METHOD OF REDUCING CONCENTRATIONS OF ONE OR MORE OF N2O and NO IN MEDIUM

Abstract

A method of reducing a concentration of N.sub.2O, NO, or a combination thereof in a medium, the method comprising: culturing a microorganism of the genus Paracoccus, a microorganism of the genus Pseudomonas, or a combination thereof in a liquid medium comprising Mg.sup.2+ ions and Fe(II)(L)-NO, N.sub.2O, or a combination thereof, wherein L is a chelating agent; and reducing NO to N.sub.2O or N.sub.2, or reducing N.sub.2O to N.sub.2.

Claims

1. A method of reducing a concentration of N.sub.2O, NO, or a combination thereof in a medium, the method comprising: culturing a microorganism of the genus Paracoccus, a microorganism of the genus Pseudomonas, or a combination thereof in a liquid medium comprising Mg.sup.2+ ions and Fe(II)(L)-NO, N.sub.2O, or a combination thereof, wherein L is a chelating agent; and reducing NO to N.sub.2O or N.sub.2, or reducing N.sub.2O to N.sub.2.

2. The method of claim 1, wherein the microorganism of the genus Paracoccus comprises Paracoccus versutus, Paracoccus denitrificans, Paracoccus pantothrophas, Paracoccus ferrooxidans, Paracoccus denitrificans, or a combination thereof.

3. The method of claim 1, wherein the microorganism of the genus Pseudomonas comprises Pseudomonas stutzeri, Pseudomonas putida, Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas mendocina, or a combination thereof.

4. The method of claim 1, wherein the liquid medium comprises about 0.1 millimolar to about 7.5 millimolar of Mg.sup.2+ ions.

5. The method of claim 1, wherein the L is ethylenediamine, diethylenetriamine, triethylenetetraamine, hexamethylenetetraamine, N-(2-hydroxyethyl)ethylenediamine-triacetic acid, ethylenediamine-tetraacetic acid, iminodiacetic acid, nitrilotriacetic acid, or diethylenetriaminepentaacetic acid.

6. The method of claim 1, wherein the liquid medium enables growth of the microorganism of the genus Paracoccus or growth of the microorganism of the genus Pseudomonas.

7. The method of claim 1, wherein the culturing is performed under anaerobic conditions.

8. The method of claim 1, wherein the culturing is performed at a temperature of about 25° C. to about 40° C.

9. The method of claim 1, wherein the culturing comprises culturing of the microorganism of the genus Paracoccus alone, culturing of the microorganism of the genus Pseudomonas alone, or culturing of a mixture of the microorganism of the genus Paracoccus and the microorganism of the genus Pseudomonas.

10. The method of claim 1, wherein the method further comprises forming of Fe(II)EDTA-NO by contacting NO.sub.2 or NO with a liquid medium comprising Fe(II)EDTA, and the culturing is performed at the same time as the forming of Fe(II)EDTA-NO or after the forming of Fe(II)EDTA-NO.

11. The method of claim 1, wherein the concentration of Fe(II)(L)-NO in the liquid medium is about 0.1 millimolar to about 20 millimolar.

12. The method of claim 1, wherein the liquid medium further comprises an electron donor.

13. The method of claim 12, wherein the electron donor is methanol, ethanol, acetate, glucose, or a combination thereof.

14. The method of claim 1, wherein the liquid medium is a buffer.

15. The method of claim 1, wherein the liquid medium comprises an LB medium, an M9 medium, a phosphate buffer, a Tris buffer, or a combination thereof.

16. The method of claim 1, wherein the N.sub.2O or the NO is derived from a waste gas or a wastewater.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0020] FIG. 1 is a graph of relative N.sub.2O retention rate (percent, %) versus test sample, which shows the relative retention rate of N.sub.2O in a gas layer in a glass serum bottle after culturing Paracoccus versutus in a N.sub.2O-containing medium in the presence or absence of magnesium ions;

[0021] FIG. 2 is a graph of relative retention rate (%) versus test sample, which shows the relative retention rates of NO, N.sub.2O, and N.sub.2 in a gas layer in a glass serum bottle after culturing Paracoccus versutus in a Fe(II)EDTA-NO-containing medium in the presence or absence of magnesium ions;

[0022] FIG. 3 is a graph of relative N.sub.2O retention rate (%) versus test sample, which shows relative retention rates of N.sub.2O in a gas layer in a glass serum bottle after culturing Paracoccus versutus in a 2.5 mM N.sub.2O-containing medium in the presence or absence of Mg.sup.2+, Ca.sup.2+, Mn.sup.2+, Mo.sup.2+, Cu.sup.2+, or Co.sup.2+ ions; and

[0023] FIG. 4 is a graph of relative retention rate (%) versus test sample, which shows the relative retention rates of NO, N.sub.2O, and N.sub.2 in a gas layer in a glass serum bottle after culturing Paracoccus versutus or Pseudomonas stutzeri in a Fe(II)EDTA-NO-containing medium in the presence or absence of magnesium ions.

DETAILED DESCRIPTION

[0024] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects.

[0025] As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0026] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. “At least one” is not to be construed as limiting “a” or “an.” As used herein, “a,” “an,” “the,” and “at least one” do not denote a limitation of quantity, and are intended to cover both the singular and plural, unless the context clearly indicates otherwise. For example, “an element” has the same meaning as “at least one element,” unless the context clearly indicates otherwise. “Or” means “and/or.” As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and/or “comprising,” or “includes” and/or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

[0027] “About” or “approximately” as used herein is inclusive of the stated value and means within an acceptable range of deviation for the particular value as determined by one of ordinary skill in the art, considering the measurement in question and the error associated with measurement of the particular quantity (i.e., the limitations of the measurement system). For example, “about” can mean within one or more standard deviations, or within ±30%, 20%, 10% or 5% of the stated value.

[0028] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0029] Some microorganisms of the genus Pseudomonas or the genus Paracoccus are known to biologically reduce nitrogen oxides. A denitrification process using these microorganisms makes it possible to remove almost all nitrates and nitrites. However, in such a biological denitrification process, it is difficult to convert N.sub.2O and NO compounds into molecular nitrogen, and therefore, it is difficult for a treatment system to completely remove them.

[0030] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to these exemplary embodiments.

Example 1: Effect of Magnesium Ions on N.SUB.2.O Removal by Paracoccus versutus

[0031] In this exemplary embodiment, the effect of magnesium ions on N.sub.2O removal by the bacterium Paracoccus versutus was examined. In detail, Paracoccus versutus was primarily cultured in a 250 milliliter (ml) Erlenmeyer flask containing an LB medium (10 grams per liter (g/L) Tryptone, 10 g/L NaCl, 5 g/L Yeast Extract), and then cells of the cultured microorganism were isolated by centrifugation. Next, the isolated cells were washed with an M9 medium (6.78 g/L of Na.sub.2HPO.sub.4, 3 g/L of KH.sub.2PO.sub.4, 0.5 g/L of NaCl, 1 g/L of NH.sub.4Cl, 0.1 mg/L of MnSO.sub.4, 0.1 mg/L of Na.sub.2MoO.sub.4, 0.1 mg/L of CuSO.sub.4 5H.sub.2O, and 2 mg/L of CaCl.sub.2). The washed cells were mixed with 30 ml of M9 medium in a 60 ml glass serum bottle, i.e., a serum bottle sealed with a silicone stopper and an aluminum cap, so that the cell concentration in the medium was resulted in an optical density (O.D.) of 1 (OD=1). Then, 2.5 millimolar (mM) N.sub.2O was injected using a syringe into the glass serum bottle containing the cells and the M9 medium, and the serum bottle was sealed with a silicone stopper and an aluminum cap, followed by incubation of the serum bottle at 30° C. for 7 hours with shaking. The upper gas layer in the glass serum bottle was collected, and the concentration of N.sub.2O or N.sub.2 was measured by gas chromatography mass-spectrometry (GC-MS). In this regard, an M9 medium containing no cells was used as a negative control group, an M9 medium containing cells without magnesium ions was used as a positive control group, and an M9 medium containing 1 mM magnesium ions and cells was used as an experimental group.

[0032] FIG. 1 shows a relative retention rate of N.sub.2O in the gas layer present in the glass serum bottle after culturing the Paracoccus versutus in the N.sub.2O-containing medium in the presence or absence of magnesium ions. The relative retention rate was calculated according to the following equation.

Relative retention rate (%)=(Remaining amount/Initial injection amount)×100%

[0033] Table 1 is a table showing the conversion rate of N.sub.2O to N.sub.2, through the reduction of N.sub.2O and the production of N.sub.2 in the gas layer in the serum bottle after culturing Paracoccus versutus in the N.sub.2O-containing medium in the presence, or absence, of magnesium ions.

Conversion rate (%)=((initial injection amount−remaining amount)/initial injection amount)×100%

TABLE-US-00001 TABLE 1 M9 0 mM Mg.sup.2+- 1 mM Mg.sup.2+- medium containing M9 containing M9 Sample (no cells) medium + cells medium + cells N.sub.2 0 0 100 conversion rate (%)

[0034] As shown in Table 1, when Paracoccus versutus was cultured in the M9 medium without Mg.sup.2+ (0 mM Mg.sup.2+-containing M9 medium), the conversion rate of N.sub.2O to N.sub.2 was 0%. In contrast, when Paracoccus versutus was cultured in the M9 medium with Mg.sup.2+ (1 mM Mg.sup.2+-containing M9 medium), the conversion rate of N.sub.2O to N.sub.2 was 100%. This indicates that when Paracoccus versutus was cultured in the M9 medium with Mg.sup.2+, 100% of the N.sub.2O was converted to N.sub.2.

Example 2: Effect of Magnesium Ions on NO Removal by Paracoccus versutus

[0035] In this exemplary embodiment, the effect of magnesium ions on NO removal by the bacterium Paracoccus versutus was examined. In detail, Paracoccus versutus was primarily cultured in a 250 ml Erlenmeyer flask containing an LB medium (10 g/L Tryptone, 10 g/L NaCl, 5 g/L Yeast Extract), and then cells of the microorganism were isolated by centrifugation. Next, the isolated cells were washed with an M9 medium (6.78 g/L of Na.sub.2HPO.sub.4, 3 g/L of KH.sub.2PO.sub.4, 0.5 g/L of NaCl, 1 g/L of NH.sub.4Cl, 0.1 mg/L of MnSO.sub.4, 0.1 mg/L of Na.sub.2MoO.sub.4, 0.1 mg/L of CuSO.sub.4 5H.sub.2O, 2 mg/L of CaCl.sub.2)), and the washed cells were mixed with 30 ml of M9 medium containing 5 mM ferrous ethylenediaminetetraacetate-nitric oxide (Fe(II)EDTA-NO) in a 60 ml glass serum bottle, i.e., a serum bottle sealed with a silicone stopper and an aluminum cap, so that the cell concentration in the medium resulted in an OD of 1. Then, the glass serum bottle, which contained the cells, the Fe(II)EDTA-NO, and the M9 medium, was sealed with the silicone stopper and the aluminum cap, and incubated at 30° C. for 7 hours with shaking. The upper gas layer in the glass serum bottle was collected, and the concentration of NO, N.sub.2O and N.sub.2 was measured by gas chromatography mass-spectrometry (GC-MS).

[0036] In this regard, a Fe(II)EDTA-NO-containing M9 medium without cells was used as a negative control group, a Fe(II)EDTA-NO-containing M9 medium with cells and without magnesium ions was used as a positive control group, and a Fe(II)EDTA-NO-containing M9 medium with 1 mM magnesium ions and cells was used as an experimental group.

[0037] FIG. 2 shows relative retention rates of NO, N.sub.2O, and N.sub.2 in the gas layer in the glass serum bottle after culturing Paracoccus versutus in the Fe(II)EDTA-NO-containing medium in the presence or absence of magnesium ions. The relative retention rate was calculated according to the following equation.

Relative retention rate (%)=(Remaining amount/Initial injection amount)×100%

[0038] Table 2 is a table showing the values of the relative retention rates of NO, N.sub.2O, and N.sub.2 in the gas layer in the serum bottle after culturing Paracoccus versutus in the Fe(II)EDTA-NO-containing medium in the presence or absence of magnesium ions.

TABLE-US-00002 TABLE 2 Medium 0 mM Mg.sup.2+-containing 1 mM Mg.sup.2+-containing (no cells) medium + cells medium + cells Sample NO N.sub.2O N.sub.2 NO N.sub.2O N.sub.2 NO N.sub.2O N.sub.2 Relative retention 100 0 0 0 100 0 0 0 100 rate (%, 24 hr later)

[0039] As shown in Table 2, when culturing was performed without Paracoccus versutus cells in the M9 medium without Mg.sup.2+, only NO was present, and the conversion rate of NO to either N.sub.2O or N.sub.2 was 0%. When Paracoccus versutus was cultured in the M9 medium with 0 mM Mg.sup.2+, conversion of NO into N.sub.2O was 100%, and no conversion of N.sub.2O into N.sub.2 occurred. In contrast, when Paracoccus versutus was cultured in the M9 medium with 1 mM Mg.sup.2+, 100% of NO was converted to N.sub.2.

Example 3: Effect of Divalent Cations on N.SUB.2.O Removal by Paracoccus versutus

[0040] In this exemplary embodiment, the effects of magnesium ions and five other divalent cations on NO denitrification by the bacterium Paracoccus versutus were examined. In detail, Paracoccus versutus was primarily cultured in a 250 ml Erlenmeyer flask containing an LB medium (10 g/L Tryptone, 10 g/L NaCl, 5 g/L Yeast Extract), and then cells of the microorganism were isolated by centrifugation. Next, the isolated cells were washed with an M9 medium (6.78 g/L of Na.sub.2HPO.sub.4, 3 g/L of KH.sub.2PO.sub.4, 0.5 g/L of NaCl, 1 g/L of NH.sub.4Cl, 0.1 mg/L of MnSO.sub.4, 0.1 mg/L of Na.sub.2MoO.sub.4, 0.1 mg/L of CuSO.sub.4 5H.sub.2O, 2 mg/L of CaCl.sub.2), and the washed cells were mixed with 30 ml of M9 medium in a 60 ml glass serum bottle, i.e., a serum bottle sealed with a silicone stopper and an aluminum cap, so that the cell concentration in the medium resulted in OD of 1. Then, 2.5 mM N.sub.2O was injected using a syringe into the glass serum bottle which contained the cells and the medium, and the glass serum bottle was sealed with a silicone stopper and an aluminum cap, followed by culturing at 30° C. for 7 hours with shaking. The upper gas layer in the glass serum bottle was collected, and the concentration of N.sub.2O or N.sub.2 was measured by gas chromatography mass-spectrometry (GC-MS).

[0041] In this regard, an M9 medium containing no cells was used as a negative control group, and an M9 medium containing 1 mM of Mg.sup.2+, Ca.sup.2+, Mn.sup.2+, Mo.sup.2+, Cu.sup.2+ or Co.sup.2+ ions and cells was used as an experimental group.

[0042] FIG. 3 shows the relative N.sub.2O retention rates of N.sub.2O and N.sub.2 in the gas layer in the glass serum bottle after culturing Paracoccus versutus in the presence of 2.5 mM N.sub.2O-containing medium in the presence or absence of Mg.sup.2+, Ca.sup.2+, Mn.sup.2+, Mo.sup.2+, Cu.sup.2+, or Co.sup.2+ ions. The relative retention rate was calculated according to the following equation.

Relative retention rate (%)=(Remaining amount/Initial injection amount)×100%

[0043] Table 3 is a table showing the relative retention rates of N.sub.2O and N.sub.2 in the gas layer in the serum bottle after culturing Paracoccus versutus in the N.sub.2O-containing medium in the presence or absence of Mg.sup.2+, Ca.sup.2+, Mn.sup.2+, Mo.sup.2+, Cu.sup.2+ or Co.sup.2+ ions.

TABLE-US-00003 TABLE 3 Medium Mg.sup.2+ Ca.sup.2+ Mn.sup.2+ Mo.sup.2+ Cu.sup.2+ Co.sup.2+ Medium N.sub.2O N.sub.2 N.sub.2O N.sub.2 N.sub.2O N.sub.2 N.sub.2O N.sub.2 N.sub.2O N.sub.2 N.sub.2O N.sub.2 N.sub.2O N.sub.2 Retention rate 100 0 0 100 95 0 97 0 97 0 95 0 96 0 (%, 7 hr later)

[0044] As shown in Table 3, when Paracoccus versutus cells were cultured in the M9 medium with Ca.sup.2+, Mn.sup.2+, Mo.sup.2+, Cu.sup.2+ or Co.sup.2+ ions, only N.sub.2O was present, and the conversion rate of N.sub.2O to N.sub.2 was 0%. When Paracoccus versutus was cultured in the M9 medium with 1 mM Mg.sup.2+, 100% of N.sub.2O was converted to N.sub.2.

Example 4: Effect of NO Removal by Paracoccus versutus or Pseudomonas stutzeri

[0045] In this exemplary embodiment, the effect of magnesium ions on NO removal by Paracoccus versutus or Pseudomonas stutzeri (purchased from Biological Resource Center, KCTC) was examined. In detail, Paracoccus versutus or Pseudomonas stutzeri was primarily cultured in a 250 ml Erlenmeyer flask containing an LB medium (10 g/L Tryptone, 10 g/L NaCl, 5 g/L Yeast Extract), and then the cells of the microorganism were isolated by centrifugation. Next, the isolated cells were washed with an M9 medium (6.78 g/L of Na.sub.2HPO.sub.4, 3 g/L of KH.sub.2PO.sub.4, 0.5 g/L of NaCl, 1 g/L of NH.sub.4Cl, 0.1 mg/L of MnSO.sub.4, 0.1 mg/L of Na.sub.2MoO.sub.4, 0.1 mg/L of CuSO.sub.4 5H.sub.2O, 2 mg/L of CaCl.sub.2), and the washed cells were mixed with 30 ml of 5 mM Fe(II)EDTA-NO-containing M9 medium in a 60 ml glass serum bottle, i.e., a serum bottle sealed with a silicone stopper and an aluminum cap, so that the cell concentration in the medium resulted in an OD of 1. Then, the glass serum bottle containing the cells, Fe(II)EDTA-NO, and the medium, was sealed with a silicone stopper and an aluminum cap, and incubated at 30° C. for 7 hours with shaking. The upper gas layer in the glass serum bottle was collected, and the concentration of NO, N.sub.2O, and N.sub.2 was measured by gas chromatography mass-spectrometry (GC-MS).

[0046] FIG. 4 shows the relative retention rates of NO, N.sub.2O, and N.sub.2 in the gas layer in the serum bottle after culturing Paracoccus versutus or Pseudomonas stutzeri in the Fe(II)EDTA-NO-containing medium in the presence or absence of magnesium ions. The relative retention rate was calculated according to the following equation.

Relative retention rate (%)=(Remaining amount/Initial injection amount)×100%

[0047] As shown in FIG. 4, when culturing was performed without Paracoccus versutus cells in the M9 medium without Mg.sup.2+, most of the NO was converted to N.sub.2O, and conversion to N.sub.2 was low. When Paracoccus versutus was cultured in the M9 medium with 1 mM Mg.sup.2+, conversion of NO into N.sub.2 was 100%. In contrast, when Pseudomonas stutzeri cells were cultured in the M9 medium without Mg.sup.2+, about 20% of NO was converted to N.sub.2O, and NO was hardly converted to N.sub.2. When Pseudomonas stutzeri cells were cultured in the M9 medium with 1 mM Mg.sup.2+, conversion of NO into N.sub.2O or N.sub.2 was 100%. This indicates that Mg.sup.2+ ions contribute to nitric oxide decomposition also in the Pseudomonas stutzeri strain.

[0048] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope as defined by the following claims.