 cfu/mL, the inoculation amount was 1%-2%, the EO-05 strain used the active sludge in the aerobic bio-ethylene oxide treatment cell as the culture, ethylene oxide was used as the carbon source and energy for metabolism, growth and proliferation, thus achieving the purpose of ethylene oxide treatment.

(63) The mixture in the treatment cell was continuously stirred, the temperature was controlled at 32° C.-42° C. and the treatment was for 48 hours. The results showed that the residual concentration of ethylene oxide in the treated wastewater was 20.96 mg/L with a treatment efficiency of 91.38%.

(64) 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.

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

(66) 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%, or 500% greater relative to the degradation rate of ethylene oxide in the absence of the Alcaligenes faecalis strain EO-05 or Alcaligenes faecalis strain comprising the 16S rDNA sequence of SEQ ID NO: 4.

(67) 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.