A microbial electronically regulated denitrification system based on nitrogen-doped carbon dots and its application

Abstract

This invention relates to the field of biological denitrification technology, and more particularly to a microbial electron-regulated denitrification system based on nitrogen-doped carbon dots and its application. The system comprises electron-donating microorganisms, electron-accepting microorganisms, and nitrogen-doped carbon dots. The nitrogen-doped carbon dots act as extracellular electron mediators, constructing an efficient extracellular electron transport network and reducing interspecies electron transport resistance. Simultaneously, the nitrogen-doped carbon dots can regulate intracellular electron transport chains and energy metabolism processes, promoting increased reducing power and energy levels, and optimizing metabolic patterns. By synergistically regulating electron generation, transport, and utilization under oligotrophic conditions with limited electron donors, this system achieves highly efficient denitrification, avoids nitrite accumulation, and significantly inhibits nitrous oxide (N2O) generation, while reducing dependence on external organic carbon sources and electron sources. This provides a new technical approach for improving the efficiency of deep denitrification under low-carbon conditions.

Description

Technical Field

[0001] This invention relates to the field of biological denitrification technology, and in particular to a microbial electronically regulated denitrification system based on nitrogen-doped carbon dots and its application. Background Technology

[0002] Under oligotrophic conditions and with extremely limited electron donors, the integrity of heterotrophic denitrification is significantly constrained. Since denitrification is a multi-step reduction process driven by electron donors, insufficient electron donors disrupt the distribution of electrons in the denitrification respiratory chain, leading to the obstruction of the complete reduction pathway of nitrate to nitrogen. This results in the accumulation and release of large amounts of N2O as an intermediate or final product into the atmosphere.

[0003] To address the aforementioned issues, academia and industry have proposed various technological pathways to mitigate or control N2O emissions. One conventional method involves adding readily degradable organic carbon sources such as methanol and sodium acetate to the treatment system to supplement electron donors. However, this conventional method not only significantly increases operating costs but also poses a risk of secondary pollution and lacks engineering feasibility in wide-area non-point source scenarios such as farmland, lakes, and estuaries. Another approach is to utilize interspecies electron transfer (IET) to construct an electrosynergistic system to improve electron utilization efficiency. In existing technologies, conductive materials such as activated carbon, graphene, and biochar are typically added as passive mediators to promote electron transfer between electroactive bacteria and denitrifying bacteria. However, under oligotrophic conditions, this approach still has significant shortcomings: First, conductive materials only serve as passive electron channels and cannot increase the total amount of endogenous electrons in the system; second, electron transfer efficiency is still limited by the limited contact interface and conduction kinetics; third, and most importantly, under conditions of severely limited carbon sources, microorganisms tend to use electrons for their own growth and metabolism, making it difficult to maintain stable mutualistic cooperation and failing to fundamentally solve the metabolic competition problem of electron utilization.

[0004] Therefore, in environments with low nutrition and limited electron donors, existing technologies cannot simultaneously solve problems such as insufficient electron sources within the system, low efficiency of interspecific electron transfer, and imbalance of metabolic competition. There is an urgent need for a technical solution that can reconstruct the pathways of electron generation, transfer, and utilization at the system level.

[0005] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a microbial electronically regulated denitrification system based on nitrogen-doped carbon dots and its application. By reconstructing the pathways for microbial electron generation, transfer, and utilization at the system level, the incomplete denitrification reaction caused by insufficient electron donors is alleviated, thereby promoting near-complete (>99.9%) reduction of nitrate to nitrogen and inhibiting the generation and release of N2O. This achieves a more efficient and stable electronic coupling relationship between electroactive microorganisms and denitrifying microorganisms under oligotrophic conditions, improving the overall stability and environmental functionality of the denitrification process. The efficient denitrification described in this invention is used to characterize the overall performance level of the denitrification process under electron donor-limited conditions. Its evaluation typically considers the degree of nitrate to nitrogen conversion, the accumulation of intermediate products during denitrification, and the effect of nitrous oxide emission control.

[0007] This invention provides a microbial electronically regulated denitrification system based on nitrogen-doped carbon dots. The system operates under oligotrophic conditions where electron donors are limited, specifically a carbon-to-nitrogen ratio (C / N) ≤ 3. Furthermore, the system does not rely on external inorganic electron donors or sacrificial energy donors as its primary electron source. The system comprises:

[0008] Electron-donating microorganisms are used to oxidize limited organic carbon sources under anaerobic conditions and generate and export electrons;

[0009] Electron acceptor microorganisms possess a complete denitrifying enzyme system for the stepwise reduction of nitrate to nitrogen.

[0010] Nitrogen-doped carbon dots possess both extracellular electron transport regulation and intracellular metabolic regulation functions, among which:

[0011] Extracellularly, the nitrogen-doped carbon dots act as electron mediators, constructing an electron transport network between the electron donor and electron acceptor microorganisms.

[0012] Intracellularly, the nitrogen-doped carbon dots can regulate the intracellular electron transport chain and energy metabolism process of microorganisms, so that the acquired electrons are preferentially used for the denitrification respiratory chain.

[0013] This allows the system to achieve efficient denitrification and suppress the generation of the strong greenhouse gas N2O even under oligotrophic conditions.

[0014] Preferably, the electron donor microorganism is an electroactive bacterium, and the electron acceptor microorganism is a denitrifying bacterium.

[0015] Preferably, the electron donor microorganism is selected from *Geobacterium thioreductoides* (Stoxam). Geobacter sulfurreducens (or other electroactive bacteria with extracellular electron output capabilities.)

[0016] Preferably, the electron acceptor microorganism is *Pseudomonas stearothermia* (…). Pseudomonas stutzeri (or other denitrifying bacteria with a complete denitrification pathway)

[0017] Preferably, the nitrogen-doped carbon dots simultaneously play the following roles outside the cell: adsorbing onto the surface of microbial cells as fixed conductive nodes to promote direct interspecies electron transfer; and acting as diffusible electron shuttles to transfer electrons between microorganisms through reversible redox reactions.

[0018] Preferably, the nitrogen-doped carbon dots can increase the intracellular ATP level and reducing power level of the electron acceptor microorganism, and induce the electron acceptor microorganism to preferentially use the acquired electrons for the denitrification respiratory chain.

[0019] Preferably, the concentration of the nitrogen-doped carbon dots is 50~200 mg / L. -1 .

[0020] Preferably, the nitrogen-doped carbon dots have a particle size of 1~10 nm and their surface contains pyridine nitrogen, pyrrole nitrogen and graphitic nitrogen.

[0021] Preferably, after introducing nitrogen-doped carbon dots, under the same test conditions, the charge transfer resistance of the system is reduced by at least 80% compared to the co-culture system without nitrogen-doped carbon dots.

[0022] Based on the same inventive concept, an application is provided in which the microbial electronically regulated denitrification system based on nitrogen-doped carbon points is mixed with the substance to be denitrified and then carried out in solution for a denitrification cascade reaction.

[0023] Based on the same inventive concept, this invention provides an application of the microbial electronically regulated denitrification system based on nitrogen-doped carbon dots to suppress N2O emissions and achieve efficient deep denitrification in a high-nitrogen, low-carbon environment.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) This invention designs a technical solution for oligotrophic conditions (C / N≤3) with high nitrogen and low carbon superposition. Without the need for a large amount of exogenous organic carbon, it can effectively control the denitrification process, alleviate the problem of incomplete denitrification caused by insufficient electron donors, promote the efficient reduction of nitrate to nitrogen, and reduce the risk of N2O generation and release. Under the conditions of the example, the nitrate reduction rate can reach more than 99.9%, and the N2O emission can be effectively suppressed to only 2.84 μg·mg. -1 TN;

[0026] (2) This invention achieves highly efficient denitrification utilization and reconstruction of denitrification metabolic pathways by using nitrogen-doped carbon dots to target intracellular electron transport and energy metabolism in microorganisms, with almost no increase in carbon source consumption. Compared with the control system without nitrogen-doped carbon dots, the system constructed in this invention significantly improves denitrification performance while only increasing carbon source consumption by about 3.4%. At the same time, the intracellular energy state and reducing power of microorganisms are significantly improved, with ATP content increasing by about 1.6 times and NADH content increasing by about 2.0 times. This significantly improves the utilization efficiency of limited electron resources in an electron donor-limited environment, enhances the integrity of the denitrification respiratory chain, and effectively inhibits the accumulation of N2O.

[0027] (3) This invention constructs a high-throughput extracellular electron transport network between different functional microorganisms by using nitrogen-doped carbon dots, forming a dual-mode electron transport system with both fixed conductive paths and mobile electron shuttle mechanisms, thereby significantly reducing the kinetic resistance of interspecies electron transport; the electrochemical impedance spectroscopy analysis results show that after the introduction of nitrogen-doped carbon dots, the charge transfer resistance of the system is reduced from about 21010 Ω to about 3952 Ω, a reduction of about 81.2%, realizing high-speed electron transport from the generation end to the utilization end, effectively overcoming the problem of insufficient denitrification performance caused by the limited electron transport efficiency of the organism itself;

[0028] (4) By constructing a high-throughput interspecific electron transport network outside the cell through nitrogen doping carbon dots, and guiding the directional distribution of electrons to the denitrification respiratory chain inside the cell, under oligotrophic conditions without relying on exogenous inorganic electron donors, the efficient conversion and utilization of limited organic carbon sources to denitrification electron flow is realized, so that deep denitrification can still be completed under low C / N conditions, and the generation of N2O is significantly inhibited. Attached Figure Description

[0029] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the following description is only a part of the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1Material characterization of the nitrogen-doped carbon dots (N-CDs) obtained in this invention: (a) is a transmission electron microscope image of N-CDs; (b) is the particle size distribution of N-CDs; (c) is the X-ray diffraction pattern of N-CDs; (d) is the Raman spectrum of N-CDs; (e) is the Fourier transform infrared spectrum of N-CDs; (f) is the high-resolution C 1s X-ray photoelectron spectrum of N-CDs; (g) is the high-resolution N 1s X-ray photoelectron spectrum of N-CDs; (h) is the high-resolution O 1s X-ray photoelectron spectrum of N-CDs.

[0031] Figure 2 The microbial electronically regulated denitrification system constructed in this invention and the comparative NO3 - (a) NO2 - (b) Comparison of N2O (c) concentrations;

[0032] Note: TN is the equivalent of nitrate nitrogen removed from the system.

[0033] Figure 3 EIS spectrum (a) and CV spectrum (b) of the microbial electronically regulated denitrification system constructed in this invention.

[0034] Figure 4 The microbial electronically regulated denitrification system constructed in this invention is compared with the sodium acetate consumption (a), relative ATP content (b), electron transfer efficiency (c), relative NADH content (d), and denitrifying enzyme activity (e-h) of the comparative example. Detailed Implementation

[0035] This invention proposes a microbial electronically regulated denitrification system based on nitrogen-doped carbon dots and its application. To facilitate understanding of this invention by those skilled in the art, the specific embodiments of this invention are described below with reference to the accompanying drawings.

[0036] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the field. The methods in the following embodiments, unless otherwise specified, are conventional methods in the field. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] Example 1: Preparation of nitrogen-doped carbon dots (N-CDs)

[0038] 1.1 Experimental Methods

[0039] (1) Pretreatment: The collected biomass raw materials (based on the concepts of "waste treatment" and "solid waste resource utilization", green tea is used in this embodiment) are washed with deionized water and dried to constant weight in an oven at 60 ℃. They are then pulverized using a high-speed pulverizer and passed through a 100-mesh standard sieve to obtain uniform green tea biomass powder;

[0040] (2) Hydrothermal reaction: Accurately weigh 1 g of pretreated green tea powder, mix it evenly with 20 mL of ultrapure water, and transfer it to a 50 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene. After sealing, place the reactor in a temperature-controlled oven, heat it to 180°C, and maintain the temperature at this temperature for 5 h.

[0041] (3) Preliminary separation: After the reaction is complete, allow the reaction vessel to cool naturally to room temperature. Centrifuge the resulting dark brown mixture at 6000 rpm for 5 min to remove unreacted solid residues and larger carbon spheres. Take the supernatant and filter it through a 0.22 μm microfiltration membrane to further remove small particles;

[0042] (4) Purification: The filtered filtrate is placed into a dialysis bag with a molecular weight cutoff of 1000 Da and placed in a large amount of deionized water for dialysis. The deionized water is changed every 6 hours until the water outside the dialysis bag becomes colorless, so as to completely remove small molecule impurities and inorganic salts.

[0043] (5) Finished product: The purified N-CDs solution is freeze-dried to obtain brown N-CDs powder solid, which is then sealed and stored for later use.

[0044] 1.2 Experimental Results

[0045] The obtained N-CDs powder solid was observed by transmission electron microscopy (TEM), such as... Figure 1 As shown in (a) and (b), N-CDs are monodisperse, near-spherical nanoparticles with a uniform particle size distribution and an average diameter of approximately 2.5 nm. The crystal structure of the material was characterized by X-ray diffraction (XRD), as shown... Figure 1 As shown in (c), N-CDs exhibit a broad diffuse diffraction peak at 2θ = 20°, corresponding to the (002) crystal plane of graphite, indicating the amorphous carbon characteristics of a short-range graphitized ordered structure. Raman spectroscopy further revealed the carbon framework structure, as shown... Figure 1 As shown in (d), peak D is located at 1340 cm⁻¹. -1 (Disordered sp³ carbon), G peak located at 1588 cm⁻¹ -1(Graphitized sp² carbon), the intensity ratio of the two peaks (ID / IG) is 1.04, indicating the presence of high-density defects in the carbon lattice. These defects have important functions, serving as surface reactive sites and thus enhancing charge transfer capabilities. Figure 1 As shown in (e), the Fourier transform infrared (FTIR) spectrum indicates that the N-CDs surface is rich in oxygen / nitrogen redox active functional groups, including -OH (3344.9 cm⁻¹). -1 C=O (1703.8 cm) -1 ) and C=C (1656.5 cm) -1 This is a crucial basis for its role as a tunable electronic mediator. For example... Figure 1 As shown in f~h, the X-ray photoelectron spectroscopy (XPS) N 1s spectra indicate that the nitrogen atom doping ratio is approximately between 1% and 20%, and reveal the existence of three nitrogen species with important functional significance: pyridine nitrogen (398.6 eV), pyrrole nitrogen (400.0 eV), and graphitic nitrogen (401.4 eV). These species play a key role in improving charge trapping, interfacial electron migration, and conductivity.

[0046] Example 2: Construction of a microbial electronically regulated denitrification system based on N-CDs

[0047] This microbial electron-regulated denitrification system includes electron donor microorganisms, electron acceptor microorganisms, and N-CDs, among which the electron donor microorganism is *Geobacterium thioreductoides* (…). Geobacter sulfurreducens PCA, ATCC 51573, is a typical electroactive bacterium capable of oxidizing simple organic compounds such as acetate under anaerobic conditions and exporting electrons through extracellular electron transport, acting as a "generator" of endogenous electrons in the system; the electron acceptor microorganism is the denitrifying bacterium *Pseudomonas stearothermia* (PCA, ATCC 51573). Pseudomonas stutzeri ATCC 17588), this strain is a model denitrifying bacterium, possessing a complete denitrifying enzyme system, capable of converting NO3- into nitrogen. - It is gradually reduced to N2, acting as an "electron consumer" and the executor of denitrification; N-CDs are the N-CDs obtained in Example 1. This material can act as an electron mediator to connect isolated microorganisms outside the cell, and can also affect intracellular electron flow and metabolic pathways.

[0048] The specific steps are as follows:

[0049] (1) Preparation of simulated oligotrophic wastewater culture medium: Each liter of deionized water contains 0.180 g / L potassium nitrate (KNO3). -1 Sodium acetate (CH3COONa) 0.096 g / L -12.880 g of disodium hydrogen phosphate (Na2HPO4), 0.480 g of potassium dihydrogen phosphate (KH2PO4), 0.400 g of ammonium chloride (NH4Cl), 0.020 g of calcium chloride (CaCl2), and 0.020 g of magnesium sulfate (MgSO4);

[0050] (2) Microbial culture: aseptic culture separately G. sulfurreducens and P. stutzeri To mid-logarithmic growth. Collect cells by centrifugation and wash three times with sterile phosphate buffer to remove residual culture medium;

[0051] (3) System Construction: 250 mL anaerobic serum bottles were used as batch reactors, each filled with 150 mL of simulated wastewater culture medium. High-purity N2 was then introduced into each reactor for 20 minutes to completely remove dissolved oxygen and headspace oxygen. The reactors were then immediately sealed with butyl rubber stoppers and aluminum caps. The bacterial cells were then resuspended and inoculated into the reactors. P. stutzeri Initial optical density (OD) 600 The value reached 0.10. G. sulfurreducens initial OD 600 The concentration reached 0.03. N-CDs were added to the reaction system to achieve a final concentration of 100 mg·L⁻¹. -1 .

[0052] All serum bottles were placed in a thermostatically controlled rocker at 30 °C and 150 rpm and incubated in the dark. Water samples were collected anaerobically using sterile syringes at predetermined time points of 0, 1, 2, 4, 6, 8, 10, 12, and 24 hours. After 24 hours of incubation, headspace gas samples were collected using an airtight syringe.

[0053] By precisely controlling the dosage of acetate, the sole small organic carbon source, and nitrate, the sole reducing nitrogen source, the carbon-to-nitrogen ratio (C / N) of the system was strictly set to 3. This C / N ratio is far lower than that required for traditional denitrification, representing an extreme oligotrophic condition with limited electron donors.

[0054] Comparative Example 1 only P. stutzeri

[0055] Similar to the method in Example 2, except that no electron donor microorganisms and N-CDs are added, and the system contains only the electron acceptor microorganism *Pseudomonas stearothermia*. P. stutzeri .

[0056] Comparative Example 2 P. stutzeri + N-CDs (100 mg·L⁻¹) -1 )

[0057] Similar to the method in Example 2, except that no electron donor microorganisms are added, and the system contains the electron acceptor microorganism *Pseudomonas stearothermia*. P. stutzeri And N-CDs.

[0058] Comparative Example 3 P. stutzeri + G. sulfurreducens

[0059] Similar to the method in Example 2, except that N-CDs are not added, and the system contains the electron donor microorganism *Geobacterium thioretinoides*. G. sulfurreducens and electron acceptor microorganisms Pseudomonas stearothermia P. stutzeri .

[0060] Comparative Example 4 only G. sulfurreducens

[0061] Similar to the method in Example 2, except that no electron acceptor microorganisms and N-CDs are added, and the system contains the electron donor microorganism *Geobacterium thioreductoides*. G. sulfurreducens .

[0062] Comparative Example 5 G. sulfurreducens + N-CDs (100 mg·L⁻¹) -1 )

[0063] Similar to the method in Example 2, except that no electron acceptor microorganisms are added, and the system contains electron donor microorganisms, *Geotrichum thioretinobacillus*. G. sulfurreducens And N-CDs.

[0064] Example 3 Performance and Mechanism Analysis

[0065] 3.1 Experimental Methods

[0066] (1) Analysis of water quality indicators and N2O emissions: After the water sample was filtered through a 0.22 μm filter membrane, NO3 was determined by ion chromatography. - NO2 - Concentration; Acetate concentration was determined by high performance liquid chromatography; N2O concentration in headspace gas was determined by gas chromatograph equipped with an electron capture detector (ECD);

[0067] (2) Electrochemical analysis: Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were performed using a standard three-electrode system and an electrochemical workstation;

[0068] (3) Biological analysis: The concentrations of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NADH / NAD⁺) in cells were determined using commercially available kits. Electron transport system activity (ETSA) was determined using the INT reduction method. The activities of nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (N₂OR) were determined using standard methods.

[0069] 3.2 Experimental Results

[0070] (1) Analysis results of water quality indicators and N2O emissions

[0071] like Figure 2 As shown, Example 2 illustrates a microbial electronically regulated denitrification system based on N-CDs ( P. stutzeri + G. sulfurreducens (+ N-CDs) enables efficient and deep nitrogen removal under oligotrophic conditions.

[0072] Comparative Example 1 (only) P. stutzeri ):like Figure 2 In the middle (a, c), as the baseline control, this system showed NO3 after 24 hours. - -N concentration remained as high as 15.3 mg·L⁻¹. -1 The reaction essentially stalled. More importantly, its total nitrogen removal rate was as high as 232.5 μg·mg. -1 N2O from TN. This result fully confirms that under oligotrophic conditions, traditional heterotrophic denitrification becomes functionally ineffective due to the lack of electron donors, and triggers a serious risk of releasing the strong greenhouse gas N2O.

[0073] Comparative Example 2 ( P. stutzeri + N-CDs): such as Figure 2 In the system (a~c), although the addition of N-CDs accelerates NO3... - The reduction rate, but this resulted in the production of nitrite (NO2). - Severe accumulation of (peak concentration up to 11.5 mg·L⁻¹) -1 Meanwhile, N2O emissions remain at a high level (25.0 μg·mg). -1 (TN). This indicates that, without an additional electron source, simply accelerating electron transport within denitrifying bacteria disrupts the kinetic synchronicity between the steps of the denitrification process, leading to NO2... - Accumulation of intermediate products.

[0074] Comparative Example 3 ( P. stutzeri + G. sulfurreducens ):like Figure 2 In the middle (a~c), compared with Comparative Example 1, by constructing a microbial symbiotic system, NO2 - Accumulation amount (2.5 mg·L) -1 ) and N2O emissions (14.3 μg·mg -1 Both TN and NO3 were significantly improved. - The reduction is still incomplete. This clearly shows that, in the absence of efficient electron mediators, the inherent kinetic bottleneck of IET limits the overall efficiency of the system and cannot support a rapid and complete denitrification process.

[0075] like Figure 2 As shown in (a), Comparative Example 4 (only) G. sulfurreducens ) and Comparative Example 5 ( G. sulfurreducens NO3 in N-CDs - The NO3- concentration did not change significantly, indicating that NO3- concentration did not change significantly. - The reduction reaction efficiency is extremely low.

[0076] The microbial electronically regulated denitrification system based on N-CDs constructed in this invention ( P. stutzeri + G. sulfurreducens + N-CDs) overcome the problems exposed in the comparative examples above. For example Figure 2 As shown, in just 6 hours, NO3 - That is, it is almost completely removed (>99.9%), and NO2 is not present. - The accumulation of these particles indicates that electrons achieve perfect kinetic synchronization in all stages of generation, transport, and utilization. Most importantly, N2O emissions are extremely suppressed to a mere 2.84 μg mg. -1 TN showed an inhibition efficiency of over 98% compared to Comparative Example 1.

[0077] (2) Electrochemical analysis results

[0078] Electrochemical analysis confirmed that N-CDs construct a highly efficient extracellular electron transport network. Figure 3 ).

[0079] Electron spectroscopy (EIS) is used to evaluate the electron transfer capability of a system. In the Nyquist plot, the diameter of the semicircle represents the charge transfer resistance (Rct). The smaller the Rct, the faster the electron transfer rate. For example... Figure 3 As shown in (a), Comparative Example 3 ( P. stutzeri + G. sulfurreducensThe Rct of the co-culture system without N-CDs was as high as 21010 Ω, indicating a significant resistance to electron transfer between bacteria. However, after the introduction of N-CDs ( P. stutzeri + G. sulfurreducens With N-CDs, the resistance Rct drops sharply to 3952 Ω, a decrease of 81.2%. This significant reduction in Rct indicates that N-CDs greatly reduce the kinetic barrier of interspecies electron transfer, providing direct electrochemical evidence for the construction of a low-resistance conductive network (DIET) by N-CDs among bacterial communities.

[0080] CV curves revealed the redox activity of N-CDs themselves. For example... Figure 3 As shown in (b), N-CDs exhibit distinct, quasi-reversible redox peaks within the scanning voltage range, confirming that they can act as electron carriers to transfer shuttle electrons (MIET) through the transition between their oxidized and reduced states.

[0081] (3) Results of biological analysis

[0082] Biological analysis data reveals that the enhanced electron flow is efficiently translated into a leap in cellular function.

[0083] like Figure 4 As shown in (a), compared with Comparative Example 3 ( P. stutzeri + G. sulfurreducens Compared to Example 2, Example 2 P. stutzeri + G. sulfurreducens While N-CDs significantly improved denitrification efficiency (>99%), the consumption of acetate, the sole carbon source, decreased from 21.5 mg·L⁻¹. -1 Slightly increased to 22.2 mg·L -1 The increase was only about 3.4%. This "low-consumption, high-efficiency" phenomenon strongly demonstrates that the metabolic pathway of the system has been fundamentally reshaped, and denitrifying bacteria... P. stutzeri No longer relying on the decomposition of precious carbon sources to obtain energy and reducing power, it bypasses the bottleneck of traditional substrate decomposition metabolism and turns to an efficient "electro-breathing" strategy, giving priority to the use of exogenous electrons transported by N-CDs, shifting the metabolic priority from biomass accumulation to denitrification using external electron flow.

[0084] like Figure 4 As shown in (b), compared with Comparative Example 3 ( P. stutzeri + G. sulfurreducens Compared to Example 2, Example 2 P. stutzeri + G. sulfurreducensThe intracellular ATP concentration increased by approximately 1.6-fold with the addition of N-CDs. Achieving such a significant energy gain with almost no additional carbon substrate consumption further confirms that cells are efficiently utilizing exogenous electrons transferred by N-CDs to synthesize ATP.

[0085] like Figure 4 As shown in (c), Example 2 mediated by N-CDs ( P. stutzeri + G. sulfurreducens The ETSA in the +N-CDs) system compared to the control group 1 (only) of single-cell culture... P. stutzeri This represents a nearly 10-fold increase compared to the co-culture control group 3 ( P. stutzeri + G. sulfurreducens The electron flux was also significantly increased by 2.5 times, indicating that the electron flux of the entire respiratory chain was significantly activated.

[0086] N-CDs induced a significant expansion of the nicotinamide adenine dinucleotide pool, Example 2 ( P. stutzeri + G. sulfurreducens + N-CDs) Comparison ratio 3 ( P. stutzeri + G. sulfurreducens Intracellular NADH and NAD⁺ levels increased by approximately 2.0-fold and 2.2-fold, respectively. Figure 4 As shown in (d), the relative NADH level in Example 2 was significantly higher than that in Comparative Example 3, which created a highly reduced intracellular environment that provided a strong thermodynamic driving force for the denitrification cascade reaction.

[0087] like Figure 4 As shown in (e~h), Example 2 ( P. stutzeri + G. sulfurreducens In N-CDs, the activities of all four key denitrifying enzymes (NAR, NIR, NOR, and N2OR) were synergistically and significantly upregulated. This ensures that the high-flux of electrons injected by N-CDs can smoothly and completely pass through the entire denitrification chain, ultimately reducing N2O to harmless N2, thus explaining the biochemical basis for the extreme inhibition of N2O.

[0088] In summary, this invention, by introducing N-CDs, synergistically regulates the electron generation, transport, and utilization processes in microbial systems at both extracellular and intracellular levels. Extracellularly, N-CDs, with their nanoscale size, high dispersibility, and reversible redox properties, construct a high-throughput interspecies electron transport network within the microbial community, possessing both fixed conductive channels and mobile electron shuttle functions. This significantly reduces the electron transport resistance between electron donor and acceptor microorganisms, overcoming the kinetic bottleneck of traditional interspecies electron transport limitations. Intracellularly, N-CDs can enter microbial cells or regulate the intracellular electron transport chain and energy metabolism processes through transmembrane electron / energy coupling, promoting the enhancement of electron generation and output capabilities in electron donor microorganisms and guiding acceptor microorganisms to preferentially utilize acquired electrons in the denitrification respiratory chain, thereby improving the directional utilization efficiency of electrons in the denitrification reaction. Through the aforementioned synergistic effects of internal and external factors, this invention achieves the conversion of limited organic carbon sources into efficient electron flow and their rapid transfer and utilization in the complete denitrification chain under oligotrophic conditions with extremely limited electron donors. This promotes the near-complete (>99.9%) reduction of nitrate to nitrogen, reduces the risk of N2O generation and release, and effectively suppresses N2O emissions to only 2.84 μg·mg. -1 TN.

[0089] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention, such as the selection of the species of microorganisms used, the preparation method of the nitrogen-doped carbon dots, and the selection of their concentrations, should be included within the scope of protection of the claims of the present invention.

Claims

1. An application of a microbial electronically regulated denitrification system based on nitrogen-doped carbon dots to suppress N2O emissions and achieve efficient deep denitrification in high-nitrogen, low-carbon environments, characterized in that, The system operates under oligotrophic conditions where electron donors are limited, specifically a carbon-to-nitrogen ratio (C / N) ≤ 3, and the system does not rely on external inorganic electron donors as the primary electron source. The system comprises: Electron-donating microorganisms are used to oxidize limited organic carbon sources and generate and export electrons under anaerobic conditions. These electron-donating microorganisms are electroactive bacteria selected from *Geobacterium thioreductans*. Geobacter sulfurreducens ; Electron acceptor microorganisms possess a complete denitrifying enzyme system for the stepwise reduction of nitrate to nitrogen gas. These electron acceptor microorganisms are denitrifying bacteria selected from *Pseudomonas schistosomiasis*. Pseudomonas stutzeri ; Nitrogen-doped carbon dots are prepared as follows: (1) Pretreatment: The collected green tea biomass raw material was washed with deionized water, dried in an oven at 60℃ to constant weight, pulverized with a high-speed pulverizer, and passed through a 100-mesh standard sieve to obtain uniform green tea biomass powder. (2) Hydrothermal reaction: Accurately weigh 1 g of pretreated green tea powder, mix it evenly with 20 mL of ultrapure water, transfer it to a 50 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene, seal it, place the reactor in a temperature-controlled oven, heat it to 180°C, and keep it at this temperature for 5 h. (3) Preliminary separation: After the reaction is completed, the reaction vessel is allowed to cool naturally to room temperature. The dark brown mixture obtained from the reaction is centrifuged at 6000 rpm for 5 min to remove unreacted solid residues and larger carbon spheres. The supernatant is taken and filtered through a 0.22 μm microfiltration membrane to further remove small particles. (4) Purification: The filtered filtrate is placed into a dialysis bag with a molecular weight cutoff of 1000 Da and placed in a large amount of deionized water for dialysis. The deionized water is changed every 6 hours until the water outside the dialysis bag becomes colorless, so as to completely remove small molecule impurities and inorganic salts. (5) Finished product: The purified nitrogen-doped carbon dot solution is freeze-dried to obtain a brownish-yellow nitrogen-doped carbon dot powder solid, which is then sealed and stored for later use. The nitrogen-doped carbon dots possess both extracellular electron transport regulation and intracellular metabolic regulation functions, wherein: Extracellularly, the nitrogen-doped carbon dots act as electron mediators, constructing an electron transport network between the electron donor and electron acceptor microorganisms. Intracellularly, the nitrogen-doped carbon dots can regulate the intracellular electron transport chain and energy metabolism process of microorganisms, so that the acquired electrons are preferentially used for the denitrification respiratory chain. This allows the system to achieve efficient denitrification and suppress the generation of the strong greenhouse gas N2O even under oligotrophic conditions.

2. The application of the microbial electronically regulated denitrification system based on nitrogen-doped carbon dots as described in claim 1 in suppressing N2O emissions and achieving efficient deep denitrification in high-nitrogen, low-carbon environments, characterized in that: The nitrogen-doped carbon dots can increase the intracellular ATP and reducing power levels of the electron acceptor microorganisms and induce them to preferentially use the electrons they acquire for the denitrification respiratory chain.

3. The application of the microbial electronically regulated denitrification system based on nitrogen-doped carbon dots as described in claim 1 in suppressing N2O emissions and achieving efficient deep denitrification in high-nitrogen, low-carbon environments, characterized in that: The concentration of the nitrogen-doped carbon dots is 50~200 mg / L. -1 .

4. The application of the microbial electronically regulated denitrification system based on nitrogen-doped carbon dots as described in claim 1 in suppressing N2O emissions and achieving efficient deep denitrification in high-nitrogen, low-carbon environments, characterized in that: The nitrogen-doped carbon dots have a particle size of 1~10 nm and their surface contains pyridine nitrogen, pyrrole nitrogen and graphitic nitrogen.

5. The application of the microbial electronically regulated denitrification system based on nitrogen-doped carbon dots as described in claim 1 in suppressing N2O emissions and achieving efficient deep denitrification in high-nitrogen, low-carbon environments, characterized in that: After introducing the nitrogen-doped carbon dots, under the same test conditions, the charge transfer resistance of the system is reduced by at least 80% compared to the co-culture system without the nitrogen-doped carbon dots.

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