Method for producing ammonia using quantum dot-bacteria hybrid system

Abstract

The present invention relates to a method for producing ammonia using a quantum dot-microorganism hybrid, wherein bench-scale studies on the scale of several milliliters to several liters have been expanded. By developing sustainable microorganism-based ammonia production technology through the use of a fermentor, it is possible to effectively respond to the growing demand for ammonia. The present invention presents a specific embodiment expanding existing technology to the practical application stage, and is expected to be competitive as an environmentally-friendly and sustainable ammonia production technology.

Description

Ammonia production method using a quantum dot-bacteria hybrid system

[0001] The present invention relates to a method for producing ammonia using a quantum dot-microbial complex, and more specifically, to a method for producing ammonia using white light and capable of producing ammonia on a large scale without replacing a culture medium.

[0002]

[0003] Ammonia (NH3) is the second most widely produced fine chemical worldwide, after sulfuric acid. Approximately 80% of the ammonia produced is used to manufacture nitrogen fertilizers. The Haber-Bosch process, the most widely used method for ammonia production, requires high temperatures (~500°C) and high pressures (~200 bar), resulting in high energy consumption. Furthermore, hydrogen, used as a reactant in the Haber-Bosch process, is mostly produced through natural gas reforming. However, producing one ton of ammonia emits approximately 2.5 tons of carbon dioxide, making this process unsustainable.

[0004] As awareness of energy and environmental issues such as global warming grows, the world is pursuing various policies to achieve the common goal of reducing carbon emissions. Against this backdrop, ammonia is easier to store and transport than hydrogen, has relatively low operating costs, and is gaining attention as a key compound for achieving carbon neutrality as a ship fuel and hydrogen storage energy source. Consequently, ammonia demand is expected to continue to grow until 2050, necessitating the development of an environmentally friendly and sustainable ammonia production technology that can replace the existing Haber-Bosch process.

[0005] Nitrogen-fixing bacteria, known to produce ammonia in nature, fix atmospheric nitrogen and convert it into ammonium under room temperature and pressure. Nitrogen-fixing bacteria use intracellular adenosine triphosphate (ATP) as an energy source and utilize nitrogenase (MoFe protein) as a biocatalyst to transfer electrons derived from organic matter, thereby converting nitrogen molecules into ammonia. However, the relatively low ammonia conversion efficiency of nitrogenase and the difficulty of precise metabolic control limit the production of large quantities of ammonia, making it difficult to apply this process to industrial applications.

[0006]

[0007] Meanwhile, according to Korean Patent Application Nos. 10-2021-0106678 and 10-2021-0106680, a method for producing ammonia using indium phosphide-based core / mercaptopropionic acid-capped selenized zinc shell quantum dots is described, and it was confirmed that ammonia can be produced in an eco-friendly manner at low temperatures. However, there was a problem that the ammonia production amount was saturated at a reaction time of 6 hours or more, making mass production of ammonia impossible.

[0008] Accordingly, the inventors of the present invention have diligently worked to develop a method capable of solving the above problems and scaling up to mass-produce ammonia. As a result, they have discovered that using white light, rather than a 400 nm wavelength light source, enables energy absorption across the entire light spectrum, reduces the production of reactive oxygen species, and increases continuous ammonia production. Furthermore, they have confirmed culture conditions that do not require purification of cultured bacteria or exchange of culture medium in a fermenter, and have verified effective ammonia production and scalability, thereby completing the present invention.

[0009]

[0010] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0011]

[0012] Summary of the invention

[0013] The purpose of the present invention is to enable industrial-scale ammonia production by expanding the cultivation of the quantum dot-microorganism complex and the ammonia production scale in a method for producing ammonia using a quantum dot-microorganism complex.

[0014]

[0015] In order to achieve the above object, the present invention provides an ammonia production method comprising the steps of (a) culturing a microorganism that endogenously expresses a nitrogen fixation enzyme or has a nitrogen fixation enzyme introduced from outside in a nutrient medium containing inorganic nanoparticle quantum dots under dark conditions to produce a quantum dot-microorganism complex; and (b) producing ammonia by irradiating white light, wherein steps (a) and (b) are performed in a single fermenter.

[0016]

[0017] Figure 1 is a transmission electron microscope (TEM) observation data of quantum dots synthesized for the production of quantum dot-nitrogen-fixing bacteria hybrids.

[0018] Figure 2 shows absorption and emission spectra of quantum dots synthesized for the production of quantum dot-nitrogen-fixing bacteria hybrids.

[0019] Figure 3 is a simple schematic illustrating the principle of ammonia production by a quantum dot-nitrogen-fixing bacteria hybrid.

[0020] Figure 4 is a simple schematic diagram of a method for fabricating a quantum dot-nitrogen-fixing bacteria hybrid.

[0021] Figure 5 is a graph comparing the difference in ammonia production of quantum dot-nitrogen fixing bacteria hybrids according to the type of light source.

[0022] Figure 6 is a graph comparing the changes in cell density of quantum dot-nitrogen-fixing bacteria hybrids according to the type of light source.

[0023] Figure 7 is a graph comparing the amount of reactive oxygen species produced by quantum dots according to the type of light source.

[0024] Figure 8 is a graph showing the results of a quantum dot-bacteria hybrid system producing ammonia under phosphate buffered saline conditions.

[0025] Figure 9 is a graph comparing the ammonia production of quantum dot-bacteria hybrids under controlled medium conditions depending on the presence or absence of added sugars (sucrose, glucose, fructose) or magnesium.

[0026] Figure 10 is a schematic diagram illustrating the process of ammonia production using the increase in bacterial biomass and electrons generated from quantum dots.

[0027] Figure 11 is a graph comparing the cell density of nitrogen-fixing bacteria according to the dissolved oxygen content of the culture medium in the fermenter.

[0028] Figure 12 is a schematic diagram of a two-step process for ammonia production using a quantum dot-nitrogen fixing bacteria hybrid in a fermenter.

[0029] Figure 13 is a graph showing the results of ammonia production over time using a quantum dot-nitrogen fixing bacteria hybrid in a fermenter.

[0030] Figure 14 is a graph comparing the TOF results of ammonia production using a quantum dot-nitrogen fixing bacteria hybrid in a fermenter.

[0031]

[0032] Detailed description of the invention and preferred embodiments

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0034]

[0035] This study aimed to improve the ammonia conversion efficiency of nitrogen-fixing bacteria and enhance ammonia productivity by constructing a hybrid system of quantum dots, which are semiconductor nanoparticles, and nitrogen-fixing bacteria. In particular, to utilize the constructed quantum dot-bacteria hybrid system in an industrial ammonia production process, a process of verifying scalability is essential. In the present invention, nitrogen-fixing bacteria were mass-cultivated using a fermenter, a large-scale microbial culture system, and the scalability of the quantum dot-bacteria hybrid complex for ammonia production was verified.

[0036] The present invention is based on the results of previously filed applications (Application Nos.: 10-2021-0106678, 10-2021-0106680) in which quantum dots, which are inorganic nanoparticles, were introduced into existing wild-type nitrogen-fixing bacteria to improve the ammonia production capacity of nitrogen-fixing bacteria, and the scalability of a fermentation process capable of industrial mass production of ammonia was confirmed through a quantum dot-bacteria hybrid system.

[0037] The previously filed invention used a 400 nm wavelength light source to induce photoexcitation of quantum dots, but suffered from ammonia production saturation after reaction times exceeding 6 hours. The present invention modifies the light source by using white light, enabling energy absorption across the entire light spectrum and reducing the production of reactive oxygen species, resulting in sustained increases in ammonia production. Furthermore, the culture conditions were optimized to reduce the time and cost of purifying bacteria cultured in liter-scale reactors or exchanging the culture medium. During this process, the effect of carbon sources, such as sucrose, on ammonia production was investigated and it was discovered that the presence of a carbon source promoted biomass formation while decreasing ammonia productivity. The present invention describes an effective ammonia production method, dividing the first stage into mass cultivation and the second stage into photochemical reactions induced by light while simultaneously depleting the carbon source. Therefore, the present invention further explains the ammonia production mechanism of the quantum dot-bacteria hybrid system by controlling the light source and culture medium conditions through this two-step process, and verifies effective ammonia production and scalability.

[0038]

[0039] Accordingly, the present invention relates to an ammonia production method, comprising, in one aspect, (a) a step of culturing a microorganism that endogenously expresses a nitrogen fixation enzyme or has a nitrogen fixation enzyme introduced externally in a nutrient medium containing inorganic nanoparticle quantum dots under dark conditions to produce a quantum dot-microorganism complex; and (b) a step of producing ammonia by irradiating white light, wherein steps (a) and (b) are performed in a single fermenter.

[0040] In the present invention, the volume of the fermenter may be, but is not limited to, 500 mL to 100 L. Preferably, it may be a single fermenter having a volume of 1 L to 50 L, more preferably 1 L to 10 L, and most preferably 5 L, but is not limited thereto.

[0041] In the present invention, it may be characterized in that step (b) is performed at a point in time when sucrose is depleted in the nutrient medium of step (a).

[0042] In the present invention, the step (b) may be characterized by directly irradiating white light to the fermenter in which the quantum dot-microorganism complex is generated in the step (a).

[0043] In the present invention, the quantum dot may be characterized by including an indium phosphide core / zinc selenide shell (InP / ZnSe), and having a particle size of 4 to 6 nm.

[0044] In the present invention, the core and shell may each be characterized by a diameter of 2 nm to 3 nm, but are not limited thereto.

[0045] In the present invention, the quantum dot may be characterized in that a hydrophilic ligand is introduced.

[0046] In the present invention, the hydrophilic ligand may be characterized by being selected from the group consisting of mercaptopropionic acid (MPA), L-glutathione (GSH), mercaptoacetic acid, mercaptobutanoic acid, mercaptopentanoic acid, mercaptohexanoic acid, mercaptoheptanoic acid, mercaptooctanoic acid, mercaptononanoic acid, mercaptodecanoic acid, mercaptoundecanoic acid, mercaptododecanoic acid and L-cysteine, and preferably, mercaptopropionic acid, but It is not limited.

[0047] In the present invention, the microorganism is Clostridium sp., Klebsiella pneumoniae, Paenibacillus polymyxa, Bacillus macerans, Escherichia intermedia, Azotobacter agilis, Azotobacter armeniacus, Azotobacter beijerinckii, Azotobacter chroococcum, Azotobacter nigricans, Azotobacter paspali, Azotobacter salinestris, Azotobacter tropicalis, It may be characterized by being selected from the group consisting of Azotobacter vinelandii, Rhizibium sp., Achromobacter, Azorhizobium sp., Frankia sp., Pseudomonas sp., Bacillus sp., Nitrobacter sp., Fusarium oxysporum, Cylindrocaropn tonkinese, Bipolaris sorokiniana, and Cyanobacteria sp. Preferably, the microorganism may be Azotobacter vinelandii, but is not limited thereto.

[0048] In the present invention, the nutrient medium may be characterized as being Burk's medium, but is not limited thereto.

[0049] In one embodiment of the present invention, nitrogen-fixing bacteria, Azotobacter vinelandii (KCTC 2426), known to produce ammonia by fixing atmospheric nitrogen under room temperature and pressure conditions, and quantum dots, semiconductor nanoparticles whose optical properties vary depending on particle size, were used. The quantum dots developed for application to the ammonia production system are core-shell structured quantum dots based on indium phosphide (InP), and the particle size is approximately 4-6 nm (Fig. 1). In the present invention, a core-shell structured quantum dot with improved charge extraction efficiency was synthesized by layering a zinc selenide (ZnSe) shell on an indium phosphide core (Fig. 2). Quantum dots have the property of generating electrons and holes when exposed to light, and exhibit the characteristic of emitting light when the generated electron-hole pairs recombine. On the other hand, the generated electron-hole pairs can be used in redox reactions and contribute to chemical reactions by being extracted without combining, and in the present invention, the generated electron-hole pairs were applied to ammonia production by utilizing this characteristic (Fig. 3).

[0050] The surface ligands of the indium phosphide and zinc selenide core-shell quantum dots synthesized in the present invention are exchanged with mercaptopropionic acid (3-Mercaptopropionic acid, MPA) to enable aqueous dispersion. The aqueous quantum dots are sterilized using a Minisart syringe filter with a particle pore size of 0.2 μm and then added to a medium. By inoculating bacteria into the medium containing quantum dots, nitrogen-fixing bacteria exposed to the quantum dots incorporate the quantum dots during the growth process, thereby forming a quantum dot-bacteria hybrid system in which the quantum dots are bound to the interior of the nitrogen-fixing bacteria (Fig. 4).

[0051] When irradiated with 400 nm light, ammonia production in the quantum dot-bacteria hybrid system was observed to be more than four times higher than in the dark. However, the ammonia production of the quantum dot-bacteria hybrid system saturates after a reaction time of more than 6 hours. Light with a wavelength of 400 nm is known to generate reactive oxygen species in cells, and the possibility that the generated reactive oxygen species may cause cell damage has been raised. In the present invention, ammonia production using white light was observed to reduce cell damage, and it was confirmed that ammonia production did not reach saturation and continued to increase for up to 12 hours compared to when irradiated with 400 nm wavelength light (Fig. 5). To explain that the decrease in ammonia productivity is due to the production of reactive oxygen species, the bacterial cell density was compared according to the light irradiation time for each light source, and it was confirmed that the decrease in cell density was significantly less under white light irradiation conditions than under 400 nm wavelength light (Fig. 6). In addition, superoxide anion (O2), an active oxygen species, was generated under different light source irradiation conditions. ●- ) was compared to explain that cell damage due to the production of reactive oxygen species is the cause of the limitation in ammonia productivity (Fig. 7).

[0052] The goal of the present invention is to develop a single-pot system that continuously performs bacterial growth and ammonia production through light irradiation in a 5 L fermenter. To eliminate the steps of harvesting and purifying cells from the fermenter and establish an effective ammonia production system, white light was selected as the light source, minimizing cell damage and enabling effective photoexcitation of quantum dots.

[0053] Prior to the ammonia production step, nutrient supply within the fermenter is essential for high-density bacterial culture. However, the quantum dot-bacteria hybrid system produced little ammonia under nutrient medium conditions, even under light irradiation. In contrast, ammonia production by the quantum dot-bacteria hybrid system was observed under phosphate-buffered saline (PBS) conditions, which aim to maintain pH and cell activity without nutrients (Fig. 8). However, in PBS treated with carbon sources such as sucrose, glucose, and fructose, almost no ammonia production was observed. Although carbon supply promotes bacterial biomass formation, it induces a metabolism opposite to ammonia production.

[0054] In nutrient media for culturing Azotobacter vinellandi, the quantum dot-bacteria hybrid system did not produce ammonia. However, when the carbon source in the nutrient media was limited, an increase in ammonia production by the hybrid system was observed (Fig. 9). This explains that bacterial biomass formation and ammonia production do not increase simultaneously, and confirms that the present invention can induce ammonia production using media from which the carbon source has been removed.

[0055] Nitrogen-fixing bacteria are known to produce ammonia using adenosine triphosphate-based charge transfer, and magnesium (Mg) in the nutrient medium directly affects the activity of adenosine triphosphate, thereby affecting the overall activity of nitrogenase. The present invention observed increased ammonia production in a quantum dot-bacteria hybrid system under magnesium-limited culture conditions, which demonstrates that quantum dots can replace the role of magnesium-adenosine triphosphate (MgATP), which is important for the activity of nitrogenase (Fig. 9). Therefore, the quantum dot-bacteria hybrid system can enhance ammonia production under carbon-free conditions, and demonstrates that quantum dots can effectively activate nitrogenase by replacing the existing energy source, magnesium-adenosine triphosphate (Fig. 10).

[0056] In one embodiment of the present invention, based on the light source optimization and culture medium optimization described above, experiments were conducted to mass-culture quantum dot-bacteria hybrids in a fermenter and to increase ammonia productivity using them. The core idea of ​​the present invention is to separate the cultivation of quantum dot-bacteria hybrids and ammonia production into two steps, enabling them to be performed in a single batch.

[0057] The first step is to inoculate wild-type Azotobacter vinellandi onto a nutrient medium containing quantum dots and cultivate the quantum dot-bacteria hybrid under dark conditions. Maintaining a constant dissolved oxygen content in the culture medium while increasing cell density is crucial for bacterial culture in a fermenter. In the present invention, we confirmed that bacteria grew to the highest cell density under conditions of 10% dissolved oxygen (Fig. 11).

[0058] A second step of ammonia production was performed by comparing the sucrose content of the culture medium during the fermentation process and irradiating white light at the point of sucrose depletion (Fig. 12). It was observed that the sucrose content decreased during bacterial growth, while the optical density at 600 nm, which supports cell density, increased. Furthermore, irradiating white light at the point of sucrose depletion confirmed increased ammonia production (Fig. 13).

[0059] In the present invention, the quantum dot-bacteria hybrid system achieved the production of 7.81 mg / L of ammonia over a reaction time of 45 hours in a 5 L reactor using a fermenter. This result represents an ammonia conversion efficiency of 11.96 s-1, which is approximately 6 times higher than the ammonia production conversion efficiency (turnover frequency, TOF) of adenosine triphosphate, iron protein, and purified nitrogenase (Fig. 14).

[0060] The ammonia production capacity through fermentation of the quantum dot-bacteria hybrid system was confirmed to have a titer of 7.81 mg / L, a yield of 7.41 mol / L, and a productivity of 0.174 mg / L / h.

[0061]

[0062] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0063]

[0064] Example 1: Quantum Dot Synthesis

[0065] Example 1-1: Synthesis of indium phosphide core quantum dots

[0066] In a three-necked round-bottomed flask, indium acetate (0.45 mmol), oleic acid (1.35 mmol), and 1-octadecene (27 mL) were placed and heated at 120°C under vacuum conditions (<200 mTorr) for 2 h to form indium oleate. The inside of the flask was changed to an inert atmosphere, 2.2 mL of trioctylphosphine was injected, and the mixture was heated for an additional 30 min under a low-pressure atmosphere, and the mixture of indium oleate and tris-trimethylsilylphosphine was cooled to room temperature.

[0067] 0.9 mL of a trioctylphosphine solution containing 0.33 M tris-trimethylsilylphosphine was injected into a flask at room temperature, and the temperature was raised to 300°C to synthesize indium phosphide core quantum dots. The particle size was confirmed to be approximately 4-6 nm (Fig. 1).

[0068]

[0069] Example 1-2: Synthesis of indium phosphide / zinc selenide core-shell structure quantum dots

[0070] Zinc oleate and selenated trioctylphosphine (2 M), prepared in an inert atmosphere, were sequentially injected into the indium phosphide core solution at a reaction temperature of 300°C. At this time, in order to synthesize shells in single-layer units, zinc oleate and selenated trioctylphosphine were injected in calculated amounts, and the selenated trioctylphosphine solution was slowly injected dropwise. After heating each shell for 30 minutes, and after all shells were synthesized and completed, the temperature of the flask was lowered to room temperature to obtain quantum dots with an indium / zinc selenide core-shell structure (Fig. 2).

[0071] By irradiating quantum dots with light, electron and hole pairs generated can be extracted and used in oxidation-reduction reactions. In the present invention, electron and hole pairs generated by quantum dots were applied to ammonia production (Fig. 3).

[0072]

[0073] Example 1-3: Surface ligand exchange of quantum dots

[0074] In Example 1-2, the synthesized quantum dot solution (5 mL) in the flask was mixed with toluene (5 mL), ethanol (20 mL), and acetone (10 mL), and the quantum dots were precipitated through centrifugation (10,000 rpm, 5 min), and then dispersed in toluene (5 mL) again. After repeating the purification process using ethanol and acetone three times, the purified quantum dots were dispersed in toluene (5 mL). A reaction solution for ligand exchange was prepared by dissolving mercaptopropionic acid (0.2 M) and tetramethylammonium hydroxide (0.35 M) in methanol. The ligand exchange solution (5 mL) and the quantum dot solution dispersed in toluene (5 mL) were mixed and stirred for 30 minutes, after which acetone (15 mL) and hexane (15 mL) were added, and centrifugation (10,000 rpm, 5 min) was performed. The precipitated quantum dots were dispersed in a ligand exchange solution (5 mL), stirred for 30 minutes, and then purified using acetone and hexane, followed by dispersion in methanol. Finally, the quantum dots were dispersed in water through purification, sterilized using a 0.2 μm pore syringe filter (Minisart), and used in the present invention.

[0075]

[0076] Example 2: Fermentation of a quantum dot-bacteria hybrid system

[0077] By inoculating bacteria onto a medium containing quantum dots, nitrogen-fixing bacteria exposed to quantum dots incorporate quantum dots during the growth process, forming a quantum dot-bacteria hybrid system in which quantum dots are bound to the interior of the nitrogen-fixing bacteria (Fig. 4).

[0078]

[0079] Example 2-1: Cultivation of quantum dot-nitrogen-fixing bacteria hybrids (flask)

[0080] Azotobacter vinelandii (KCTC 2426) was cultured in Burk's medium (MgSO4 0.200 g / L, K2HPO4 0.800 g / L, KH2PO4 0.200 g / L, CaSO4 0.130 g / L, FeCl3 0.00145 g / L, Na2MoO4 0.000253 g / L, Sucrose 20.000 g / L) modified with iron trichloride (FeCl3) and sodium molybdate (Na2MoO4). Azotobacter vinelandii was cultured at 30°C and 200 rpm. Hybrid cells were prepared by culturing quantum dots and Azotobacter vinelandii simultaneously. Bacteria were inoculated into Burk's medium containing sterilized quantum dots and cultured at 30°C with shaking at 200 rpm. The concentration of quantum dots in the culture medium was set at 50 nM. The grown cells were washed three times with phosphate-buffered saline (PBS) at low temperature. The optical density at 600 nm (OD) was measured using a TECAN Infinite 200PRO. 600 ) was measured to adjust the cell density to 2.0, and 2 mL of the cell suspension was exposed to light. For the flask-scale experiment, an OSRAM DULUX L 36W / 864 lamp (6500K, 53 mW) was used as an LED panel.

[0081]

[0082] Example 2-2: Cultivation of quantum dot-nitrogen-fixing bacteria hybrid (fermenter)

[0083] Azotobacter vinellandi was cultured in a bioreactor fermenter. A 5-L fermenter was prepared with sterilized 1.8 L of Burk's medium, and quantum dots (50 nM) and bacteria (200 mL) were simultaneously inoculated. The quantum dot-bacteria hybrid cells were cultured under dark conditions. Unlike flask-scale fermentation, several factors require optimization for microbial growth in the fermenter, including pH, dissolved oxygen (DO), sucrose concentration, and foam formation. The pH was maintained at 7.50 using 1 M NaOH. The fermentation was performed at 30°C, and the dissolved oxygen (DO) level was maintained at 10% by controlling the air flow rate and stirring speed. During fermentation, the cell density was monitored as the optical density at 600 nm using a UV-Vis spectrophotometer. The bacterial fermentation process was performed using a BioTron (Liflux GX) device from Hanil Science. Sucrose concentration was measured using an equipment equipped with HPLC (1515 isocratic HPLC pump, Waters), a refractive index detector (2414, Waters), and a MetaCarb 87H column (Agilent).

[0084]

[0085] Example 2-3: Ammonia production by quantum dot-nitrogen-fixing bacteria hybrid (fermenter)

[0086] Bacterial growth was observed using a UV-Vis spectrophotometer, and no further bacterial growth was observed at the point where sucrose was depleted. At this time, the ammonia production reaction through the quantum dot-nitrogen-fixing bacteria hybrid was observed by irradiating the fermenter with white light. The ammonia concentration in the cell culture was measured using the indophenol blue indicator reaction. The cell culture was diluted with phosphate-buffered saline, the supernatant was collected, and a calibration curve was created by preparing a standard ammonium ion solution of a known concentration. The indophenol blue indicator was added to each standard solution and the supernatant of the sample, and the reaction was performed for 2 hours at room temperature, after which the absorbance was measured at 655 nm.

[0087] Ultimately, the original goal was to develop a system that could simultaneously produce ammonia and continuously grow bacteria using a culture medium of nitrogen-fixing bacteria.

[0088]

[0089] Example 3: Light source optimization for ammonia production

[0090] When irradiated with 400 nm light, ammonia production was observed to be more than four times higher than in the dark. However, ammonia production in the QD-bacteria hybrid system reached saturation after a reaction time of 6 hours or longer. Light with a wavelength of 400 nm is known to generate reactive oxygen species (ROS) in cells, and the possibility that these generated ROS could cause cell damage has been raised.

[0091] In this example, ammonia production using white light was observed to reduce cell damage, and it was confirmed that ammonia production continued to increase for up to 12 hours without being saturated compared to when irradiated with light of 400 nm wavelength (Fig. 5). To explain that the decrease in ammonia productivity is an effect due to the production of reactive oxygen species, the cell density of bacteria according to the light irradiation time for each light source was compared, and it was confirmed that the decrease in cell density was significantly less under white light irradiation conditions than under light of 400 nm wavelength (Fig. 6). In addition, under different light source irradiation conditions, the production of reactive oxygen species, superoxide anion (O2), was increased. ●- ) was compared to confirm that cell damage due to the production of reactive oxygen species was the cause of the limitation in ammonia productivity (Fig. 7).

[0092] The goal of the present invention is to develop a single-pot system that continuously performs bacterial growth and ammonia production through light irradiation in a 5 L fermenter. To eliminate the steps of harvesting and purifying cells from the fermenter and establish an effective ammonia production system, white light was selected as the light source, minimizing cell damage and enabling effective photoexcitation of quantum dots.

[0093]

[0094] Example 4: Optimization of culture conditions for ammonia production

[0095] Prior to ammonia production, a high-density bacterial culture requires a nutrient supply within the fermenter. Nitrogen-fixing bacteria were observed to consume sucrose, a carbon source, during growth, and when sucrose is depleted, the bacterial culture tends to reach a stationary phase.

[0096] When ammonia production using a quantum dot-bacteria hybrid system was performed in a 2 mL reaction volume using a test tube, almost no ammonia was produced under nutrient medium conditions despite light irradiation. However, ammonia production was observed under phosphate-buffered saline conditions, which maintain pH and cell activity without nutrients (Fig. 8). Therefore, this suggests that additional sucrose addition during bacterial culture does not contribute to enhanced ammonia production.

[0097] Furthermore, by observing that ammonia production increased when sucrose, a carbon source, was removed from Burk's medium containing sucrose, we determined that sucrose, a carbon source contained in the bacterial culture, inhibited ammonia production in the quantum dot-bacteria hybrid cell (Fig. 9). This explains why bacterial biomass formation and ammonia production do not increase simultaneously, and suggests that ammonia production can be induced using a medium from which carbon sources have been removed.

[0098] Furthermore, nitrogen-fixing bacteria are known to produce ammonia using adenosine triphosphate-based charge transfer, and magnesium (Mg) in the nutrient medium directly influences the activity of adenosine triphosphate, thereby affecting the overall activity of nitrogenase. Accordingly, we observed an increase in ammonia production in the quantum dot-bacteria hybrid system under magnesium-limited culture conditions, and as a result, we confirmed that quantum dots can replace the role of magnesium-adenosine triphosphate (MgATP), which is important for the activity of nitrogenase (Fig. 9).

[0099] As a result, the quantum dot-bacteria hybrid system can enhance ammonia production under carbon-free conditions, and quantum dots can effectively activate nitrogenase by replacing the existing energy source, magnesium-adenosine triphosphate (Fig. 10).

[0100]

[0101] Example 5: Optimization of the fermentation process for mass production of ammonia.

[0102] Based on the light source optimization of Example 3 and the culture medium optimization of Example 4, we aimed to develop a method for mass-cultivating quantum dot-bacteria hybrids in a fermenter and increasing ammonia productivity using the same.

[0103] Since ammonia production was confirmed in Burk's medium excluding sucrose in Example 4, ammonia production was observed using a fermenter under sucrose-depleted culture conditions. To simultaneously cultivate nitrogen-fixing bacteria and produce ammonia in a single fermenter, a strategy was developed to divide the fermentation process into two stages.

[0104] In the first step, quantum dots were injected into Burk's medium containing sucrose, which is necessary for bacterial growth, and wild-type Azotobacter vinnellii was inoculated and cultured to form quantum dot-bacteria hybrid complexes while depleting the sucrose in the culture medium. Maintaining a constant dissolved oxygen content in the culture medium while increasing cell density is crucial for bacterial culture in a fermenter, and it was confirmed that bacteria grew to the highest cell density under conditions of 10% dissolved oxygen (Fig. 11).

[0105] In the second step, when the quantum dot-bacteria hybrid cells had sufficiently grown, i.e., when the sucrose content of the culture medium was depleted during the fermentation process, ammonia was produced by irradiating the cells with white light (Fig. 12). It was observed that the sucrose content decreased during bacterial growth, while the optical density at 600 nm, which supports cell density, increased. Furthermore, irradiating the cells with white light at the point of sucrose depletion confirmed that ammonia production increased (Fig. 13). Therefore, it can be seen that ammonia can be successfully produced in a bench-scale culture system using a fermenter.

[0106] The quantum dot-bacteria hybrid system according to this example achieved the production of 7.81 mg / L of ammonia over a reaction time of 45 hours in a 5 L reactor using a fermenter. This is a 6-fold increase in the turnover frequency (TOF) of ammonia production compared to that of adenosine triphosphate, Fe protein, and purified nitrogenase, reaching 11.96 s. -1 It shows the ammonia conversion efficiency (Fig. 14).

[0107] The ammonia production capacity through fermentation of the quantum dot-bacteria hybrid system was confirmed to have a titer of 7.81 mg / L, a yield of 7.41 mol / L, and a productivity of 0.174 mg / L / h.

[0108]

[0109] The quantum dot-microbe complex, or quantum dot-bacteria hybrid system, according to the present invention suggests the possibility of effective industrial-scale ammonia production. The Haber-Bosch process, widely known as the conventional ammonia production process, entails energy and environmental issues, and the need for alternative technologies has long been emphasized. However, its high ammonia conversion efficiency has prevented its replacement. While efforts have been made to develop an eco-friendly and sustainable ammonia production system, these efforts have largely been limited to laboratory-scale research. The present invention, by expanding this research to bench-scale, is expected to hasten the establishment of alternative technologies.

[0110]

[0111] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for producing ammonia comprising the following steps: (a) a step of culturing a microorganism that endogenously expresses a nitrogen fixing enzyme or has a nitrogen fixing enzyme introduced externally in a nutrient medium containing inorganic nanoparticle quantum dots under dark conditions to create a quantum dot-microorganism complex; and (b) a step of generating ammonia by irradiating white light; Here, an ammonia production method characterized in that steps (a) and (b) are performed in a single fermenter.

2. A method for producing ammonia, characterized in that step (b) is performed at a point in time when sucrose is depleted in the nutrient medium of step (a) in paragraph 1.

3. A method for producing ammonia, characterized in that in the first paragraph, the quantum dot comprises an indium phosphide core / zinc selenide shell (InP / ZnSe), and the particle size is 4 to 6 nm.

4. A method for producing ammonia, characterized in that the quantum dot in paragraph 1 has a hydrophilic ligand introduced thereto.

5. A method for producing ammonia, characterized in that in paragraph 4, the hydrophilic ligand is selected from the group consisting of mercaptopropionic acid (MPA), L-glutathione (GSH), mercaptoacetic acid, mercaptobutanoic acid, mercaptopentanoic acid, mercaptohexanoic acid, mercaptoheptanoic acid, mercaptooctanoic acid, mercaptononanoic acid, mercaptodecanoic acid, mercaptoundecanoic acid, mercaptododecanoic acid, and L-cysteine.

6. In the first paragraph, the microorganism is Clostridium sp., Klebsiella pneumoniae, Paenibacillus polymyxa, Bacillus macerans, Escherichia intermedia, Azotobacter agilis, Azotobacter armeniacus, Azotobacter beijerinckii, Azotobacter chroococcum, Azotobacter nigricans, Azotobacter paspali, Azotobacter salinestris, Azotobacter tropicalis A method for producing ammonia, characterized in that the ammonia producing bacteria is selected from the group consisting of Azotobacter vinelandii, Rhizibium sp., Achromobacter, Azorhizobium sp., Frankia sp., Pseudomonas sp., Bacillus sp., Nitrobacter sp., Fusarium oxysporum, Cylindrocaropn tonkinese, Bipolaris sorokiniana, and Cyanobacteria sp.

7. A method for producing ammonia, characterized in that the nutrient medium in paragraph 1 is Burk's medium.

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