Production Method of Ammonia Using Quantum Dot-Bacteria Hybrid System

Abstract

The present invention relates to a method for producing ammonia using quantum dot-microbial hybrids, extending bench-scale research from a few milliliters to several liters. By developing a sustainable microbial-based ammonia production technology using a fermenter, it is possible to effectively respond to the increasing demand for ammonia. The present invention presents a concrete implementation that extends existing technology to the commercialization stage and is expected to possess competitiveness as an eco-friendly and sustainable ammonia production technology.

Description

Technology Field

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

[0002] [Related Research Projects]

[0003] [Project ID] SRFC-MA2001-07

[0004] [Department Name] Samsung Electronics Co., Ltd.

[0005] [Specialized Research Management Agency] Samsung Electronics Future Technology Development Center

[0006] [Research Project Name] Samsung Future Technology Development Project

[0007] [Research Project Title] Development of Eco-friendly Ammonia Production Technology Based on Quantum Dot-Bacteria Hybrid

[0008] [Research Period] August 2020 – July 2023 Background Technology

[0010] Ammonia (NH3) is the second most produced fine chemical globally after sulfuric acid, with approximately 80% of the produced ammonia being used in the manufacture of nitrogen fertilizers. The Haber-Bosch process, currently the most widely used method for ammonia production, has the disadvantage of requiring high energy because it demands high temperatures (~500°C) and high pressures (~200 bar). Additionally, the hydrogen used as a reactant in the Haber-Bosch process is mostly produced through natural gas reforming, which has the limitation of low sustainability as it emits about 2.5 tons of carbon dioxide to produce 1 ton of ammonia.

[0011] As global awareness of energy and environmental issues such as global warming increases, various policies are being pursued to achieve the common goal of reducing carbon emissions. Against this backdrop, ammonia is attracting attention as an important compound for realizing carbon neutrality as a ship fuel and hydrogen storage energy source, as it is easier to store and transport than hydrogen and has relatively lower technical operating costs. Accordingly, the demand for ammonia is expected to continue increasing until 2050, and there is a need to develop environmentally friendly and sustainable ammonia production technologies capable of replacing the existing Haber-Bosch process.

[0012] Nitrogen-fixing bacteria, known to produce ammonia in nature, fix atmospheric nitrogen and convert it into ammonium under conditions of room temperature and pressure. These bacteria utilize intracellular adenosine triphosphate (ATP) as an energy source and convert nitrogen molecules into ammonia by transferring electrons derived from organic matter using nitrogenase (MoFe protein) as a biological catalyst. However, due to the relatively low ammonia conversion efficiency of nitrogenase and the difficulty of precise metabolic regulation, there are limitations in producing large quantities of ammonia, which presents distinct restrictions for its application in industrial-scale processes.

[0014] Meanwhile, according to Korean patent applications No. 10-2021-0106678 and No. 10-2021-0106680, a method for producing ammonia using zinc selenide shell quantum dots capped with indium phosphide-based core / mercaptopropionic acid is described, and it was confirmed that ammonia can be produced in an eco-friendly manner at low temperatures, but there was a problem in that the ammonia production volume became saturated after a reaction time of 6 hours or more, making it impossible to mass-produce ammonia.

[0015] Accordingly, the inventors of the present invention have made diligent efforts to solve the aforementioned problems and develop a method capable of mass-producing ammonia by scaling up. As a result, by using white light instead of a light source with a wavelength of 400 nm, they enabled energy absorption across the entire light spectrum and reduced the generation of reactive oxygen species, thereby increasing continuous ammonia production. Furthermore, by confirming culture conditions in the fermenter that eliminate the need for purifying cultured bacteria or exchanging the culture medium, and verifying effective ammonia production and scalability, the present invention was completed.

[0017] The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to those skilled in the art to which the present invention belongs. Prior art literature

[0019] Republic of Korea Patent Application 10-2021-0106678 Republic of Korea Patent Application 10-2021-0106680

[0020] S. Koh, Y. Choi, I. Lee, GM Kim, J. Kim, YS Park, SY Lee, DC Lee, "Light-Driven Ammonia Production by Azotobacter vinelandii Cultured in Medium Containing Colloidal Quantum Dots", Journal of the American Chemical Society, 2022, Vol. 144, no. 24, pp. 10798-10808, DOI: 10.1021 / jacs.2c01886 The problem to be solved

[0021] The objective of the present invention is to enable industrial-scale ammonia production in a method for producing ammonia using a quantum dot-microbial complex by expanding the scale of quantum dot-microbial complex cultivation and ammonia production. means of solving the problem

[0023] To achieve the above objective, the present invention provides a method for producing ammonia comprising: (a) a step of producing a quantum dot-microorganism complex by culturing microorganisms in which nitrogen fixation enzymes are intrinsically expressed or externally introduced in a nutrient medium containing inorganic nanoparticle quantum dots under dark conditions; and (b) a step of producing ammonia by irradiating with white light, wherein steps (a) and (b) are performed in a single fermenter. Effects of the invention

[0025] The quantum dot-microbial complex, or quantum dot-bacterial hybrid system, according to the present invention has demonstrated the potential for effective industrial-scale ammonia production. The Haber-Bosch process, widely known as a conventional ammonia production process, entails energy and environmental issues, and the need for alternative technologies has long been emphasized; however, the reality is that it has not been replaced due to its high ammonia conversion efficiency. Consequently, while the development of an eco-friendly and sustainable ammonia production system has been undertaken, it has remained at the laboratory level. The present invention is expected to accelerate the establishment of alternative technologies by expanding this research to a bench scale. Brief explanation of the drawing

[0027] Figure 1 shows transmission electron microscope (TEM) observation data of quantum dots synthesized for the fabrication of quantum dot-nitrogen-fixing bacteria hybrids. Figure 2 shows the absorption and emission spectra of quantum dots synthesized for the fabrication of quantum dot-nitrogen-fixing bacteria hybrids. Figure 3 is a simple diagram explaining the principle of ammonia production in a quantum dot-nitrogen-fixing bacteria hybrid. Figure 4 is a simple schematic of the method for fabricating quantum dot-nitrogen-fixing bacteria hybrids. 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. Figure 6 is a graph comparing the change in cell density of quantum dot-nitrogen-fixing bacteria hybrids according to the type of light source. Figure 7 is a graph showing the results of comparing the amount of active oxygen species generated by quantum dots according to the type of light source. Figure 8 is a result graph showing that the quantum dot-bacteria hybrid system generates ammonia under phosphate-buffered physiological saline conditions. Figure 9 is a graph comparing the ammonia production of quantum dot-bacterial hybrids under medium conditions controlled by the addition of sugars (sucrose, glucose, fructose) or magnesium. Figure 10 is a diagram illustrating the process of ammonia production using electrons generated from quantum dots and an increase in bacterial biomass. Figure 11 is a graph showing the results of comparing the cell density of nitrogen-fixing bacteria according to the dissolved oxygen content of the culture medium in a fermenter. Figure 12 is a schematic diagram of a two-step process for ammonia production using quantum dot-nitrogen-fixing bacteria hybrids in a fermenter. Figure 13 is a graph showing the results of ammonia production over time using a quantum dot-nitrogen-fixing bacteria hybrid in a fermenter. Figure 14 is a graph comparing the TOF of ammonia production results using a quantum dot-nitrogen-fixing bacteria hybrid in a fermenter. Specific details for implementing the invention

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0030] The aim was 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, verifying scalability is essential for utilizing the constructed quantum dot-bacteria hybrid system in industrial ammonia production processes. In this invention, nitrogen-fixing bacteria were cultured in large quantities using a fermenter, a large-scale microbial culture system, and the scalability of the quantum dot-bacteria hybrid complex for ammonia production was verified.

[0031] Based on the results of a previous application (Application Nos.: 10-2021-0106678, 10-2021-0106680) that improved the ammonia production capacity of nitrogen-fixing bacteria by introducing quantum dots, which are inorganic nanoparticles, into existing wild-type nitrogen-fixing bacteria, the present invention confirmed the scalability of a fermenter process capable of industrial mass production of ammonia through a quantum dot-bacteria hybrid system.

[0032] In the previously filed invention, photoexcitation of quantum dots was induced using a light source with a wavelength of 400 nm; however, there was a problem in that ammonia production saturated after a reaction time of more than 6 hours. In this invention, by modifying the light source using white light, energy absorption across the entire light spectrum was enabled, and the generation of reactive oxygen species was reduced, thereby achieving a continuous increase in ammonia production. Furthermore, culture medium conditions were optimized to reduce time and cost associated with purifying bacteria cultured in a liter-scale reactor 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 carbon sources promotes biomass formation while reducing ammonia productivity. This invention describes an effective ammonia production method by dividing the process into a first stage of mass cultivation and a second stage in which a photochemical reaction induced by light proceeds simultaneously with the depletion of the carbon source. Accordingly, this invention further explains the ammonia production mechanism of a quantum dot-bacteria hybrid system in detail by controlling the conditions of the light source and culture medium through this two-stage process, and verifies effective ammonia production and scalability.

[0034] Accordingly, in one aspect, the present invention relates to a method for producing ammonia comprising: (a) a step of producing a quantum dot-microorganism complex by culturing a microorganism in which a nitrogen-fixing enzyme is intrinsically expressed or an externally introduced nitrogen-fixing enzyme is introduced in a nutrient medium containing inorganic nanoparticle quantum dots under dark conditions; and (b) a step of producing ammonia by irradiating with white light, wherein steps (a) and (b) are performed in a single fermenter.

[0035] In the present invention, the volume of the fermenter may be characterized as being 500 mL to 100 L, but is not limited thereto. Preferably, it may be a single fermenter of 1 L to 50 L, more preferably 1 L to 10 L, and most preferably 5 L, but is not limited thereto.

[0036] In the present invention, the step (b) may be performed at the time when sucrose is depleted in the nutrient medium of step (a).

[0037] In the present invention, step (b) may be characterized by directly irradiating white light onto the fermenter in which the quantum dot-microorganism complex is generated in step (a).

[0038] In the present invention, the quantum dots may be characterized by comprising an indium phosphide core / zinc selenide shell (InP / ZnSe) and having a particle size of 4 to 6 nm.

[0039] In the present invention, the core and shell may each be characterized by having a diameter of 2 nm to 3 nm, but are not limited thereto.

[0040] In the present invention, the quantum dots may be characterized by having a hydrophilic ligand introduced therein.

[0041] In the present invention, the hydrophilic ligand may be characterized as 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 may be characterized as being mercaptopropionic acid, but is not limited thereto. no.

[0042] In the present invention, the microorganism is Clostridium sp. ( Clostridium sp . ), Klebsiella pneumonia ( Klebsiella pneumoniae ), Phenibacillus polymixa( Paenibacillus polymyxa ), Bacillus macegans (B acillus macerans ), Essericia Intermedia ( Escherichia intermedia ), Azotobacter agilus( Azotobacter agilis ), Azotobacter Armeniacus ( Azotobacter armeniacus ), Azotobacter beijerinche ( Azotobacter beijerinckii ), Azotobacter crucocum( Azotobacter chroococcum ), Azotobacter nigricas( Azotobacter nigricans ), Azotobacter pasparli( Azotobacter paspali ), Azotobacter salinestris( Azotobacter salinestris ), Azotobacter tropicalis( Azotobacter tropicalis ), Azotobacter vinellandi( Azotobacter vinelandii ), Rigidizium sp.( Rhizibium sp . ), Acromobacter( Achromobacter ), Azorizobium( Azorhizobium sp . ), Francia sp.( Frankia sp . ), Pseudomonas sp.( Pseudomonas sp . ), Bacillus sp., Nitrobacter sp. Nitrobacter sp. ), Fusarium oxysporum ( Fusarium oxysporum ), Cylindrocaroff Tonkinese( Cylindrocaropn tonkinese ), non-polaris sorokiniana( Bipolaris sorokiniana ), and cyanobacteria sp.( Cyanobacteria sp . It may be characterized by being selected from a group consisting of ). Preferably, the microorganism is Azotobacter vinellandi ( Azotobacter vinelandii It may be, but is not limited to.

[0043] In the present invention, the nutrient medium may be characterized as being Burk's medium, but is not limited thereto.

[0044] In one embodiment of the present invention, a nitrogen-fixing bacterium, Azotobacter vinellandi, known to produce ammonia by fixing atmospheric nitrogen under conditions of room temperature and pressure ( Azotobacter vinelandii Quantum dots, which are semiconductor nanoparticles whose optical properties vary depending on particle size, were utilized (KCTC 2426). The quantum dots developed for application in an ammonia production system are indium phosphide (InP)-based core-shell structured quantum dots with a particle size of approximately 4-6 nm (Fig. 1). In this invention, a core-shell structured quantum dot with improved charge extraction efficiency was synthesized by stacking a zinc selenide (ZnSe) shell onto an indium phosphide core (Fig. 2). Quantum dots have the characteristic of generating electrons and holes when exposed to light, and they exhibit the characteristic of emitting light when the generated electron-hole pairs recombine. Conversely, the generated electron-hole pairs are extracted without recombining and can be used in redox reactions to contribute to chemical reactions; in this invention, these characteristics were utilized to apply the generated electron-hole pairs to ammonia production (Fig. 3).

[0045] 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 water dispersion. The water-dispersed quantum dots are sterilized using a Minisart syringe filter with a particle pore size of 0.2 μm and then added to a culture medium. By inoculating bacteria into the medium containing the quantum dots, nitrogen-fixing bacteria exposed to the quantum dots incorporate the quantum dots during the growth process, and a quantum dot-bacteria hybrid system is formed in which the quantum dots are bound inside the nitrogen-fixing bacteria (Fig. 4).

[0046] When irradiated with 400 nm light, ammonia production in the quantum dot-bacterial hybrid system is observed to be more than four times higher than under dark conditions. However, results show that the ammonia production of the quantum dot-bacterial hybrid system saturates after a reaction time of 6 hours or longer. It is known that light with a wavelength of 400 nm generates reactive oxygen species (ROS) in cells, and the possibility has been raised that these generated ROS may cause cell damage. In this invention, to reduce cell damage, ammonia production using white light was observed, and results were confirmed showing that ammonia production did not saturate and continued to increase up to 12 hours compared to when irradiated with 400 nm light (Fig. 5). To explain that the decrease in ammonia productivity is an effect of ROS generation, bacterial cell density according to irradiation time was compared for each light source, and it was confirmed that the decrease in cell density was significantly less under white light irradiation conditions compared to 400 nm light (Fig. 6). Furthermore, under different light source irradiation conditions, the reactive oxygen species superoxide anion (O2 ·- By comparing the production amounts of ), it was explained that cell damage caused by the generation of reactive oxygen species is the cause of the limitation in ammonia productivity (Fig. 7).

[0047] The objective of the present invention is to develop a one-pot system in which the growth of bacteria and the production of ammonia through light irradiation are continuously carried out in a 5 L-scale fermenter. In order to eliminate the steps of harvesting and purifying cells in the fermenter and to establish an effective ammonia production system, white light capable of minimizing cell damage and enabling effective photoexcitation of quantum dots was adopted as the light source.

[0048] Prior to the ammonia production stage, the supply of nutrients within the fermenter is essential for high-density bacterial culture. However, the quantum dot-bacterial hybrid system produced almost no ammonia under nutrient medium conditions despite light irradiation. On the other hand, ammonia production by the quantum dot-bacterial hybrid system was observed under phosphate buffered saline (PBS) conditions, which are intended to maintain pH and cell activity without nutrients (Fig. 8). However, almost no ammonia production was observed in phosphate buffered saline treated with carbon sources such as sucrose, glucose, and fructose, and although the supply of carbon sources promotes bacterial biomass formation, it triggers metabolism opposite to that of ammonia production.

[0049] In the nutrient medium for culturing Azotobacter vinellandi, the quantum dot-bacterial hybrid system did not produce ammonia, but when the carbon source in the nutrient medium was restricted, an increase in the ammonia production of the hybrid system was observed (Fig. 9). This explains that the biomass formation and ammonia production of bacteria do not increase simultaneously, and it was confirmed that the present invention can induce ammonia production using a medium from which the carbon source has been removed.

[0050] 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 nitrogen-fixing enzymes. The present invention observed an increase in ammonia production in a quantum dot-bacterial hybrid system under magnesium-limited culture conditions, which demonstrates that quantum dots can replace the role of magnesium-adenosine triphosphate (MgATP), which is critical for nitrogen-fixing enzyme activity (Fig. 9). Therefore, the quantum dot-bacterial hybrid system can enhance ammonia production under conditions without a carbon source, and demonstrates that quantum dots can effectively activate nitrogen-fixing enzymes by replacing magnesium-adenosine triphosphate, the existing energy source (Fig. 10).

[0051] In one embodiment of the present invention, experiments were conducted on the mass cultivation of quantum dot-bacterial hybrids in a fermenter and the increase in ammonia productivity using the same, based on the light source optimization and culture medium optimization described above. The core idea of ​​the present invention is to divide the process into two stages in order to perform the cultivation of quantum dot-bacterial hybrids and ammonia production in a single batch.

[0052] The first step is to inoculate wild-type Azotobacter vinellandi into a nutrient medium infused with quantum dots and to culture the quantum dot-bacterial hybrid under dark conditions. For bacterial culture in a fermenter, it is important to maintain a constant amount of dissolved oxygen in the culture medium and increase cell density, and in the present invention, it was confirmed that bacteria grow to the highest cell density under conditions of 10% dissolved oxygen (Fig. 11).

[0053] During the fermentation process, the sucrose content of the culture medium was compared, and a second step of ammonia production was performed by irradiating white light at the point when the sucrose was depleted (Fig. 12). During bacterial growth, the sucrose content decreased, and the optical density at 600 nm, which is a supporting factor for cell density, increased. It was observed that the production of ammonia increased by irradiating white light at the point when the sucrose was depleted (Fig. 13).

[0054] In this invention, the quantum dot-bacterial hybrid system achieved the production of 7.81 mg / L of ammonia over a reaction time of 45 hours in a 5 L-scale reactor using a fermenter. This result represents a turnover frequency (TOF) of 11.96 s⁻¹, which is approximately six times higher than the ammonia production turnover efficiency (TOF) of adenosine triphosphate, Fe protein, and purified nitrogen-fixing enzymes. -1 It shows the ammonia conversion efficiency (Fig. 14).

[0055] The ammonia production capacity through fermentation of the quantum dot-bacteria hybrid system was confirmed to be 7.81 mg / L titer, 7.41 mol / L yield, and 0.174 mg / L / h productivity.

[0057] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0059] Examples 1: quantum dots synthesis

[0060] Examples 1-1: Indium phosphide Core quantum dots synthesis

[0061] Indium acetate (0.45 mmol), oleic acid (1.35 mmol), and 1-octadecine (27 mL) were placed in a 3-neck round-bottom flask and heated at 120°C under vacuum conditions (<200 mTorr) for 2 hours to form indium oleate. The inside of the flask was switched to an inert atmosphere and 2.2 mL of trioctylphosphine was injected, then heated for an additional 30 minutes under a low-pressure atmosphere, and the mixture of indium oleate and tris-trimethylsilylphosphine was cooled to room temperature.

[0062] 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, and it was confirmed that the particle size was approximately 4-6 nm (Fig. 1).

[0064] Examples 1-2: Indium phosphide / selenide zinc core-shell structure quantum dots synthesis

[0065] Zinc oleate and trioctylphosphine selenide (2 M), prepared in an inert atmosphere, were sequentially injected into an indium phosphide core solution at a reaction temperature of 300°C. At this time, to synthesize shells in single-layer units, zinc oleate and trioctylphosphine selenide were injected in calculated amounts, and the trioctylphosphine selenide solution was injected slowly drop by drop. After heating each shell for 30 minutes, once 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).

[0066] Electron-hole pairs generated by irradiating light onto quantum dots can be extracted and used in oxidation-reduction reactions, and in the present invention, electron-hole pairs generated by quantum dots were applied to ammonia production (Fig. 3).

[0068] Examples 1-3: quantum dots Surface ligand exchange

[0069] In Examples 1-2, the quantum dot solution (5 mL) synthesized in the flask was mixed with toluene (5 mL), ethanol (20 mL), and acetone (10 mL), and the quantum dots were precipitated by centrifugation (10,000 rpm, 5 min), and then dispersed again in toluene (5 mL). 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 (5 mL) dispersed in toluene were mixed and stirred for 30 minutes, after which acetone (15 mL) and hexane (15 mL) were added and centrifuged (10,000 rpm, 5 min). The precipitated quantum dots were dispersed in a ligand exchange solution (5 mL), stirred for 30 minutes, and then dispersed in methanol after repeating the purification process using acetone and hexane. Finally, the quantum dots were dispersed in water through purification and sterilized using a syringe filter (Minisart) with 0.2 μm pores for use in the present invention.

[0071] Examples 2: quantum dots -bacteria hybrid fermentation of the system

[0072] By inoculating bacteria into a medium containing quantum dots, nitrogen-fixing bacteria exposed to quantum dots incorporate the quantum dots during the growth process, and a quantum dot-bacteria hybrid system is formed in which the quantum dots are bound inside the nitrogen-fixing bacteria (Fig. 4).

[0074] Examples 2-1: quantum dots - Nitrogen fixation bacteria hybrid Culture (flask)

[0075] Azotobacter vinellandi ( Azotobacter vinelandii (KCTC 2426) was used as a modified medium by additionally adding iron trichloride (FeCl3) and sodium molybdate (Na2MoO4) to 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) from Himedia. Azotobacter vinellandi was cultured under conditions of 30°C and 200 rpm. Hybrid cells were prepared by simultaneously culturing quantum dots and Azotobacter vinellandi. Bacteria were inoculated into Burk's medium containing sterile quantum dots and cultured at 30°C with stirring at 200 rpm. The concentration of quantum dots in the culture medium was set to 50 nM. Grown cells were washed three times with phosphate-buffered saline (PBS) at low temperature. Optical density (OD) at 600 nm was measured using a TECAN Infinite 200PRO. 600 After adjusting the cell density to 2.0 by measuring ), 2 mL of cell suspension was exposed to light. For flask-scale experiments, an OSRAM DULUX L 36W / 864 lamp (6500K, 53 mW) was used with an LED panel.

[0077] Examples 2-2: quantum dots - Nitrogen fixation bacteria hybrid Cultivation (Fermenter)

[0078] Azotobacter vinellandi was cultured in a fermenter, which is a bioreactor. 1.8 L of sterile Burk's medium was prepared in a 5 L volume fermenter, and quantum dots (50 nM) and bacteria (200 mL) were simultaneously inoculated to culture quantum dot-bacterial hybrid cells under dark, light-free conditions. Unlike flask-scale fermentation, several factors require optimization for microbial growth in a fermenter, allowing for the control of pH, dissolved oxygen (DO), sucrose concentration, and foam formation. The pH was maintained at 7.50 using 1 M NaOH. The fermentation process was carried out at 30°C, and the dissolved oxygen (DO) level was maintained at 10% by controlling the air flow rate and stirring speed. During fermentation, cell density was monitored using a UV-Vis spectrophotometer to determine optical density at 600 nm. The bacterial fermentation process was performed using Hanil Science's BioTron instrument, the Liflux GX. Sucrose concentration was measured using equipment equipped with an HPLC (1515 isocratic HPLC pump, Waters), a refractive index detector (2414, Waters), and a MetaCarb 87H column (Agilent).

[0080] Examples 2-3: quantum dots - Nitrogen fixation bacteria hybrid Ammonia production (fermenter)

[0081] Bacterial growth was observed using a UV-Vis spectrophotometer, and no further bacterial growth was observed when sucrose was depleted. At this point, the ammonia production reaction via quantum dot-nitrogen-fixing bacteria hybrids was observed by irradiating the fermenter with white light. The indophenol blue indicator reaction was used to measure the ammonia concentration in the cell culture medium. The cell culture medium was diluted with phosphate-buffered physiological saline to collect the supernatant, and a calibration curve was constructed using standard ammonium ion solutions of known concentrations. Indophenol blue indicator was added to the supernatants of each standard solution and the sample, and after reacting at room temperature for 2 hours, the absorbance was measured at 655 nm.

[0082] Ultimately, the original goal is to develop a system that enables the continuous growth of bacteria and the simultaneous production of ammonia using a culture medium of nitrogen-fixing bacteria.

[0084] Examples 3: Light Source Optimization for Ammonia Production

[0085] In the case of ammonia production in a quantum dot-bacterial hybrid system, irradiation with 400 nm light results in a more than fourfold increase in ammonia production compared to dark conditions. However, the ammonia production of the quantum dot-bacterial hybrid system saturates after a reaction time of 6 hours or more. Light with a wavelength of 400 nm is known to generate reactive oxygen species (ROS) in cells, and the possibility has been raised that the generated reactive oxygen species may cause cell damage.

[0086] In this embodiment, ammonia production using white light was observed to reduce cell damage, and results were confirmed showing that ammonia production continued to increase for up to 12 hours without saturation compared to when irradiated with 400 nm wavelength light (Fig. 5). To explain that the decrease in ammonia productivity is an effect of reactive oxygen species generation, bacterial cell density according to irradiation time was compared for each light source, and it was confirmed that the decrease in cell density was significantly smaller under white light irradiation conditions compared to 400 nm wavelength light (Fig. 6). In addition, under different light source irradiation conditions, the reactive oxygen species superoxide anion (O2 ·- By comparing the production amounts of ), it was confirmed that cell damage caused by the generation of reactive oxygen species is the cause of the limitation in ammonia productivity (Fig. 7).

[0087] The objective of the present invention is to develop a one-pot system in which the growth of bacteria and the production of ammonia through light irradiation are continuously carried out in a 5 L-scale fermenter. In order to eliminate the steps of harvesting and purifying cells in the fermenter and to establish an effective ammonia production system, white light capable of minimizing cell damage and enabling effective photoexcitation of quantum dots was adopted as the light source.

[0089] Examples 4: Optimization of Culture Medium Conditions for Ammonia Production

[0090] Prior to ammonia production, the supply of nutrients within the fermenter is essential for high-density bacterial culture. It was confirmed that nitrogen-fixing bacteria consume sucrose, a carbon source, during the growth process, and it was observed that the bacterial culture tends to reach a stationary phase when sucrose is depleted.

[0091] When ammonia production was carried out in a quantum dot-bacterial hybrid system in a 2 mL reaction volume using a test tube, almost no ammonia was produced under nutrient medium conditions despite light irradiation, whereas ammonia production was observed under phosphate-buffered saline conditions intended to maintain pH and cell activity without nutrients (Fig. 8). Therefore, this suggests that the additional injection of sucrose during the bacterial culture process does not contribute to the improvement of ammonia production capacity.

[0092] In addition, by observing that ammonia production increased when sucrose, a carbon source, was removed from Burk's medium containing sucrose, it was determined that sucrose, a carbon source included in the bacterial culture medium, inhibits ammonia production in quantum dot-bacterial hybrid cells (Fig. 9). This explains that bacterial biomass formation and ammonia production do not increase simultaneously and suggests that ammonia production can be induced using a medium from which the carbon source has been removed.

[0093] In addition, it is known that nitrogen-fixing bacteria 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 nitrogen-fixing enzymes. Accordingly, an increase in ammonia production in a quantum dot-bacterial hybrid system was observed under magnesium-limited culture conditions, and as a result, it was confirmed that quantum dots can replace the role of magnesium-adenosine triphosphate (MgATP), which is important for the activity of nitrogen-fixing enzymes (Fig. 9).

[0094] As a result, the quantum dot-bacterial hybrid system can improve ammonia production under conditions without a carbon source, and the quantum dots can effectively activate nitrogen fixation enzymes by replacing magnesium-adenosine triphosphate, the existing energy source (Fig. 10).

[0096] Examples 5: Optimization of Fermentation Process for Mass Ammonia Production

[0097] 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 cultivation of quantum dot-bacteria hybrids in a fermenter and a method to increase ammonia productivity using the same.

[0098] Since it was confirmed in Example 4 that ammonia was produced in Burk's medium excluding sucrose, ammonia production was observed using a fermenter under sucrose-depleted culture conditions. In order to carry out the cultivation of nitrogen-fixing bacteria and the production of ammonia simultaneously in a single fermenter, a strategy was devised to divide the fermentation process into two stages.

[0099] In the first step, quantum dots were injected into Burk's medium containing sucrose, which is necessary for bacterial growth, and wild-type Azotobacter vinellandi were inoculated and cultured to deplete the sucrose in the culture medium while forming a quantum dot-bacteria hybrid complex. It is important to maintain a constant amount of dissolved oxygen in the culture medium and increase cell density for bacterial culture in the fermenter, and it was confirmed that bacteria grew to the highest cell density under conditions of 10% dissolved oxygen (Fig. 11).

[0100] In the second step, when the quantum dot-bacteria hybrid cells had grown sufficiently, that is, when the sucrose content of the culture medium was compared during the fermentation process, light (white light) was irradiated at the point when the sucrose was depleted to produce ammonia (Fig. 12). It was observed that while the bacteria were growing, the sucrose content decreased, and the optical density at 600 nm, which is a supporting factor for cell density, increased. It was confirmed that the production of ammonia increased when white light was irradiated at the point when the sucrose was depleted (Fig. 13). Therefore, it can be seen that ammonia can be successfully produced in a bench-scale culture system using a fermenter.

[0101] The quantum dot-bacteria hybrid system according to the present embodiment achieved a result of producing 7.81 mg / L of ammonia in a 5 L-scale reactor using a fermenter for a reaction time of 45 hours. This represents a turnover frequency (TOF) of 11.96 s⁻¹, which is approximately six times higher than the ammonia production turnover efficiency (TOF) of adenosine triphosphate, Fe protein, and purified nitrogen-fixing enzymes. -1 It shows the ammonia conversion efficiency (Fig. 14).

[0102] The ammonia production capacity through fermentation of the quantum dot-bacteria hybrid system was confirmed to be 7.81 mg / L titer, 7.41 mol / L yield, and 0.174 mg / L / h productivity.

[0104] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A method for producing ammonia comprising the following steps: (a) a step of producing a quantum dot-microorganism complex by culturing microorganisms in which nitrogen fixation enzymes are intrinsically expressed or externally introduced in a nutrient medium containing inorganic nanoparticle quantum dots under dark conditions; and (b) a step of producing ammonia by irradiating with white light, wherein steps (a) and (b) are performed in a single fermenter. Claim 2 A method for producing ammonia according to claim 1, characterized by performing step (b) at the time when sucrose is depleted in the nutrient medium of step (a). Claim 3 A method for producing ammonia according to claim 1, wherein the quantum dots comprise an indium phosphide core / zinc selenide shell (InP / ZnSe) and the particle size is 4 to 6 nm. Claim 4 A method for producing ammonia according to claim 1, wherein the quantum dots are characterized by having a hydrophilic ligand introduced therein. Claim 5 A method for producing ammonia according to claim 4, wherein 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. Claim 6 In paragraph 1, the microorganism is Clostridium sp. ( Clostridium sp . ), Klebsiella pneumonia ( Klebsiella pneumoniae ), Phenibacillus polymixa( Paenibacillus polymyxa ), Bacillus macegans (B acillus macerans ), Essericia Intermedia ( Escherichia intermedia ), Azotobacter agilus( Azotobacter agilis ), Azotobacter Armeniacus ( Azotobacter armeniacus ), Azotobacter beijerinche ( Azotobacter beijerinckii ), Azotobacter crucocum( Azotobacter chroococcum ), Azotobacter nigricas( Azotobacter nigricans ), Azotobacter pasparli( Azotobacter paspali ), Azotobacter salinestris( Azotobacter salinestris ), Azotobacter tropicalis( Azotobacter tropicalis ), Azotobacter vinellandi( Azotobacter vinelandii ), Rigidizium sp.( Rhizibium sp . ), Acromobacter( Achromobacter ), Azorizobium( Azorhizobium sp . ), Francia sp.( Frankia sp . ), Pseudomonas sp.( Pseudomonas sp. ), Bacillus sp., Nitrobacter sp. Nitrobacter sp . ), Fusarium oxysporum ( Fusarium oxysporum ), Cylindrocaroff Tonkinese( Cylindrocaropn tonkinese ), non-polaris sorokiniana( Bipolaris sorokiniana ), and cyanobacteria sp.( Cyanobacteria sp . A method for producing ammonia characterized by being selected from a group consisting of ). Claim 7 A method for producing ammonia according to claim 1, wherein the nutrient medium is Burk's medium.

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