METHOD OF REDUCING NITROGEN OXIDE CONCENTRATION IN SAMPLE, BIOREACTOR, AND PLUG FLOW REACTOR

Abstract

A method of reducing a concentration of a nitrogen oxide, the method comprising: contacting a microorganism with a nitrogen oxide-containing sample to reduce the concentration of the nitrogen oxide in the sample, wherein the contacting comprises contacting the microorganism with Fe(II)(L)-NO.sub.x in a bioreactor, wherein the Fe(II)(L)-NO.sub.x is a complex in which a chelating agent, Fe.sup.2+, and NO.sub.x are chelated, wherein L is the chelating agent, and wherein NO.sub.x is a nitrogen oxide ligand.

Claims

1. A method of reducing a concentration of a nitrogen oxide, the method comprising: contacting a microorganism with a nitrogen oxide-containing sample to reduce the concentration of the nitrogen oxide in the sample, wherein the contacting comprises contacting the microorganism with Fe(II)(L)-NO.sub.x in a bioreactor, wherein the Fe(II)(L)-NO.sub.x is a complex in which a chelating agent, Fe.sup.2+, and NO.sub.x are chelated, wherein L is the chelating agent, and wherein NO.sub.x is a nitrogen oxide ligand.

2. The method of claim 1, wherein L is ethylenediamine, diethylenetriamine, triethylenetetraamine, hexamethylenetetramine, N-(2-hydroxyethyl)ethylenediaminetriacetic acid, ethylenediaminetetraacetic acid, iminodiacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, or a combination thereof.

3. The method of claim 1, wherein the microorganism is a single microorganism or a combination of different microorganisms.

4. The method of claim 3, wherein the single microorganism comprises a recombinant microorganism of the genus Escherichia, and the combination of different microorganisms is a microbial collection derived from activated sludge or sewage.

5. The method of claim 4, wherein the recombinant microorganism of the genus Escherichia comprises a genetic modification that increases expression of a nosZ gene encoding a nitrous oxide reductase NosZ in the recombinant microorganism, a nosR gene encoding NosR, a nosD gene encoding NosD, a nosF gene encoding NosF, a nosY gene encoding NosY, and an apbE gene encoding ApbE, wherein the nosZ gene, the nosR gene, the nosD gene, the nosF gene, the nosY gene, and the apbE gene are derived from a microorganism of the genus Pseudomonas, the genus Paracoccus, or a combination thereof.

6. The method of claim 1, wherein the bioreactor is a plug flow reactor comprising a plurality of carriers.

7. The method of claim 6, wherein the plug flow reactor comprises two or more compartments, wherein each of the two or more compartments are separated from each other by a porous plate comprising a plurality of pores.

8. The method of claim 7, wherein the two or more compartments comprises: a first compartment comprising a bacterium that reduces NO.sub.2 and/or NO to N.sub.2, and a second compartment comprising a bacterium that reduces Fe(III) to Fe(II).

9. The method of claim 8, wherein the sample flows within the bioreactor in the direction from the first compartment to the second compartment.

10. The method of claim 8, wherein the bacterium that reduces NO.sub.2 and/or NO to N.sub.2 is a microorganism of the family Rhodocyclaceae, Zoogloeaceae, Rhodobacteraceae, Clostridiaceae, or a combination thereof, and the bacterium that reduces Fe(III) to Fe(II) is a microorganism of the family Clostridiaceae, Shewanellaceae, Geobacteraceae, Rhodobacteraceae, Pseudomonadaceae, or a combination thereof.

11. The method of claim 1, wherein the contacting further comprises flowing the sample through the bioreactor.

12. The method of claim 1, wherein the Fe(II)(L)-NO.sub.x is Fe(II)(EDTA)-NO.

13. The method of claim 11, wherein the sample flowing out from the bioreactor is recirculated back into the bioreactor.

14. The method of claim 11, wherein the sample flowing out from the bioreactor is recirculated back into the bioreactor by recombining with the nitrogen oxide-containing sample.

15. The method of claim 1, wherein the bioreactor is fluidly connected to a wastewater-containing vessel or a Fe(III)(EDTA)-containing vessel.

16. The method of claim 1, further comprising introducing wastewater or Fe(III)(EDTA) into the bioreactor.

17. A plug flow reactor for reducing a concentration of a nitrogen oxide in a nitrogen oxide-containing sample, the plug flow reactor comprising: two or more compartments separated by a porous plate, wherein the porous plate comprises a plurality of pores; a plurality of carriers to which a microorganism is adsorbed, wherein the plurality of carriers are disposed in each of the two or more compartments; an inlet through which a nitrogen oxide-containing sample is introduced into the plug flow reactor; and an outlet through which the reacted nitrogen oxide-containing sample flows out from the plug flow reactor, wherein the outlet is fluidly connected to the inlet so that a discharged sample is recirculated to the plug flow reactor through the inlet.

18. The plug flow reactor of claim 17, wherein the inlet is fluidly connected to a vessel generating Fe(II)(L)-NO.sub.x or a vessel comprising Fe(II)(L)-NO.sub.x.

19. The plug flow reactor of claim 17, wherein the outlet and the inlet are fluidly connected to each other through a vessel generating Fe(II)(L)-NO.sub.x or a vessel comprising Fe(II)(L)-NO.sub.x.

20. The plug flow reactor of claim 18, wherein the vessel generating Fe(II)(L)-NO.sub.x or the vessel comprising Fe(II)(L)-NO.sub.x each comprises an inlet through which the nitrogen oxide-containing sample is introduced.

21. The plug flow reactor of claim 18, wherein the vessel generating Fe(II)(L)-NO.sub.x or the vessel comprising Fe(II)(L)-NO.sub.x comprises a gas outlet that discharges N.sub.2O, N.sub.2, or a combination thereof.

22. The plug flow reactor of claim 18, wherein the vessel generating Fe(II)(L)-NO.sub.x or the vessel comprising Fe(II)(L)-NO.sub.x comprises a fluid outlet for regulating a fluid level.

23. The plug flow reactor of claim 17, wherein the plug flow reactor is fluidly connected to: a vessel containing wastewater or a vessel comprising Fe(III)(EDTA).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The above and other aspects, features, and advantages of certain exemplary embodiments of the disclosure will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0074] FIG. 1 is a diagram showing an example of a plug flow reactor system according to one or more embodiments;

[0075] FIG. 2 shows concentrations of Fe(II)(EDTA), Fe(III)(EDTA), and Fe(II)(EDTA)-NO at an inlet, a lower compartment, an upper compartment, and an outlet, when operated at a flow rate of 0.9 liters per hour (L/h) and a hydraulic retention time (HRT) of 1.06 hours;

[0076] FIG. 3 shows concentrations of Fe(II)(EDTA), Fe(III)(EDTA), and Fe(II)(EDTA)-NO at an inlet, a lower compartment, an upper compartment, and an outlet, when operated at a flow rate of 1.8 L/h and HRT of 0.53 hours; and

[0077] FIG. 4 shows concentrations of Fe(II)(EDTA), Fe(III)(EDTA), and Fe(II)(EDTA)-NO at an inlet, a lower compartment, an upper compartment, and an outlet, when operated at a flow rate of 0.5 L/h and HRT 2.0 hours.

DETAILED DESCRIPTION

[0078] Reference will now be made in further detail to exemplary embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present exemplary embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the exemplary embodiments are merely described in further detail below, and by referring to the figures, to explain certain aspects of the present detailed description. As used herein, the term and/or includes any and all combinations of at least one 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.

[0079] The terminology used herein is for the purpose of describing one or more exemplary embodiments only and is not intended to be limiting. As used herein, the singular forms a, an, and the are intended to include the plural forms as well, unless the context clearly indicates otherwise. The term or means and/or. 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.

[0080] It will be understood that, although the terms first, second, third etc. may be used herein to describe various elements, components, regions, layers, and/or sections, these elements, components, regions, layers, and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, a first element, component, region, layer, or section discussed below could be termed a second element, component, region, layer, or section without departing from the teachings of the present embodiments.

[0081] Exemplary embodiments are described herein with reference to cross section illustrations that are schematic illustrations of idealized embodiments. As such, variations from the shapes of the illustrations as a result, for example, of manufacturing techniques and/or tolerances, are to be expected. Thus, embodiments described herein should not be construed as limited to the particular shapes of regions as illustrated herein but are to include deviations in shapes that result, for example, from manufacturing. For example, a region illustrated or described as flat may, typically, have rough and/or nonlinear features. Moreover, sharp angles that are illustrated may be rounded. Thus, the regions illustrated in the figures are schematic in nature and their shapes are not intended to illustrate the precise shape of a region and are not intended to limit the scope of the present claims.

[0082] It will be understood that when an element is referred to as being on another element, it can be directly in contact with the other element or intervening elements may be present therebetween. In contrast, when an element is referred to as being directly on another element, there are no intervening elements present.

[0083] 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 general inventive concept 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.

[0084] 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%, 5% of the stated value.

[0085] The term increase in expression, as used herein, refers to a detectable increase in the expression of a given gene. The increase in expression means that a gene expression level in a genetically modified (e.g., genetically engineered) cell is greater than the expression level of a comparative cell of the same type that does not have a given genetic modification (e.g., original or wild-type cell). For example, a gene expression level of a genetically modified cell may be increased by about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 30% or greater, about 50% or greater, about 60% or greater, about 70% or greater, or about 100% or greater than an expression level of a non-engineered cell of the same type, i.e., a wild-type cell or a parent cell. A cell having an increased expression of a protein or an enzyme may be identified by using any method known in the art.

[0086] The term copy number increase may be caused by introduction or amplification of a gene in a cell, and encompasses a cell which has been genetically modified to include a gene that does not naturally exist in a non-engineered cell. The introduction of the gene may be mediated by a vehicle such as a vector. The introduction of the gene may be a transient introduction in which the gene is not integrated into a genome of the cell, or an introduction that results in integration of the gene into the genome of the cell. The introduction may be performed, for example, by introducing a vector into the cell, the vector including a polynucleotide encoding a target polypeptide, and then, replicating the vector in the cell, or integrating the polynucleotide into the genome of the cell. The term copy number increase may be an increase the copy number of a gene or genes encoding one or more polypeptides constituting a complex, and which together, exhibit nitrous oxide reductase activity.

[0087] The introduction of the gene may be performed via a known method, such as transformation, transfection, or electroporation. The gene may be introduced with or without the use of a vehicle. The term vehicle, as used herein, refers to a nucleic acid molecule that is able to deliver other nucleic acids linked thereto, to a cell. In view of a nucleic acid sequence mediating introduction of a specific gene, the term vehicle may be used interchangeably with a vector, a nucleic acid construct, or a cassette. The vector may include, for example, a plasmid vector, a virus-derived vector, but is not limited thereto. The plasmid includes a circular double-stranded DNA sequence to which additional DNA encoding a gene of interest, may be linked. The vector may include, for example, a plasmid expression vector (e.g., a bacterial plasmid), a virus expression vector, such as a replication-defective retrovirus, an adenovirus, an adeno-associated virus, or a combination thereof. In an aspect, the vector may be a bacterial plasmid including a bacterial origin of replication and selectable marker.

[0088] The genetic modification disclosed herein may be performed by any suitable molecular biological method known.

[0089] The term genetic modification, as used herein, refers to an artificial alteration in a constitution or structure of a genetic material of a cell.

[0090] Hereinafter, the exemplary embodiments will be described in more detail through examples. However, these examples are intended to illustrate the presented exemplary embodiments, and the scope of the present disclosure is not limited to these examples.

Example 1: Removal of Nitrogen Oxide in Sample by Using Plug Flow Reactor and Mixed Microbial Collection

[0091] In the present example, nitrogen oxide included in a sample was reduced and removed by using a plug flow reactor as shown in FIG. 1. FIG. 1 is a diagram showing an example of a plug flow reactor system. The plug flow reactor system is as described hereinabove.

[0092] The plug flow reactor includes reaction compartments 940 and 950, each of which was a cylinder having a diameter of about 5 cm and a height of about 25 cm, with a total diameter:height ratio of about 1:10, and cylindrical buffer compartments 950 and 970, each of which with a diameter of about 5 cm and a height of about 10 cm. The reaction compartments 940 and 950 each had an internal reaction space of about 0.5 liters (L). The buffer compartments 960 and 970 each had an internal reaction space of about 0.2 L.

[0093] Each of the two compartments were filled with a plurality of disc-shaped carriers made of high-density polyethylene (HDPE) with an average diameter of about 0.8 cm. The disc-shaped carriers had a structure in which five plates are radially extended from a point near the center of the disk to form five pores.

[0094] At least some of microorganisms in the compartments were attached to the carriers to form biofilms. A process of forming a biofilm by attaching microorganisms to a carrier and growing the same was carried out by allowing activated sludge to adhere to the carriers by introducing sewage containing activated sludge derived from a sewage treatment facility at a flow rate of about 5 milliliters per minute (mL/min) while stirring through the upper outlet 980 of the plug flow reactor 900, and circulating at 25? C. for 24 hours. After removing the sewage through the inlet into the reactor 900 at a flow rate of about 5 mL/min to replace the sewage excluding the activated sludge with artificial sewage containing nutrients and Fe(III)(EDTA), artificial sewage was introduced at a flow rate of about 5 mL/min at 25? C. for 72 hours to form a biofilm by connecting the vessel 200 generating Fe(II)(EDTA)-NO, which is needed for the recirculation process, to the reactor outlet 980 and the inlet 900.

[0095] After inducing biofilm formation, it was confirmed that most of Fe(III) was reduced to Fe(II) by measuring the concentration of Fe(III) in the reactor 900. That is, most of the introduced Fe(III)(EDTA) was reduced by the iron reducing microorganisms to exist in the form of Fe(II)(EDTA).sup.2.

[0096] After that, the pre-connected Fe(II)(EDTA)-NO generating vessel 200 was adjusted to hold 500 mL of an aqueous solution including Fe(II)(EDTA).sup.2?, and a gas, in which 99.999 vol % N.sub.2 gas and NO (50 vol % in N.sub.2) were mixed at a ratio of 100:1, was continuously purged in the vessel 200 at a flow rate of about 500 mL/min to contact with Fe(II)(EDTA).sup.2? to form Fe(II)(EDTA)-NO, and then, the Fe(II)(EDTA)-NO-containing solution was introduced into the reactor 900 from the vessel 200 through the inlet 900. In this case, NO concentration in the incoming mixed gas was about 3,000 parts per million by volume (ppmv) to about 10,000 ppmv, which was three times or more greater than about 100 ppmv to about 1,000 ppmv, which was a NO concentration range in an exhaust gas actually discharged from a power plant. This Fe(II)(EDTA)-NO containing solution was used as an experimental group of the nitrogen oxide containing solution. In this regard, the formation process of Fe(II)(EDTA)-NO from nitrogen oxide seemed to be according to the following Reaction Scheme 1, but should not limited thereto:

[Fe(II)(EDTA)].sup.2?+NO.sub.2(aq)<->[Fe(II)(EDTA)-NO].sup.2?Reaction Scheme 1

[0097] As a result, concentration of the produced [Fe(II)(EDTA)-NO].sup.2? (which is used interchangeably with Fe(II)(EDTA)-NO for convenience herein) was about 3.0 millimolar (mM).

[0098] Next, a solution containing about 1.0 mM of [Fe(II)(EDTA)-NO].sup.2? in the Fe(II)(EDTA)-NO generating vessel 200 was introduced through the inlet 900 of the plug flow reactor. In this regard, flow rates were about 0.9 L/h and about 0.53 L/h, and hydraulic retention times (HRTs) of the fluid in each compartment were 1.06 hours and 0.53 hours, respectively. The HRT was calculated by Equation 1:

HRT=reactor volume/flow rateEquation 1

[0099] In addition, in order to supply organic matter to microorganisms during the process, artificial wastewater was intermittently introduced from an artificial wastewater-containing vessel 600 into the buffer section 960 of the plug flow reactor. The artificial wastewater was artificial sewage made to simulate organic carbon and organic nitrogen and other components of sewage in sewage.

[0100] In addition, in order to supplement Fe(II)(EDTA) lost during the process, an aqueous solution containing 5 mM of Fe(III)(EDTA) was intermittently introduced at the same flow rate as the artificial wastewater from the Fe(III)(EDTA)-containing vessel 610 to the buffer compartment 960 of the plug flow reactor. As a result, the aqueous solution containing 5 mM of Fe(III)(EDTA) was two-fold diluted with the wastewater to obtain an aqueous solution containing 2.5 mM of Fe(III)(EDTA).

[0101] During the process operation, the Fe(II)(EDTA)-NO concentration in the solution flowing into the reactor and the Fe(II)(EDTA)-NO concentration in the solution flowing out from the reactor were measured. The Fe(II)(EDTA)-NO concentration in the solution flowing into the reactor and the Fe(II)(EDTA)-NO concentration in the solution flowing out from the reactor were measured by taking samples in the reaction compartments 940 and 950 from sampling ports 912 and 912, respectively.

[0102] The concentration of the Fe(II)(EDTA)-NO in the solution was measured by measuring absorbance at a wavelength of 425 nm by using a spectrophotometer. The NO concentrations at the inlet 210 and the outlet 500 of the Fe(II)(EDTA)-NO generating vessel 200 were measured by using a gas chromatograph-mass spectrometer. The concentrations of Fe(II) and total Fe were measured by using a spectrophotometer at a wavelength of 562 nm by using the Ferrozine assay. In this regard, [Total FeFe(II)] concentration is Fe(III) concentration.

[0103] In addition, N.sub.2O concentration in the collected sample was measured. N.sub.2O concentration in the sample was measured by sampling 10 mL of the reactor solution by filtering through a 0.22 micron filter in a 27 mL vial filled with N.sub.2, then stirring the sample for 15 minutes in order that the dissolved N.sub.2O is equilibrated with the headspace, and then by using an HP6890 Series gas chromatograph equipped with an HPPLOT/Q column.

[0104] In addition, the oxygen concentration in the reactor was measured during the process operation. The oxygen concentration was measured by attaching sensor spots to the inlet 900 of the reactor, at the bottom of the lower compartment 940, and on top of the upper compartment 950, rather than by measuring oxygen concentration in the solution, and by using a non-contact method using the principle that a fiber-optic oxygen sensor of a FireStingO.sub.2 device emits near-infrared (NIR) light in response to oxygen. That is, oxygen was measured by using sensor spots inside the reactor and an optical fiber sensor outside the reactor.

[0105] Oxygen concentration of the solution flowing into the reactor and the oxygen concentration of the solution flowing out from the reactor were measured by using oxygen dots 914 in the reaction compartments 940 and 950, respectively. The oxygen dots 914 are sensor spots, which are capable of measuring oxygen concentration in the solution. Oxygen concentration was measured by using a FireStingO.sub.2Optical Oxygen Meter.

[0106] Table 1 shows results of measuring Fe(II)(EDTA)-NO concentration in the solution flowing into the reactor and Fe(II)(EDTA)-NO concentration in the solution flowing out from the reactor. In Table 1, in the process with operating times of 24 hours and 72 hours (hereinafter Process Operation 1), the sample inflow flow rate was about 0.9 L/h, the hydraulic retention time (HRT) of the fluid in each compartment was 1.06 hours, and the process with operating times of 168 hours and 192 hours (hereinafter Process Operation 2) had a sample inflow flow rate of about 1.8 L/h and hydraulic retention time (HRT) of the fluid in each compartment of 0.53 hours. That is, Process Operation 2 had twice the flow rate, while HRT was 0.5 times shorter, compared to Process Operation 1.

TABLE-US-00001 TABLE 1 Operation Inflow Outflow NO removal time concentration concentration efficiency (hours) (mM) (mM) (%) 24 2.51 0.86 65.7 72 0.99 0.24 75.8 168 1.35 0.16 88.1 192 1.30 0.18 86.2

[0107] As shown in Table 1, in the case of Process Operation 1, up to 75.8% of NO removal efficiency could be achieved. In addition, in the case of Process Operation 2, up to 88.1% of NO removal efficiency could be achieved. In addition, even when a sample containing about 3,000 ppmv to about 10,000 ppmv of high concentration NO was introduced into the plug flow reactor, the growth of microorganisms and the denitrification and iron reduction reactions were smoothly performed. FIG. 2 shows concentrations of Fe(II)(EDTA), Fe(III)(EDTA), and Fe(II)(EDTA)-NO at the inlet, lower compartment, upper compartment, and outlet, when operated at a flow rate of 0.9 L/h and HRT of 1.06 hours. In FIG. 2, A and B were operated with operation times of 24 hours and 72 hours, respectively.

[0108] FIG. 3 shows concentrations of Fe(II)(EDTA), Fe(III)(EDTA), and Fe(II)(EDTA)-NO at the inlet, lower compartment, upper compartment, and outlet, when operated at a flow rate of 1.8 L/h and HRT of 0.53 hours. In FIG. 3, A and B were operated with operating times of 168 hours and 192 hours, respectively.

[0109] In addition, as a result of measuring oxygen concentrations in the reactor solution, oxygen concentration in the buffer compartment 960 was 0.9?0.2%, in the lower section 940 was 0.4?0.1%, and in the upper section 950 was 0%.

[0110] In addition, as a result of measuring N.sub.2O concentration in the reactor solution, in the case of Process Operation 1, N.sub.2O concentration discharged through the outlet was 0.9 micromoles per hour (?mole/h), and in the case of Process Operation 2, N.sub.2O concentration discharged through the outlet was 3.6 ?mole/h. Therefore, when NO in a more realistic NO concentration range of about 100 ppmv to about 1,000 ppmv is introduced into the reactor, the N.sub.2O concentration discharged through the outlet is expected to be lowered to a value close to zero.

Example 2: Removal of Nitrogen Oxide in Sample by Using Plug Flow Reactor and Recombinant E. coli

[0111] In the present example, nitrogen oxide contained in a sample was reduced and removed by using a plug flow reactor as shown in FIG. 1, except for using the medium containing vessel 600 instead of the artificial wastewater containing vessel 600, without going through a recirculation process. In this regard, the microorganisms used for the biological reaction were recombinant E. coli (DE3) in which a NosZ gene, which encodes a nitrous oxide (N.sub.2O) reductase, was inserted. The recombinant E. coli (DE3) is disclosed in US Patent Publication No. US2022/0177896A1, the content of which are incorporated herein in their entirety.

[0112] The plug flow reactor included two reaction compartments 940 and 950, each of which is a cylinder having a diameter of 5 cm and a height of 25 cm, with a diameter: height ratio of 1:5, and cylindrical buffer compartments 960 and 970, each of which with a diameter of 5 cm and a height of 10 cm. The reaction compartments 940 and 950 each have an internal reaction space of 0.5 L. The buffer compartments 960 and 970 each have an internal reaction space of 0.2 L.

[0113] Each of the two compartments was filled with a plurality of disk-shaped carriers made of high-density polyethylene (HDPE) with a diameter of about 0.8 cm. The disk-shaped carriers have a structure in which five plates are radially extended from a point near the center of the disk to form five pores.

[0114] At least some of the microorganisms in the compartments were attached to the carriers to form biofilms. The process of forming a biofilm by attaching microorganisms to a carrier and growing the same was by inoculating the recombinant E. coli (DE3) into nutrient-rich yeast extract tryptone (YT) medium (tryptone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L, and CuCl.sub.2 67.2 mg/L), culturing the same at 35? C. under aerobic conditions for 24 hours, introducing the obtained culture through the inlet of the plug flow reactor at a flow rate of 5 mL/min, and supplying 2 g/L of glucose at a flow rate of 0.3 L/h at 25? C. for 72 hours to form a biofilm.

[0115] First, a 6 mM solution of Fe(II)(EDTA)-NO was added to water prepared externally in the Fe(II)(EDTA)-NO-containing vessel 200, and a solution containing about 6.0 mM Fe(II)(EDTA)-NO was introduced through the inlet 900 of the plug flow reactor from the container 200, at a flow rate of 0.21 L/h. In this regard, hydraulic retention time (HRT) of the fluid in each compartment was 2.0 hours.

[0116] In addition, medium was continuously introduced into the buffer compartment 960 of the plug flow reactor from the medium-containing vessel 600 at a flow rate of 0.21 L/h, in order to supply organic matter to the microorganisms during the process. The medium is 2? M9 medium containing 2 g/L glucose. M9 medium includes 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, 0.2 g/L of MgCl.sub.2 6H.sub.2O, and 1 mL of 1000? trace metal (0.5 g/L of MnSO.sub.4, 0.1 g/L of Na.sub.2MoO.sub.4, 0.1 g/L of CuSO.sub.4 5H.sub.2O, and 2 g/L of CaCl.sub.2)).

[0117] During the process operation, the Fe(II)(EDTA)-NO concentration in the solution flowing into the reactor and the Fe(II)(EDTA)-NO concentration in the solution flowing out from the reactor were measured. The Fe(II)(EDTA)-NO concentration in the solution flowing into the reactor and the Fe(II)(EDTA)-NO concentration in the solution flowing out from the reactor were measured by taking samples in the reaction compartments 940 and 950 from sampling ports 912 and 912, respectively.

[0118] The concentration of the Fe(II)(EDTA)-NO in the solution was measured by measuring absorbance at a wavelength of 425 nm by using a spectrophotometer. The NO concentrations at the inlet 210 and the outlet 500 of the Fe(II)(EDTA)-NO generating vessel 200 were measured by using a gas chromatograph-mass spectrometer. The concentrations of Fe(II) and total Fe were measured by using a spectrophotometer at a wavelength of 562 nm by using the Ferrozine assay. In this regard, [Total FeFe(II)] concentration is Fe(III) concentration.

[0119] In addition, N.sub.2O concentration in the collected sample was measured. N.sub.2O concentration in the sample was measured by sampling 10 mL of the reactor solution by filtering through a 0.22 ?m filter in a 27 mL vial filled with N.sub.2, then stirring the sample for 15 minutes in order that the dissolved N.sub.2O was equilibrated with the headspace, and then by using an HP6890 Series gas chromatograph equipped with an HPPLOT/Q column.

[0120] In addition, oxygen concentration in the reactor was measured during the process operation. The oxygen concentration was measured by attaching sensor spots to the inlet 900 of the reactor, the bottom of the lower compartment 940, and the top of the upper compartment 950, rather than by measuring oxygen concentration in the solution, and by using a non-contact method using the principle that a fiber-optic oxygen sensor of a FireStingO.sub.2 device emits near-infrared (NIR) light in response to oxygen. That is, oxygen was measured by using sensor spots inside the reactor and an optical fiber sensor outside the reactor.

[0121] Oxygen concentration of the solution flowing into the reactor and the oxygen concentration of the solution flowing out from the reactor were measured by using oxygen dots 914 in the reaction compartments 940 and 950, respectively. The oxygen dots 914 are sensor spots, which are capable of measuring oxygen concentration in the solution. Oxygen concentration was measured by using a FireStingO.sub.2Optical Oxygen Meter.

[0122] Table 2 shows results of measuring the maximum concentration of N.sub.2O generated and measured in the central portion of the upper compartment 950 of the reactor, N.sub.2O concentration in the solution flowing out through the outlet, and N.sub.2O removal efficiency. Table 3 shows the NO removal efficiency as a result of the process.

TABLE-US-00002 TABLE 2 Operation Maximum concentration Outflowing N.sub.2O N.sub.2O removal time of generated N.sub.2O concentration efficiency (hours) (?M) (?M) (%) 72 96 90 6.3 96 131 121 7.8

TABLE-US-00003 TABLE 3 Outflowing Operation Inflowing Fe-EDTA-NO Fe-EDTA-NO NO removal time concentration concentration efficiency (hours) (mM) (mM) (%) 72 1.02 0.69 32.3 96 0.53 0.23 56.6

[0123] As shown in Tables 2 and 3, even when recombinant E. coli with enhanced denitrification ability were used as microorganisms of a single kind, and a chemically defined medium was used as the medium, the NO in the fluid could be removed. FIG. 4 is a diagram showing concentrations of Fe(II)(EDTA), Fe(III)(EDTA), and Fe(II)(EDTA)-NO at the inlet 900, the lower compartment 940, the upper compartment 950, and the outlet 980, when operated at a flow rate of 0.5 L/h and HRT of 2.0 hours. In FIG. 4, A and B were operated with operation times of 72 hours and 96 hours, respectively.

[0124] In addition, as a result of measuring oxygen concentrations in the bioreactor solution, all were 0%.

[0125] It should be understood that exemplary embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each exemplary embodiment should typically be considered as available for other similar features or aspects in other exemplary embodiments. While one or more exemplary 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 of the disclosure as defined by the following claims.