A method and device for denitrification of microalgae p450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation

Abstract

The application provides a microalgae P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification method and device. A photocatalyst is added to a mixed flora with anaerobic ammonia oxidation as the core, photoelectrons are excited to induce in-situ enrichment of microalgae, and a photocatalysis-coupled bacteria-algae hybrid system is constructed. A "starvation-micro-dosing" cycle feeding strategy is adopted to realize interspecies feeding of heterotrophic bacteria with algal glycogen as the only carbon source, synthesize salicylic acid to promote loading of microalgae P450 enzyme cofactors (iron porphyrin and Fe-S cluster), and combine "segmented light distribution" to improve NADPH generation and its enrichment in the P450 localization area, thereby completing efficient activation of the P450 enzyme. Under the condition that ammonia nitrogen is the only influent, hydroxylamine is generated from ammonia nitrogen through P450 enzyme catalysis, thereby forming a microalgae P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification mode. The application breaks through the bottleneck of traditional dependence on scarce nitrite in sewage, and has the advantages of high denitrification efficiency, greenhouse gas emission reduction and the like.

Description

Technical Field

[0001] This invention belongs to the field of water treatment technology, specifically relating to a non-nitrite-dependent anaerobic ammonia oxidation denitrification method and apparatus mediated by microalgae P450 enzyme. Background Technology

[0002] Excessive nitrogen emissions are one of the main drivers of eutrophication in global water bodies, severely weakening the resilience and self-purification capacity of aquatic ecosystems. Anaerobic ammonium oxidation (anammox) is considered a core biological process for sustainable nitrogen removal due to its low carbon consumption and lack of the need for external organic carbon. Its biochemical essence is: using hydroxylamine (NH₂OH) as an essential substrate for hydrazine synthase (HZS), it converts ammonium nitrogen (NH₄⁺) into nitrogen. + Nitrite (NO2) is oxidized to hydrazine (N2H4) and ultimately reduced to nitrogen (N2). In the traditional pathway, nitrite (NO2) is oxidized to hydrazine (N2H4) and then reduced to nitrogen (N2). - ) is a key precursor for the formation of NH2OH (see equations (1)–(3)). Therefore, anammox bacteria require sufficient substrate (NO2) - and NH4 + NO2 is supplied to maintain its strong metabolic activity and life processes. However, in actual wastewater, NO2... - The widespread scarcity of NH2OH limits its supply and restricts subsequent anammox nitrogen metabolism. Therefore, eliminating the dependence of anammox processes on nitrite and developing efficient anammox denitrification technologies that do not involve nitrite is an important issue for promoting the engineering of anammox-based biological denitrification processes.

[0003] NO2 - + 5H+ + 4e - → NH2OH + H2O (1)

[0004] NH2OH + NH4 + + 2e - + 2H+ → N2H4 +H2O (2)

[0005] N2H4 → N2↑ + 4H + + 4e - (3)

[0006] Cytochrome P450 enzymes have a non-NO2 monooxygenation catalytic property. - This provides a new approach to the development of anammox-dependent technology. Currently, the NH bond monooxygenation reaction based on P450 enzymes has been confirmed by multiple studies, namely, using molecular oxygen (O2) as an oxidant to catalyze the NH bond under electron-driven conditions and generate the corresponding hydroxylation product N-OH. This provides a new approach for the development of NH4+, a typical NH-bonded compound in wastewater. +-N provides a theoretical basis for P450 enzymes to become substrates and be catalyzed to produce NH2OH. Since microalgae naturally possess the ability to photosynthesize oxygen and generate reducing power, and their genomes encode a large number of P450 family members, they are considered preferred expression hosts for P450 enzymes. This also provides a basis for constructing microalgal P450 enzyme-driven non-NO2... - The dependent anammox process provides both theoretical and practical feasibility.

[0007] However, although algal genomes encode a broad spectrum of the P450 family, their transcriptional expression activity remains low (<5%), hindering their function and application. Mechanistically, P450 monooxygenation requires the iron porphyrin active site, relies on NADPH for reducing power, and achieves electron transfer through reduction chaperone proteins containing Fe–S clusters. This indicates that the activation of microalgal P450 enzyme catalytic function mainly depends on: (1) sufficient NADPH supply; (2) efficient synthesis of core structural components (iron porphyrin and iron-sulfur clusters). However, the conserved light-harvesting range of microalgae makes their photosynthetic efficiency usually <1%, far below the theoretical value of 13%, which limits the full synthesis of intracellular NADPH and ATP. In addition, the plant hormone salicylic acid is an important siderophore signaling regulator, playing a key role in regulating the synthesis of cellular iron porphyrin and iron-sulfur clusters. The algal cell genome lacks genes encoding key enzymes such as PAL in the plant salicylic acid synthesis pathway, which prevents the induction of iron porphyrin and iron-sulfur cluster synthesis, resulting in low P450 enzyme protein loading rates. These combined limitations restrict the construction and functional performance of microalgal P450 enzyme catalytic systems, hindering the engineering of non-nitrite-dependent anammox processes mediated by microalgal P450 enzymes. Therefore, there is an urgent need to develop an efficient and rapid activation strategy for microalgal P450 enzymes to overcome the challenges of constructing microalgal P450 enzyme-driven non-NO2... - Technical bottlenecks in the anammox-dependent process.

[0008] In this regard, the close collaboration between microalgae and heterotrophic bacteria under the influence of photogenerated electrons can provide a solution for stimulating the monooxygenation catalytic function of P450 enzymes. On the one hand, combining microalgal photosynthesis with photocatalytic materials possessing efficient light-harvesting and photoelectrochemical response properties, and constructing a "photocatalysis-microalgal electron channel," can achieve the directional injection of photogenerated electrons and the continuous regeneration of intracellular NADPH, thereby providing stable reducing power support for P450 enzyme reactions. On the other hand, compared to autotrophic bacteria, heterotrophic bacteria possess a more complex gene pool for more diverse biosynthesis. They have been reported to perform efficient salicylic acid synthesis when using glycogen as a substrate for carbon metabolism. Therefore, under the influence of photogenerated electrons, microalgae are expected to obtain salicylic acid provided by heterotrophic bacteria to regulate the synthesis of iron porphyrins and iron-sulfur clusters, thereby synergistically enhancing the expression, loading, and functional activation of P450 enzymes.

[0009] However, current technologies still lack a high-density activation pathway for P450. Furthermore, there is a lack of unified solutions regarding the controlling factors, stable operating engineering boundary conditions, and scale-up feasible device implementation pathways for this type of photo-microalgae-heterotrophic bacteria collaborative system. Therefore, there is an urgent need for a photocatalytically driven, closed-loop controlled, and scalable non-nitrite-dependent anammox denitrification method and device centered on microalgae P450, which is feasible on an engineering scale. This would fundamentally solve the key bottleneck of insufficient nitrite supply in current anammox processes, improve its adaptability and stability in mainstream low-C / N wastewater, and provide theoretical support and a technical platform for a deeper understanding and regulation of the bacterial-algae collaborative relationship. This is of great significance for the future construction of carbon-neutral oriented smart urban wastewater treatment plants. Summary of the Invention

[0010] This invention aims to fundamentally solve the key technical bottleneck of existing anammox processes' dependence on scarce nitrite in wastewater, and thus provide a non-nitrite-dependent anaerobic ammonia oxidation denitrification method mediated by microalgae P450 enzymes. This technology innovatively integrates photocatalytic materials, microalgae electron flow reconstruction mechanisms, and a P450 enzyme-induced hydroxylamine generation module to establish an alternative anammox electron pathway that does not require nitrite supply. By constructing a synergistic system of microalgae-heterotrophic bacteria-anammox bacteria in the reactor, precise coupling and feedback control of electron flow, carbon flow, and signal flow are achieved. This allows ammonia nitrogen in wastewater to be converted into NH2OH via photogenerated electrons and mediated by microalgae P450 enzymes, thus providing a key intermediate for the anammox reaction and achieving a completely NO2-free process. - A highly efficient nitrogen removal pathway involving -N. This invention also provides a non-nitrite-dependent anaerobic ammonia oxidation nitrogen removal apparatus for implementing the aforementioned microalgal P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation nitrogen removal method and apparatus.

[0011] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0012] A non-nitrite-dependent anaerobic ammonia oxidation denitrification method mediated by microalgal P450 enzymes, characterized by comprising the following steps:

[0013] Step 1: Construct a photocatalytically coupled anammox-bacterial-algae hybrid system to prepare for the domestication and formation of a microalgae P450 enzyme-mediated non-nitrite anammox denitrification mode: Add a photocatalyst to a reactor inoculated with anaerobic ammonia-oxidizing bacteria, start 24 hours of continuous light, stimulate photogenerated electrons and induce algae to accumulate and grow in situ on the surface of the anammox bacterial sludge, thus completing the construction of the photocatalytically coupled anammox-bacterial-algae hybrid system.

[0014] Furthermore, the inoculated sludge must meet the following conditions:

[0015] A. The volume of the inoculated sludge accounts for 40% to 60% of the total reactor volume;

[0016] B. The total suspended solids in the initial mixed liquor formed after stirring are 4000-7000 mg / L; and the mass ratio of volatile suspended solids to total suspended solids is 0.4-0.6; the specific anammox activity (SAA) of the inoculated sludge reaches 0.05 g N / g VSS / day or higher, to ensure that the added sludge has a sufficient and active anammox microbial community.

[0017] Furthermore, the nitrogen in the wastewater in step 1 includes NO2. - -N, NH4 + -N,NH4 + -N concentration ranges from 35-80 mg / L, NO2 - -N concentration ranges from 50 to 120 mg / L;

[0018] Furthermore, the illumination in step 1 uses full-spectrum irradiation, with a light emission range of 380-780 nm and a light intensity range of 150-350 μmol photons·m. -2 ·s -1 ;

[0019] Furthermore, the photocatalysts mentioned in step 1 include one or more of the following: various g-C3N4, organometallic frameworks (MOFs), carbon dots (CDs), CdSe, and TiO2;

[0020] The photocatalyst is added to the influent at a concentration of 100-300 mg / L based on the working liquid volume of the reactor.

[0021] Furthermore, the conditions for completing step 1 are: continuous operation until the total nitrogen removal rate (TNRE) of the reactor is greater than or equal to 85%, and the chlorophyll a content in the sludge rises to more than 2 mg / L.

[0022] Step 2: Entering the microalgae P450 enzyme activation stage, which is divided into stage A and stage B:

[0023] Phase A: Maintaining the light parameters in step 1, and without any other external carbon source input, glycogen is added to the reactor using a "starvation-micro-feeding cycle" to limit carbon source availability and guide heterotrophic bacteria to establish a metabolic pathway with glycogen as the sole substrate.

[0024] Furthermore, the "starvation-micro-feeding cyclical feeding" strategy described in step 2, stage A, specifically involves: quantitative glycogen addition to the reactor every 6 hours, with the initial glycogen addition controlled at 0.2 g COD / g NH4 based on the influent mass ratio. +-N was added, followed by a 6-hour starvation incubation period before starting the next cycle; the glycogen dosage in the next cycle was reduced by 0.05 g COD / g NH4 compared to the previous cycle. + -N, and so on. Each cycle consists of 4 cycles, and this process is repeated for 2-8 cycles before entering stage B of acclimatization.

[0025] Furthermore, in the "starvation-micro-feeding cyclical feeding" strategy described in step 2, the glycogen dosage is controlled at 0.05–0.2 g COD / g NH4 based on the influent mass ratio. + -N;

[0026] Phase B: Subsequently, periodic segmented light distribution was set up and activated to enhance the conversion efficiency of photogenerated electrons into intracellular NADPH in microalgae; glycogen addition was stopped simultaneously to construct an interspecific feeding pathway for heterotrophic bacteria with microalgal glycogen as the sole carbon source and enhance SA secretion, strengthen the synthesis and loading of iron porphyrin and Fe-S clusters, and complete the high-density activation of microalgal P450 enzymes.

[0027] Furthermore, the periodic segmented light distribution strategy described in step 2 is as follows: relying on a deep-penetrating tunable spectral light field system, the daily light exposure follows a cycle of "alternating red and blue light pulses (2s red light / 2s blue light) for 2 hours: full-spectrum irradiation for 3 hours: darkness for 1 hour," with two light cycles per day. Glycogen addition is simultaneously stopped to construct an interspecific feeding pathway for heterotrophic bacteria using microalgal glycogen as the sole carbon source and enhance SA secretion, thereby strengthening the synthesis and loading of iron porphyrin and Fe-S clusters. The above light cycle is repeated until the microalgal P450 enzyme activity reaches 50 μmol / h / mg protein or higher, completing the high-density activation of the microalgal P450 enzyme.

[0028] Furthermore, during the activation process of the microalgae P450 enzyme in step 2, when the enzyme activity of the sludge P450 enzyme, as measured by the ELISA kit, is less than 20% higher than the enzyme activity level before photocatalysis was initiated, the illumination period is automatically extended by 2 light cycles until the activity reaches the set threshold of 50 μmol / h / mg protein.

[0029] Furthermore, the conditions for completing the high-density activation of microalgae P450 enzyme in step 2 are as follows: the P450 enzyme activity reaches above 50 μmol / h / mg protein as monitored by an ELISA kit, and at the same time, the NH2OH concentration in the reactor sludge layer rises to the level of the influent NH4+. + -N content is over 40%;

[0030] Furthermore, the periodic segmented light distribution strategy described in step 2 is designed based on the precise matching and dynamic regulation of the light absorption characteristics of the photocatalytic material and the microalgal photosystem. It aims to maximize the bioavailability of photogenerated electrons and efficiently drive the activation process of microalgal P450 enzymes. Pulsed red-blue light alternation (2s red light / 2s blue light) precisely matches the optimal absorption bands of microalgal PSI and PSII (approximately 460nm for blue light and 630nm for red light), enhancing the coupling efficiency between primary and cyclic electron flows in photosynthesis, thereby significantly increasing the intracellular NADPH synthesis rate. Simultaneously, it strengthens the electron reception and transfer capabilities of electron transport-related proteins in microalgae, enhancing the conversion efficiency of photocatalytically excited photogenerated electrons into NADPH within the microalgal cell, thus providing a stable and sufficient electron donor for the P450 enzyme. The full-spectrum continuous irradiation stage further simulates the natural sunlight environment, synergistically enhancing the algal carbon storage capacity and strengthening glycogen production under the regulation of photogenerated electrons, thereby supporting the subsequent interspecies feeding process of heterotrophic bacteria. The dark phase can promote the release of algal glycogen, expression of P450-related proteins, and loading of cofactors, forming a "metabolic window" that is dynamically complementary to the aforementioned light reaction chain, thus promoting the synchronous transfer of energy and metabolism within the system.

[0031] Furthermore, in step 2, stage B, exogenous inducing factors, including one or more of benzoic acid, L-tryptophan, and methyljasmonate, need to be added simultaneously to enhance the expression of the SA signaling pathway and promote the synthesis and loading of iron porphyrin and iron-sulfur clusters required by microalgal P450 enzymes.

[0032] Furthermore, the exogenous inducing factor is added at a mass concentration of 10 mg / L based on the working liquid volume of the reactor.

[0033] Step 3: Adjust the lighting mode to alternate between 12 hours of light and 12 hours of darkness. Provided the total nitrogen removal rate in the effluent remains above 90% for 5 consecutive days, gradually reduce the nitrite concentration in the influent. Specifically: initially reduce the nitrite concentration in the influent from the initial concentration to 25 mg / L. Subsequently, while maintaining a stable total nitrogen removal rate of ≥90% in each round, continue reducing the nitrite concentration by 5–10 mg / L each time, until it reaches 0 mg / L. This is done without an external carbon source and using NH4+. + The reactor was continuously operated under the condition that -N was the only nitrogen source, and eventually a non-nitrite-dependent anaerobic ammonia oxidation denitrification mode mediated by microalgae P450 enzymes was formed.

[0034] Furthermore, the criteria for determining the formation of the microalgal P450 enzyme-mediated non-nitrite-dependent anaerobic ammonium oxidation denitrification mode are: zero external carbon source addition, NH4 + -N is the only nitrogen element. The total nitrogen removal rate (TNRE) of the reactor is greater than or equal to 95%, and the P450 enzyme activity is at least 30% higher than at the end of step 2.

[0035] A photocatalytically driven, non-nitrite-dependent anaerobic ammonia oxidation denitrification device with microalgae P450 enzyme as its core comprises: a raw water storage and pretreatment unit, a photocatalytically coupled anaerobic ammonia oxidation reactor, a deep-penetration tunable spectral light field system, an effluent and sampling module, and a central control and monitoring module arranged sequentially; the photocatalytically coupled anaerobic ammonia oxidation reactor adopts a sequencing batch reactor (SBR).

[0036] Furthermore, the deep-penetrating tunable spectral light field system is set around the reactor and uses a lateral partitioned dimming layout to achieve periodic alternating irradiation of red light, blue light and the full spectrum. Combined with the light guide rod, the pulsed light is directed to the interface region between microalgae and anammox sludge, thereby increasing the local photogenerated electron density and improving the P450 activation efficiency.

[0037] The advantages of the non-nitrite-dependent anaerobic ammonia oxidation denitrification method and apparatus mediated by microalgae P450 enzymes described in this invention are as follows:

[0038] 1. This invention provides a photocatalytically driven high-density activation strategy for microalgal P450 enzymes, and innovatively constructs a non-nitrite-dependent anammox denitrification mode mediated by microalgal P450 enzymes, breaking through the rigid dependence of traditional anammox processes on nitrite supply.

[0039] By constructing a photocatalytic-anammox hybrid algae-bacterial system, and utilizing a dual strategy of "minimum glycogen carbon flow guidance + segmented light distribution regulation" to excite photogenerated electrons, the efficiency of photogenerated electron reception, transport, and conversion to NADPH in the microalgal photoreaction chain was optimized. Simultaneously, through glycogen-induced interspecific feeding between microalgae and heterotrophic bacteria, a salicylic acid-mediated regulatory pathway was established, significantly promoting the enrichment of NADPH required for intracellular P450 enzymes in microalgae and the biosynthesis of key cofactors iron porphyrin and Fe–S clusters, thereby achieving high-density, functionalized expression of P450 enzymes. Ultimately, the activated microalgal P450 enzymes can efficiently catalyze NH4+. + -N monooxygenation generates NH2OH, which directly provides HZS enzyme substrates for anammox bacteria and hydroxylamine substrates for anammox bacteria, bypassing the dependence on NO2--N and forming a simplified anammox pathway with ammonia nitrogen as the sole nitrogen source, thus achieving rapid and efficient anaerobic denitrification.

[0040] Furthermore, in the traditional anammox metabolic pathway involving NO2--N, NO2--N, in addition to acting as an electron acceptor for nitrogen removal, also acts as an electron donor, oxidizing to NO3- byproducts under the catalysis of NXR enzymes. This results in approximately 11% of the nitrogen in the system being converted to NO3- as a byproduct. -The presence of -N residues limits the theoretical nitrogen removal efficiency of this technology to around 89%. Importantly, this byproduct not only reduces total nitrogen removal efficiency but also necessitates the addition of additional carbon sources or coupling with a denitrification system for further removal, increasing the processing burden and cost. This invention proposes a non-nitrite pathway anammox denitrification mechanism based on microalgal P450 enzyme catalysis. This mechanism directly provides the hydroxylamine intermediate required by anammox through activated microalgal P450 enzymes, eliminating the fundamental need for NO2-. Simultaneously, photocatalytically excited photogenerated electrons create an electron-rich environment for anammox bacteria, eliminating the need for NXR enzymes to oxidize NO2--N to provide electrons. Therefore, this novel pathway eradicates NO3- from a metabolic mechanism perspective. - The generation of -N byproducts significantly improves the total nitrogen removal efficiency and effluent water quality stability of the system, and reduces the dependence on subsequent advanced treatment processes, providing a new path for the anammox process in the direction of low-carbon and high-efficiency nitrogen removal.

[0041] 2. The microalgae P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification method provided by this invention has both CO2 and N2O greenhouse gas emission reduction effects, and possesses significant carbon neutrality potential. Wastewater treatment plants are considered one of the world's largest sources of greenhouse gas (GHG) emissions, accounting for 2%–3% of total emissions. Among these, non-CO2 greenhouse gas emissions, mainly N2O, account for an even higher proportion, reaching 4.6%–5.2%, with a global warming potential 265 times that of CO2. Against this backdrop, developing low-carbon denitrification technologies with greenhouse gas emission reduction potential is particularly important for promoting carbon neutrality. To address this, the "minimum glycogen carbon flow guidance + segmented light distribution regulation" dual strategy proposed in this invention can specifically acclimatize heterotrophic bacteria in the anammox algae-microalgae hybrid system, establishing an interspecies carbon feeding relationship between microalgae and heterotrophic bacteria. This allows the heterotrophic bacteria to use microalgal glycogen as the sole carbon source for growth and metabolism, and to utilize intracellular carbon flow for SA synthesis rather than entering the TCA cycle for decomposition. This significantly weakens the CO2 release capacity of the heterotrophic bacteria and avoids the indirect greenhouse gas release caused by added organic carbon in traditional heterotrophic bacteria cultivation processes. Furthermore, the introduction of photogenerated electrons under photocatalytic coupling significantly enhances the carbon fixation capacity of the autotrophic organisms in the system. Therefore, the system exhibits a significant CO2 emission reduction advantage under photocatalytically enhanced autotrophic carbon fixation and weakened heterotrophic bacteria CO2 release. Regarding N2O control, this invention innovatively constructs a P450 enzyme-driven nitrite-free anammox metabolic pathway, using microalgal P450 enzymes to catalyze NH4+. + The addition of oxygen alone produces NH2OH, which replaces NO2. - As an anammox substrate, it cuts off NO2 at its source. - and its subsequent oxidation product NO3 - The generation chain. Since the generation of N2O depends on NO2... -By eliminating the accumulation of nitrogen-containing intermediates such as NO, this pathway completely avoids N2O generation at the substrate level, eliminating the potential emission risk of N2O in traditional anammox systems. Based on this, this invention provides an effective solution for developing sustainable wastewater treatment technologies that are dually oriented towards deep denitrification and greenhouse gas emission reduction.

[0042] 3. The microalgae P450 enzyme activation strategy provided by this invention enables the oxygen produced by microalgae photosynthesis to be used in situ as a substrate for NH2OH synthesis under the mediation of intracellular P450 enzymes. This avoids the inhibition of anammox bacteria by excessive dissolved oxygen in traditional algae coupling processes with anammox as the core, enhances the stability of process operation, and broadens the application scenarios of the process.

[0043] 4. The microalgal P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification method provided by this invention couples a photocatalyst with microorganisms, and the excited photogenerated electrons can directly provide additional reducing power for the WLP pathway of anammox bacteria's growth metabolism. Therefore, unlike the traditional method where anammox relies on NO2- oxidation via NXR enzymes to provide electrons for its growth metabolism WLP, the method provided by this invention enables anammox bacteria to achieve autotrophic growth dependent on photocatalytically supplied electrons and enhances the growth and metabolic activity of the bacteria and algae. Therefore, anammox can achieve rapid enrichment and activity enhancement under this environment, ensuring efficient denitrification and modular engineering deployment of the process in full-scale reactors.

[0044] 5. The microalgal P450 enzyme-mediated non-nitrite-dependent anaerobic ammonium oxidation denitrification method and apparatus provided by this invention facilitates bidirectional and multidimensional interspecies communication between microalgae and heterotrophic bacteria, breaking through the traditional unidirectional and unstable cooperative mode of microalgae-heterotrophic bacteria, and significantly improving the interspecies synergy and operational stability of the microbial-microbe coupled denitrification system. Specifically, in traditional microbial-algae systems, high-molecular-weight hydrophobic compounds such as lipids are the main carbon fixation products, and their transmembrane expulsion faces a significant energy barrier, making them difficult to use as a carbon source for heterotrophic bacteria and hindering the establishment of a microalgae-heterotrophic bacteria symbiotic relationship. This invention introduces high-flux photogenerated electrons, thereby triggering the glycogen carbon sink mechanism of microalgae, guiding the intracellular carbon flow of microalgae into the glycogen synthesis pathway, and strengthening microalgal glycogen synthesis; at the same time, the involvement of photogenerated electrons helps to activate the activity of the microalgal electron transport chain and further promote ATP synthesis, thereby providing sufficient energy for the active transmembrane expulsion of glycogen, breaking the energy barrier, and promoting efficient glycogen expulsion. Based on this, heterotrophic bacteria and microalgae establish interspecies material exchange based on glycogen. Furthermore, heterotrophic bacteria readily enhance the synthesis and secretion of intracellular salicylic acid (SA) during glycogenolysis, thereby using cross-species signaling of SA to feed back into the key structures (iron porphyrin and Fe-S clusters) of P450 enzymes synthesized by microalgae. Ultimately, this leads to bidirectional, multi-dimensional interspecies communication between microalgae and heterotrophic bacteria, enhancing the "adhesiveness" of their spatial symbiosis and functional synergy, achieving closed-loop resource utilization, and contributing to the maintenance of system homeostasis and environmental robustness in applications.

[0045] 6. Start-up efficiency is crucial to the engineering deployment capability and application feasibility of the process in WWTPs. Traditional microbial-algae coupling systems often employ direct addition of microalgae or in-situ enrichment through pure light cultivation, resulting in low algae enrichment efficiency, weak adhesion, and poor coupling depth with sludge, with start-up cycles generally exceeding 25 days. The microbial-algae coupling method provided in this patent utilizes photocatalytically excited photogenerated electrons, which can effectively improve the photosynthetic efficiency of microalgae and significantly enhance the Calvin cycle carbon fixation growth of microalgae, thereby achieving rapid in-situ enrichment on the surface of microalgae sludge. This will significantly shorten the spatial distance between microbes and algae, forming a close complementary metabolic relationship, thus completing the synergistic enrichment and stable operation of functional algae and core bacterial communities in a shorter time, demonstrating the process's rapid deployment capability and start-up responsiveness under complex site conditions.

[0046] 7. The microalgae P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification method described in this invention is simple to operate, low in cost, and environmentally friendly, possessing industrialization potential. This invention uses light energy as the sole energy source for cultivation, ensuring sustainability.

[0047] To make the technical solution of the microalgae P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification method and apparatus of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0048] like Figure 1 The diagram shown is a schematic diagram of the microalgae P450 enzyme-mediated non-nitrite-dependent anaerobic ammonium oxidation denitrification device of the present invention.

[0049] The attached figures are labeled as follows:

[0050] 1-Raw water storage and pretreatment unit; 2-Glycogen storage tank; 3-Glycogen metering micropump; 4-Photocatalytic coupled anaerobic ammonia oxidation reactor; 5-Multi-channel LED module side-emitting light guide rod; 6-Side-emitting light guide panel; 7-Real-time multi-parameter detector; 8-Signal sensor-transmitter; 9-Outlet water tank; 10-Automatic parameter control console. Detailed Implementation

[0051] The microalgae P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification device used in this embodiment, such as... Figure 1 As shown, the following components are arranged sequentially: raw water storage and pretreatment unit 1, glycogen storage tank 2, glycogen metering micropump 3, photocatalytically coupled anaerobic ammonia oxidation reactor 4, multi-channel LED module side-emitting light guide rod 5, side-emitting light guide panel 6, real-time multi-parameter detector 7, signal sensor-transmitter 8, outlet tank 9, and automated parameter control console 10. The glycogen storage tank and glycogen metering micropump are connected to the photocatalytically coupled anaerobic ammonia oxidation reactor. The photocatalytically coupled anaerobic ammonia oxidation reactor is a sequencing batch reactor (SBR). In this embodiment, the effective volume of the reactor is 4.5L. The main body is made of corrosion-resistant polypropylene (PP), and the inner wall is covered with an inert material to prevent metal ions from interfering with the activity of microalgae P450. Multiple sealed flange interfaces are provided on the top for installing light guide components, cables, and sensors. The reactor simultaneously enriches anammox bacteria, microalgae, and functional heterotrophic bacteria, constructing a bacteria-algae-catalytic material composite system. The outlet of the photocatalytically coupled anaerobic ammonia oxidation reactor is connected to the outlet tank 9.

[0052] The reactor's inner wall is fitted with multi-channel LED module side-emitting light guide rods, which, together with the side-emitting light guide panel, form a deep-penetrating tunable spectrum light field system. A 63W full-spectrum LED module is used, with a group of red (λ = 620–680nm), blue (λ = 450–480nm), and full-spectrum combination LEDs evenly spaced every 6cm, providing pulse dimming and band switching capabilities. The light guide rods are made of high-transmittance polymethyl methacrylate (PMMA) with a light-enhancing coating to improve the uniform distribution of light energy within the reactor. The light guide rods are attached to the reactor's inner wall using high-strength nano-adsorption patches, while the outer light guide panel is fixed using a transparent acrylic bracket. Together, the light guide rods and the light guide panel form a surround lighting structure, achieving highly uniform, deep-penetrating light distribution that precisely matches the microalgal pigment absorption band, thereby improving photogenerated electron excitation and energy conversion efficiency.

[0053] The glycogen supply system consists of the glycogen storage tank 2 and the metering micropump 3. The glycogen storage tank is a 3L sealed polyethylene tank with an internal stirring paddle to prevent sedimentation; it is externally fitted with a temperature-controlled heating belt (20–40℃) to maintain the fluidity of the glycogen source. The metering micropump 3 is a high-precision peristaltic pump driven by a stepper motor, with an error controlled within ±2%; it is equipped with a reflux pipe and a check valve to prevent accidental addition. The glycogen storage tank 2 and the metering micropump 3 are connected to the reactor inlet via a silicone hose (Φ4mm), and the feed flow rate is intelligently and synchronously adjusted by the control console 10 according to the light cycle setting.

[0054] The real-time multi-parameter detector has multiple built-in sensors and probes, which are respectively set at the reactor inlet and outlet and key internal nodes, integrating DO, ORP, and NO2. - NO3 - NH4 + Sensors for pH, temperature, etc., with a sampling frequency of 0.5Hz, are used to monitor the reactor status in real time. The data is then linked with the automated control module to dynamically adjust light intensity, sugar source input, and operating cycle. A real-time multi-parameter detector 7 is connected to a signal transmitter 8, converting the signals into electrical signals and sending them to the automated parameter control console 10, enabling indirect judgment and feedback control of the bacterial and algal metabolic state. The automated parameter control console 10 is the core control unit of the device, integrating a PLC control system and a host computer software platform. It supports setting parameters such as segmented light distribution strategies, glycogen addition frequency and flow rate, and SBR operating cycle (influent–reaction–sedimentation–discharge), and can adaptively adjust based on real-time sensor data.

[0055] The following specific embodiments illustrate a non-nitrite-dependent anaerobic ammonia oxidation denitrification method mediated by microalgae P450 enzymes based on the above-mentioned device.

[0056] Example 1

[0057] First, a photocatalytically coupled anammox-bacterial-algal hybrid system was constructed to prepare for the formation of a microalgal P450 enzyme-mediated non-nitrite anammox denitrification mode. The specific method is as follows:

[0058] (1) A mixed sludge with anammox as the core was inoculated into a photocatalytically coupled anammox reactor. The sludge mixture concentration was approximately 5.50 g / L, and the inoculated sludge volume accounted for 50% of the total reactor volume. Simulated wastewater was used as the influent. The reactor was operated at a temperature of 34.5-35.5℃, and the influent pH was adjusted to 7.5 via a pH pre-adjustment reactor. The single cycle time of the SBR reaction process was 12 h, including 10 min of influent, 680 min of stirring, 15 min of sedimentation, 10 min of effluent, and 5 min of idle time. The influent was simulated wastewater, and the composition of the simulated wastewater was as follows: ammonia nitrogen 45±5 mg·L. -1 Nitrite nitrogen 55±5 mg·L -1 KHCO3 125 mg·L -1 KH2PO4 54 mg·L -1 EDTA 5 mg·L -1 and trace element solution I 2 mL·L -1 Trace element solution II 1 mL·L -1 The solvent is water.

[0059] Trace element solution I composition: NaCl 500 mg·L -1 KCl 700 mg·L -1 CaCl2·2H2O 700mg·L -1 MgSO4·7H2O 500mg·L -1 .

[0060] Trace element solution II composition: CuSO4·5H2O 0.25 mg·L -1 ZnSO4·7H2O 0.43 mg·L -1 CoCl2·6H2O, 0.24 mg·L -1 MnCl2·4H2O 0.99 mg·L -1 NaMoO4·2H2O 0.22mg·L -1 NiCl2·6H2O, 0.19 mg·L -1 NaSeO4 0.11 mg·L -1 H3BO3 0.014mg·L -1 .

[0061] After operating under the above method for 4 days, the total nitrogen removal rate of the reactor was about 75% and the SAA measurement value was 0.053 g N / g VSS / day, indicating that the reactor was operating stably.

[0062] 200 mg / L of modified g-C3N4 photocatalyst material was further added to the reactor along with the influent. The synthesis method and steps of this material are as follows: 5.0 g of melamine and 1.5 g of thiourea were weighed and thoroughly mixed in a mortar for 30 min. The mixed powder was transferred to a heat-resistant ceramic crucible and 2 mL of graphene oxide suspension (corresponding to approximately 4 mg of GO) was added. The mixture was stirred until a wet paste was formed, and then placed in a 60℃ forced-air drying oven for 12 h until completely dehydrated. The dried precursor was then heated to 550℃ in a muffle furnace at a rate of 5℃ / min, held at that temperature for 2 h, and then cooled to room temperature. Finally, it was purified by acid washing to obtain a light yellow powder of modified g-C3N4 nanosheet photocatalyst material. The reactor illumination system was started to provide full-spectrum illumination, thereby efficiently exciting photogenerated electrons and inducing in-situ enrichment of microalgae. The light-dark time ratio was set to 24 h:0 h, and the light intensity was 250 μmol photons·m. -2 ·s -1 The influent substrate composition and pH were kept consistent with previous levels. After 7 days of continuous operation, microalgae gradually accumulated and grew on the surface of the anammox sludge, with chlorophyll a content rising to 2.14 mg / L and TNRE increasing to 88%. The photocatalytically coupled anammox-bacterial-algae hybrid system was successfully constructed and entered the microalgae P450 enzyme activation stage.

[0063] (2) Entering the microalgal P450 enzyme activation stage, this stage is divided into stage A and stage B:

[0064] Phase A: Maintaining the light parameters in (1), and without any other external carbon source input, start the reactor glycogen addition system and use a "starvation-micro-feeding cyclic feeding" strategy to add glycogen to the reactor. The "starvation-micro-feeding cyclic feeding" strategy is as follows: a micro-quantitative glycogen addition is performed on the reactor every 6 hours, and after the addition, the reactor is switched to a 6-hour starvation culture before entering the next cycle; the glycogen addition amount in the next cycle is reduced by 0.05 g COD / g NH4 compared to the previous cycle. + -N, and so on. One cycle consists of 4 cycles, with the glycogen dosage controlled at 0.05–0.2 g COD / g NH4 based on the influent mass ratio. + -N;

[0065] Table 1. Glycogen dosage per cycle (4 cycles) of the "starvation-microfeeding cyclical feeding" strategy.

[0066]

[0067] The results showed that after 5 cycles, i.e., up to day 12, the heterotrophic bacteria were successfully guided to establish a metabolic pathway with glycogen as the sole substrate, and then entered the B-stage domestication.

[0068] Phase B: After inducing the glycogen metabolism pathway, periodic segmented light distribution is further initiated to enhance the conversion efficiency of photogenerated electrons into intracellular NADPH in microalgae. The light distribution strategy employed in this phase is as follows: relying on a deep-penetration tunable spectral light field system, the daily illumination follows a cycle of "alternating red and blue light pulses (2s red / 2s blue) for 2 hours: full-spectrum illumination for 3 hours: darkness for 1 hour," with two cycles per day, resulting in 14 hours of illumination and 2 hours of darkness daily. The dominant wavelength of the LED light source is adjusted to 630±10nm for red light and 460±10nm for blue light, and matched with the band gap response range (420–680nm) of modified g-C3N4 to enhance the excitation efficiency of microalgae PSI / PSII and the release rate of photocatalytically generated electrons. Glycogen addition is simultaneously stopped, constructing a heterotrophic interspecies feeding pathway with microalgal glycogen as the sole carbon source, and the synthesis and loading of iron porphyrin and Fe–S clusters are enhanced through SA signaling. To further promote the activation level of P450 enzyme, trace amounts of exogenous inducing factor methyljasmonate were added simultaneously from day 13 to day 16, with a daily dosage of 10 mg / L, to enhance the expression of the SA signaling pathway.

[0069] The reactor operated continuously for 20 days. The activity of microalgae P450 enzymes was detected using a CYP450 ELISA kit. The results showed that the enzyme activity reached 58.6 μmol / h / mg protein, meeting the high-density activation conditions. Simultaneously, the concentration of NH2OH in the sludge layer increased to that of the influent NH4+. + The presence of 47.3% -N further confirms the completion of microalgal P450 enzyme function activation.

[0070] (2) After the microalgae P450 enzyme activation was completed, the illumination mode was adjusted to alternate between full-spectrum light and darkness, i.e., a 12h light / 12h dark cycle, maintaining a light intensity of 250 μmol photons·m⁻¹. -2 ·s -1 During this stage, while ensuring that TNRE is not lower than 90%, the influent nitrite concentration is gradually reduced in stages, as shown in the table below:

[0071] Table 2. Changes in nitrite concentration in the influent.

[0072]

[0073] The results showed that after 30 days of reactor operation, the influent NO2 - -N content dropped to 0, and monitoring results showed that the total nitrogen removal rate (TNRE) of the system reached 96.7%. At the same time, the CYP450 enzyme activity increased by about 33% compared with day 20, reaching 78.1 μmol / h / mg protein, indicating that the non-nitrite-dependent anammox denitrification mode was successfully started and operated stably.

[0074] Furthermore, after 30 days of cultivation and acclimatization, the P450 enzyme activity in the system increased by 40.34% compared to the initial level, SA metabolism-related proteins were upregulated by 3.6 times, and NO2... - -N deletion did not affect nitrogen removal efficiency, further confirming that the P450 enzyme-driven hydroxylamine synthesis is a novel electron donor, successfully replacing NO2. - -N serves as a precursor substrate for the anammox reaction. Simultaneously, the effluent nitrate content is 0, and the carbon fixation rate reaches 5.46 mg CO2 / g MLVSS h. -1 The gene abundance of NOR, a key enzyme in N2O synthesis, was <0.0001, and N2O emissions remained below the detection limit (<0.2 μg / L). Key WLP reductases such as FDH and CODH were upregulated 2.1-fold under photogenerated electron intervention, and the accumulation level of the metabolite acetyl-CoA increased by 65%. The abundance of *Anammillaria* bacteria increased 5.41-fold (to 16.66%), and the generation time was shortened to 11.48 days (below the theoretical minimum of 15 days).

[0075] Example 2

[0076] First, a photocatalytically coupled anammox-bacterial-algal hybrid system was constructed to prepare for the formation of a microalgal P450 enzyme-mediated non-nitrite anammox denitrification mode. The specific method is as follows:

[0077] (1) A mixed sludge with anammox as the core was inoculated into a photocatalytically coupled anammox reactor. The sludge mixture concentration was approximately 6 g / L, and the inoculated sludge volume accounted for 50% of the total reactor volume. Simulated wastewater was used as the influent. The reactor was operated at a temperature of 34.5-35.5℃, and the influent pH was adjusted to 7.5 via a pH pre-adjustment reactor. The single cycle time of the SBR reaction process was 12 h, including 10 min of influent, 680 min of stirring, 15 min of sedimentation, 10 min of effluent, and 5 min of idle time. The influent was simulated wastewater, and the composition of the simulated wastewater was as follows: ammonia nitrogen 45±5 mg·L. -1 Nitrite nitrogen 55±5 mg·L -1 KHCO3 125 mg·L -1 KH2PO4 54 mg·L -1 EDTA 5 mg·L -1 and trace element solution I 2 mL·L -1 Trace element solution II 1 mL·L -1 The solvent is water.

[0078] Trace element solution I composition: NaCl 500 mg·L -1 KCl 700 mg·L -1CaCl2·2H2O 700mg·L -1 MgSO4·7H2O 500mg·L -1 .

[0079] Trace element solution II composition: CuSO4·5H2O 0.25 mg·L -1 ZnSO4·7H2O 0.43 mg·L -1 CoCl2·6H2O, 0.24 mg·L -1 MnCl2·4H2O 0.99 mg·L -1 NaMoO4·2H2O 0.22mg·L -1 NiCl2·6H2O, 0.19 mg·L -1 NaSeO4 0.11 mg·L -1 H3BO3 0.014mg·L -1 .

[0080] After operating under the above method for 4 days, the total nitrogen removal rate of the reactor was about 75% and the SAA measurement value was 0.061 g N / g VSS / day, indicating that the reactor was operating stably.

[0081] Further, 200 mg / L of organometallic framework (MOF) photocatalytic material was added to the reactor along with the influent. The MOF material used was a UiO-66-NH2-derived modified material based on divalent zirconium ions and terephthalic acid (BDC), which possesses good photocatalytic properties. The synthesis method and steps of this material are as follows: 0.65 g of ZrCl was dissolved in 50 mL of DMF and sonicated for 10 min to aid dissolution; simultaneously, 0.45 g of NH2-BDC was dissolved in another 50 mL of DMF solution, and 10 mL of acetic acid was added to control the crystal size. The two solutions were mixed and magnetically stirred for 30 min to form a homogeneous precursor solution. Subsequently, the reaction was carried out at 120°C for 24 h to promote the orderly growth of MOF crystals. After the reaction was completed, the mixture was naturally cooled to room temperature, and the precipitate was separated by centrifugation (8000 rpm, 10 min). The precipitate was washed three times each with DMF and ethanol to remove unreacted residues. Finally, the obtained pale yellow precipitate was dried in a vacuum drying oven at 60°C for 12 h to obtain the MOF photocatalytic material. The reactor's illumination system was activated to provide full-spectrum illumination, thereby efficiently exciting photogenerated electrons and inducing in-situ enrichment of microalgae. The light-dark time ratio was set to 24h:0h, and the light intensity was 250 μmol photons·m⁻¹. -2 ·s -1The influent substrate composition and pH were kept consistent with previous levels. After 5 days of continuous operation, microalgae gradually accumulated and grew on the surface of the anammox sludge, with chlorophyll a content rising to 2 mg / L and TNRE increasing to 88%. The photocatalytically coupled anammox-bacterial-algae hybrid system was completed and entered the microalgae P450 enzyme activation stage.

[0082] (2) Entering the microalgal P450 enzyme activation stage, this stage is divided into stage A and stage B:

[0083] Phase A: Maintaining the light parameters in (1), and without any other external carbon source input, start the reactor glycogen addition system and use a "starvation-micro-feeding cyclic feeding" strategy to add glycogen to the reactor. The "starvation-micro-feeding cyclic feeding" strategy is as follows: a micro-quantitative glycogen addition is performed on the reactor every 6 hours, and after the addition, the reactor is switched to a 6-hour starvation culture before entering the next cycle; the glycogen addition amount in the next cycle is reduced by 0.05 g COD / g NH4 compared to the previous cycle. + -N, and so on. One cycle consists of 4 cycles, with the glycogen dosage controlled at 0.05–0.2 g COD / g NH4 based on the influent mass ratio. + -N;

[0084] Table 1. Glycogen dosage per cycle (4 cycles) of the "starvation-microfeeding cyclical feeding" strategy.

[0085]

[0086] The results showed that after three cycles, i.e., up to day 8, the heterotrophic bacteria were successfully guided to establish a metabolic pathway with glycogen as the sole substrate, and then entered the B-stage domestication.

[0087] Phase B: After inducing the glycogen metabolism pathway, periodic segmented light distribution is further initiated to enhance the conversion efficiency of photogenerated electrons into intracellular NADPH in microalgae. The light distribution strategy employed in this phase is as follows: relying on a deep-penetration tunable spectral light field system, the daily illumination follows a cycle of "alternating red and blue light pulses (2s red / 2s blue) for 2 hours: full-spectrum illumination for 3 hours: darkness for 1 hour," with two cycles per day, resulting in 14 hours of illumination and 2 hours of darkness daily. The dominant wavelength of the LED light source is adjusted to 630±10nm for red light and 460±10nm for blue light, and matched with the band gap response range (420–680nm) of modified g-C3N4 to enhance the excitation efficiency of microalgae PSI / PSII and the release rate of photocatalytically generated electrons. Glycogen addition is simultaneously stopped, constructing a heterotrophic interspecies feeding pathway with microalgal glycogen as the sole carbon source, and the synthesis and loading of iron porphyrin and Fe–S clusters are enhanced through SA signaling. To further promote the activation level of P450 enzyme, trace amounts of exogenous inducing factor methyljasmonate were added simultaneously from day 9 to day 12, with a daily dosage of 10 mg / L, to enhance the expression of the SA signaling pathway.

[0088] The reactor operated continuously for 15 days, maintaining a total nitrogen removal rate of approximately 90%. The activity of microalgae P450 enzymes was detected using a CYP450 ELISA kit, showing an enzyme activity of 66.82 μmol / h / mg protein, meeting the high-density activation conditions. Simultaneously, an increase in NH2OH concentration was detected in the sludge layer compared to the influent NH4+ concentration. + The presence of 60.3% -N further confirms the completion of microalgal P450 enzyme function activation.

[0089] (3) After the microalgae P450 enzyme activation is completed, adjust the light mode to alternate between full-spectrum light and darkness, i.e., a 12h light / 12h dark cycle, maintaining a light intensity of 250 μmol photons·m -2 ·s -1 During this stage, while ensuring that TNRE is not lower than 90%, the influent nitrite concentration is gradually reduced in stages, as shown in the table below:

[0090] Table 2. Changes in nitrite concentration in the influent.

[0091]

[0092] The results showed that after the reactor had been running for 20 days, the influent NO2 - -N content dropped to 0, and monitoring results showed that the total nitrogen removal rate (TNRE) of the system reached 98.2%. At the same time, the CYP450 enzyme activity increased by about 38% compared with day 15, reaching 92.21 μmol / h / mg protein, indicating that the non-nitrite-dependent anammox denitrification mode was successfully started and operated stably.

[0093] Furthermore, after 20 days of cultivation and acclimatization, the P450 enzyme activity in the system increased by 50.4% compared to the end of step (2), SA metabolism-related proteins were upregulated by 3.4 times, and NO2... - -N deletion did not affect nitrogen removal efficiency, further confirming that the P450 enzyme-driven hydroxylamine synthesis is a novel electron donor, successfully replacing NO2. - -N serves as a precursor substrate for the anammox reaction. Simultaneously, the effluent nitrate content is 0, and the carbon fixation rate reaches 6.21 mg CO2 / g MLVSS h. -1 The gene abundance of NOR, a key enzyme in N2O synthesis, was <0.0001, and N2O emissions remained below the detection limit (<0.2 μg / L). The abundance of anammox bacteria increased 6.22-fold, and the generation time was shortened to 10.48 days.

[0094] It is worth noting that the MOF photocatalyst used in this embodiment, compared with the modified g-C3N4 photocatalyst, can more efficiently excite photogenerated electrons, shorten the construction time of the photocatalytically coupled anammox algae-bacterial hybrid system by 2 days, and shorten the final acclimatization time by 10 days. Furthermore, it exhibits better and higher P450 enzyme activity and content, resulting in a higher total nitrogen removal rate. Therefore, this material is a relatively preferred choice.

[0095] Comparative Example 1

[0096] This comparative example aims to verify the necessity and technical advantages of the modified g-C3N4 photocatalytic material and its induction strategy in Example 1 in the activation of microalgal P450 enzymes and the initiation of non-nitrite anammox denitrification.

[0097] (1) Construction stage of bacterial-algal hybrid system

[0098] The operating conditions in this stage are completely consistent with those in Example 1, using the same reactor configuration, inoculated sludge concentration (5.50 g / L), SBR operating cycle (12 h), and influent composition. The reactor temperature is controlled at 34.5–35.5 °C, and the pH is maintained at 7.5.

[0099] The difference lies in the fact that no modified g-C3N4 photocatalyst was added to the influent;

[0100] The reactor is still equipped with a full-spectrum illumination system with a light intensity of 250 μmol photons·m. -2 ·s -1 The illumination time was 24h:0h, but without the participation of a photocatalyst, it was impossible to excite effective photogenerated electrons for subsequent microalgae regulation.

[0101] After 4 days of operation, the TN removal rate of the system was 75%, consistent with Example 1. The SAA was 0.051 g N / g VSS / d. Microalgal growth was limited, and the chlorophyll a concentration was maintained at 0.7 mg / L, indicating that microalgae did not achieve effective enrichment without photocatalytic induction, and the bacterial-algal coupling state was not obvious.

[0102] (2) Microalgal P450 enzyme activation stage

[0103] Compared with the activation method of Example 1, the following induction measures were omitted in this comparative example: no g-C3N4 was added, so there was no photogenerated electron intervention; the starvation-micro-decreasing addition strategy was cancelled, and glycogen was added quantitatively and constantly every 6 hours at a dose of 0.2g COD / gNH + - -N; The illumination method is continuous full-spectrum irradiation without alternating red and blue pulses; No exogenous methyl jasmonic acid or other P450 activating factors are added.

[0104] By day 12, the chlorophyll a content of microalgae had only increased to 1.2 mg / L, and the P450 enzyme activity was only 22.7 μmol / h / mg protein, which was significantly lower than the level on day 12 in the example (>58.6). The NH2OH concentration in the system was less than 0.5 mg / L, indicating that a stable P450 enzyme-mediated NH2OH synthesis had not been established.

[0105] (3) Nitrite stripping and non-nitrite anammox validation stage

[0106] Starting from the 21st, NO2 will be introduced into the water. - -N concentration was gradually stripped down to 0 mg / L as in Example 1, with a 12-hour light / 12-hour dark cycle maintained and a light intensity of 250 μmol photons·m⁻¹. -2 ·s -1 .

[0107] The observation results are as follows: On day 30, the system's TNRE decreased to 71.2%, failing to maintain efficient denitrification; CYP450 enzyme activity showed no significant increase (only 24.3 μmol / h / mg protein); no significant accumulation of NH2OH was observed, and NO2... - After the -N is stripped, the anammox reaction is inhibited; NO3 in the system effluent - -N increased to 9.7 mg / L, and N2O emissions increased to 0.73 μg / L, indicating that partial denitrification was dominant; RubisCO protein abundance did not increase significantly, indicating a lack of effective carbon flux coupling between heterotrophic bacteria and microalgae, and microalgae could not provide stable substrates; anammox bacteria abundance decreased by 2.3 times.

[0108] The above experimental results indicate that, without photocatalyst coupling, the non-nitrite-dependent anaerobic ammonia oxidation denitrification mode mediated by microalgal P450 enzymes cannot be successfully activated.

[0109] Comparative Example 2

[0110] This comparative example maintained the same basic conditions as Example 1, including reactor structure, sludge inoculum size (5.50 g / L), SBR operating cycle (12 h), and temperature and pH control. The main differences were: the addition of modified g-C3N4 photocatalyst material to the influent, with the dosage adjusted to 300 mg / L; the elimination of the periodic photodistribution induction system; and the replacement of the illumination method with constant full-spectrum irradiation (400–700 nm) at a light intensity of 250 μmol photons·m⁻¹. -2 ·s -1 The illumination time was 24 hours; other aspects, such as the glycogen tapering strategy and reactor operating parameters, were consistent with the example. This comparative example was conducted simultaneously with Example 1, and the operating results are as follows:

[0111] (1) Construction stage of the bacterial-algal hybrid system:

[0112] After 4 days of operation, the TN removal rate was 75%, consistent with Example 1, and the SAA measurement value was 0.058 g N / g VSS / day, indicating stable reactor operation. Adding 300 mg / L of modified g-C3N4 photocatalyst and continuing operation until the 4th day, microalgae gradually accumulated and grew on the surface of the anammox sludge, with chlorophyll a content increasing to 2 mg / L and TNRE increasing to 87%. The photocatalytically coupled anammox-algae hybrid system was successfully constructed and entered the microalgae P450 enzyme activation stage. The system construction time for this stage was shortened by 1 day compared to Example 1, indicating that the 300 mg / L dose of modified g-C3N4 photocatalyst had a better photogenerated electron excitation effect.

[0113] (2) Microalgal P450 enzyme activation stage

[0114] Compared to Example 1, Comparative Example 2 eliminated the periodic photodistribution induction system and changed the illumination method to constant full-spectrum irradiation (400–700 nm) with a light intensity of 250 μmol photons·m. -2 ·s -1 Under a 24-hour light exposure mode, the total nitrogen removal rate was only around 88% by day 15 of reactor operation. Simultaneously, monitoring results from the CYP450 ELISA kit showed that the P450 enzyme activity was only 8.26 μmol / h / mg protein. The NH2OH concentration in the sludge layer was below 5 mg / L. These results indicate that without a segmented light distribution strategy, the system cannot achieve functional activation of microalgal P450 enzymes.

[0115] (3) Nitrite stripping and non-nitrite anammox validation stage

[0116] Starting from the 15th, NO2 will be introduced into the water. - -N concentration was gradually stripped down to 0 mg / L as in Example 1, with a 12-hour light / 12-hour dark cycle maintained and a light intensity of 250 μmol photons·m⁻¹. -2 ·s -1 The experimental results are as follows: It reduced the NO2 content in the influent. - After N-N, the anammox reaction was inhibited, and the system's TNRE significantly decreased to below 50%, failing to maintain efficient denitrification; CYP450 enzyme activity did not significantly increase; no significant accumulation of NH2OH was observed, and NO3 in the system effluent... - -N increased to 26.7 mg / L. The construction of the microalgal P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification mode failed.

[0117] Based on the above, Example 1 utilizes a three-stage segmented light distribution of "blue light – red light – dark period," corresponding to metabolic windows such as algal energy accumulation, efficient P450 synthesis, and electron flow conversion recovery, effectively regulating the intracellular energy and carbon flow direction of microalgae and forming an electron flow-NADPH-P450 activation coupling mechanism. Comparative Example 2, with constant illumination, disrupts the energy rhythm and fails to induce the formation of the P450 system.

[0118] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. A non-nitrite-dependent anaerobic ammonium oxidation denitrification method mediated by microalgae P450 enzyme, characterized in that, Includes the following steps: (1) Add a photocatalyst to a reactor inoculated with anammox bacteria, the volume of the inoculated sludge accounting for 40% to 60% of the total volume of the reactor, and the anammox activity SAA is 0.05 gN / g VSS / day or more; start 24h continuous light irradiation to excite photogenerated electrons and induce algae to accumulate and grow in situ on the surface of the anammox bacteria sludge, and complete the construction of the photocatalytically coupled anammox bacteria-algae hybrid system; (2) Maintain the light parameters in step (1) and control the influent COD / g NH4 in the reactor at a ratio of 0.05–0.2 g COD / g NH4. + Adding glycogen at a -N dosage guides the in-situ symbiotic heterotrophic bacteria in the reactor to establish a metabolic pathway with glycogen as the sole substrate. Subsequently, periodic segmented light distribution is set and activated to enhance the conversion efficiency of photogenerated electrons to intracellular NADPH in microalgae, while glycogen addition is simultaneously stopped. This constructs an interspecific feeding pathway for heterotrophic bacteria using microalgal glycogen as the sole carbon source and enhances salicylic acid secretion. Under salicylic acid (SA) signal regulation, the synthesis and loading of iron porphyrin and Fe-S clusters in microalgae are strengthened, completing high-density activation of microalgal P450 enzymes. The conditions for successful high-density activation of microalgal P450 enzymes are: P450 enzyme activity reaching above 50 μmol / h / mg protein as monitored by an ELISA kit, and simultaneously, the NH2OH concentration in the reactor sludge layer rising to the level of the influent NH4+. + -N content is over 40%; (3) Adjust the lighting mode to alternate between 12 hours of light and 12 hours of darkness. Provided the total nitrogen removal rate of the system effluent is above 90% for 5 consecutive days, gradually reduce the nitrite concentration in the influent. Specifically, initially reduce the nitrite concentration in the influent to 25 mg / L. Subsequently, while maintaining a stable total nitrogen removal rate of ≥90% in each round, continue reducing the nitrite concentration by 5–10 mg / L each time until it reaches 0 mg / L. This is done without an external carbon source and using NH4+. + The reactor was continuously operated under the condition that -N was the only nitrogen source, and eventually a non-nitrite-dependent anaerobic ammonia oxidation denitrification mode mediated by microalgae P450 enzymes was formed.

2. The microalgal P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification method according to claim 1, characterized in that, Includes the following steps: (1) Adding a photocatalyst to a reactor inoculated with anaerobic ammonia-oxidizing bacteria reduces the nitrogen content of the wastewater to NO2. - -N, NH4 + -N,NH4 + -N concentration ranges from 35-80 mg / L, NO2 - -N concentration range: 50-120 mg / L; 24-hour continuous illumination with full-spectrum irradiation, i.e., light emission range: 380–780 nm, light intensity range: 150-350 μmol photons·m -2 ·s -1 The process involves stimulating photogenerated electrons and inducing algae to accumulate and grow in situ on the surface of anammox bacteria sludge; the process continues until the total nitrogen removal rate (TNRE) of the reactor is greater than or equal to 85%, and the chlorophyll a content of the sludge rises to more than 2 mg / L, thus completing the construction of the photocatalytically coupled anammox bacteria-algae hybrid system. (2) Entering the microalgal P450 enzyme activation stage, this stage is divided into stage A and stage B: Phase A: Under the premise of no other external carbon source input, glycogen is added to the reactor using a "starvation-micro-dosing cyclic feeding" method. This "starvation-micro-dosing cyclic feeding" method involves quantitative glycogen addition to the reactor every 6 hours, with the initial glycogen addition controlled at 0.2 g COD / g NH4 based on the influent mass ratio. + -N, after addition, followed by 6 hours of starvation incubation before entering the next cycle; the glycogen addition amount in the next cycle is reduced by 0.05g COD / g NH4 compared to the previous cycle. + -N, and so on; The process is repeated 2-8 times, with 4 cycles constituting one cycle, before entering the B stage of acclimatization. Phase B: Set up and activate periodic segmented light distribution to enhance the conversion efficiency of photogenerated electrons into intracellular NADPH in microalgae. The periodic segmented light distribution strategy is as follows: relying on a deep-penetrating tunable spectral light field system, the daily light irradiation follows a cycle of "alternating red and blue light pulses, i.e., 2s red light / 2s blue light irradiation for 2 hours: full spectrum irradiation for 3 hours: darkness for 1 hour", with 2 light cycles per day; glycogen addition is stopped simultaneously; the cyclic light irradiation is repeated until the high-density activation threshold of microalgae P450 enzyme is reached, i.e., enzyme activity of 50 μmol / h / mg protein or above. (3) Adjust the lighting mode to 12h: 12h full-spectrum light and dark alternation, gradually reducing the nitrite content in the influent until the nitrite concentration in the influent is 0 mg / L, and add NH4+ without external carbon source injection. + Under the condition that -N is the only nitrogen element, the reactor is continuously operated until the total nitrogen removal rate (TNRE) is greater than 95% and the P450 enzyme activity is at least 30% higher than at the end of step (2). The non-nitrite-dependent anaerobic ammonia oxidation denitrification mode mediated by microalgae P450 enzyme is started and completed.

3. The microalgal P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification method according to claim 2, characterized in that, The photocatalysts mentioned in step (1) include one or more of g-C3N4, metal-organic frameworks (MOFs), carbon dots (CDs), CdSe, and TiO2.

4. The microalgal P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification method according to claim 3, characterized in that, The concentration of the photocatalyst, based on the working liquid volume of the reactor, ranges from 100 to 300 mg / L.

5. The microalgal P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification method according to claim 2, characterized in that, When the increase in sludge P450 enzyme activity measured by an ELISA kit is less than 20% compared to the enzyme activity level before photocatalysis was started, the illumination period is automatically extended by 2 light cycles until the activity increase reaches the set threshold of 50 μmol / h / mg protein or more.

6. The microalgal P450 enzyme-mediated non-nitrite-dependent anaerobic ammonia oxidation denitrification method according to claim 2, characterized in that, Phase B also includes the simultaneous addition of exogenous inducing factors at a mass concentration of 10 mg / L based on the working liquid volume of the reactor, including one or more of benzoic acid, L-tryptophan, and methyljasmonate.

7. A photocatalytically driven, non-nitrite-dependent anaerobic ammonia oxidation denitrification device with microalgal P450 enzymes as its core, characterized in that, include: The system consists of a raw water storage and pretreatment unit, a photocatalytically coupled anaerobic ammonia oxidation reactor, a deep-penetration tunable spectral light field system, an effluent and sampling module, and a central control and monitoring module, arranged sequentially. The photocatalytically coupled anaerobic ammonia oxidation reactor is a sequencing batch reactor (SBR) connected to the deep-penetration tunable spectral light field system. The deep-penetration tunable spectral light field system is located around the reactor and uses a lateral, zoned dimming configuration to achieve periodic alternating irradiation with red, blue, and full-spectrum light. Combined with a light guide rod, the pulsed light is directed to the interface region between microalgae and anammox sludge, increasing the local photogenerated electron density and improving the P450 activation efficiency.

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