Establishment method of catalyst / microorganism coupling system and application method of catalyst / microorganism coupling system in synthesizing ammonia through light-driven reduction of nitrate

Abstract

The invention relates to an establishment method of a catalyst / microorganism coupling system and an application method of the catalyst / microorganism coupling system for synthesizing ammonia through light-driven reduction of nitrate, and the method comprises the following steps: firstly, enabling shewanella to grow in an LB culture medium until the logarithmic phase OD600 is 0.2-0.5; then, adding the g-C3N4-CQDs (carbon quantum dots) to construct an S.oneidens-g-C3N4-CQDs system; and centrifugally collecting and re-suspending in NaCl. The invention provides a method for biologically converting nitrate into ammonia by utilizing light driving, aiming at the problems that although the nitrate can be removed by a traditional denitrification technology, nitrogen resources are wasted, greenhouse gas N2O is discharged and an organic carbon source is depended, and energy consumption and cost are relatively high in a traditional Adaer-Bosch method ammonia synthesis technology. The method realizes enzymatic reduction reaction under the condition of no organic carbon source, and directionally reduces nitrate into ammonia by virtue of the synergistic effect of photocatalysis and microorganisms, so that the dual targets of pollution treatment and resource utilization are realized.

Description

Technical Field

[0001] The present invention belongs to the cross - field of materials, biology, environmental governance and energy conversion technologies, mainly relates to the synergistic technology of photocatalysis and biological treatment, and specifically relates to a technical system and method for synthesizing ammonia by driving the biological conversion of nitrate with photoelectrons. Background Art

[0002] Nitrate pollution has become a severe challenge in global water environment governance. With the intensive development of modern agriculture, the problem of nitrate leaching caused by excessive application of nitrogen fertilizer has become increasingly prominent. The nitrate exceeding standard rate of groundwater in the North China Plain of China has reached 38.6%, and the concentration in some local areas exceeds 300 mg / L. This kind of pollution not only comes from agricultural non - point sources, but the nitrate concentration in industrial wastewaters such as electroplating and pharmaceutical industries can reach 2000 - 5000 mg / L, while the nitrate residual in the effluent of urban sewage treatment plants is usually 10 - 30 mg / L, forming a compound pollution situation. The threat of nitrate pollution to human health cannot be ignored either. Infants drinking water with excessive nitrate will cause methemoglobinemia, and its pathogenic mechanism has been confirmed by epidemiological studies: when the nitrate concentration in drinking water exceeds 10 mg / L, the risk of infant illness increases significantly. Long - term exposure to nitrate - polluted environment is also positively correlated with the incidence of digestive tract cancer. Research shows that for every 1 mg / L increase in the nitrate concentration of drinking water, the relative risk of gastric cancer increases by 11%.

[0003] Currently, the main technical routes for nitrate treatment include electrocatalytic reduction and biological denitrification processes. Electrocatalytic reduction of nitrate to synthesize ammonia drives the nitrate reduction reaction by applying external electric energy. However, this method relies on noble metal catalysts (such as Ni, Fe) and high - purity electrode materials, with high costs, and requires continuous electric energy input, making it difficult to be applied on a large scale. Traditional biological denitrification processes such as the A / O process require aeration and an additional carbon source (such as methanol), with high costs. Traditional ammonia synthesis technologies mainly rely on thermal catalysis. For example, the Haber - Bosch process uses nitrogen and hydrogen as raw materials to synthesize ammonia at high temperature and high pressure (400 °C, 250 atm), with problems such as high energy consumption (accounting for more than 1% of the global total energy consumption) and large CO2 emissions. This process cannot directly use nitrate as a raw material and is contrary to the goal of green and low - carbon.

[0004] Li et al. (Cell Reports Physical Science, 2023, 101433) constructed a microbial electrosynthesis system, using Shewanella oneidensis MR - 1 to form a biofilm on the carbon felt cathode, reversing its classical Mtr electron transfer path, using the electrode as an electron donor, and reducing nitrate to ammonia through the dissimilatory nitrate reduction (DNRA) pathway. However, this technology needs to continuously apply voltage to maintain the reaction, with high energy consumption, and the electron transfer efficiency of the biofilm is affected by the charge transfer resistance, requiring long - term (>7 days) domestication.

[0005] Cheng et al. (Environmental Science & Technology, 2017, 51: 12948 - 12955) constructed a photo - electrochemically driven denitrification system (PEDeN), coupling a TiO2 photoanode with a biocathode, and using ultraviolet light - excited photogenerated electrons to drive the reduction of nitrate to N2. However, TiO2 only responds to ultraviolet light (accounting for 4% of the solar spectrum), requires a high - energy light source, and has a low actual solar energy utilization rate; the denitrification pathway is forced to direct N2 generation and cannot be flexibly regulated to convert to ammonia, limiting the resource value of ammonia.

[0006] In view of the problems that traditional denitrification technologies can remove nitrates but result in nitrogen resource waste, emission of greenhouse gas N2O, and dependence on organic carbon sources, and that traditional Haber - Bosch ammonia synthesis technologies have high energy consumption and costs, etc., the present invention provides a method for the photocatalytic biological conversion of nitrate to ammonia, realizing an enzymatic reduction reaction under the condition of no organic carbon source. By means of the synergistic effect of photocatalysis and microorganisms, nitrate is directionally reduced to ammonia, thereby achieving the dual goals of pollution treatment and resource utilization. Summary of the Invention

[0007] In view of the above problems, the present invention provides a method for the photocatalytic biological conversion of nitrate to ammonia. For the first time, a graphite - phase carbon nitride - carbon quantum dots (g - C3N4 - CQDs) photocatalyst is coupled with Shewanella oneidensis MR - 1 to form a material - biological reaction system, and through the mechanism of transmembrane inward transfer of photoelectrons, ammonia synthesis without organic carbon, low energy consumption, and high selectivity is achieved.

[0008] A method for establishing a catalyst / microorganism coupling system, using Shewanella oneidensis to grow first in Luria Bertani medium at a temperature maintained at 20 - 35 °C and an oscillation speed of 100 - 200 r / min until the optical density (OD) reaches the logarithmic growth phase of 0.2 - 0.5; then adding 5 mg - 20 mg of the prepared g - C3N4 - CQDs to construct the S. oneidensis - g - C3N4 - CQDs system; after incubating in the dark for 6 - 18 h, washing the cells three times with 0.9% NaCl, collecting by centrifugation and resuspending in 5 mL of 0.9% NaCl, with a centrifugation speed of 5000 - 10000 rpm and a centrifugation time of 5 - 10 minutes. 600 For 0.2 - 0.5; then add 5 mg - 20 mg of the prepared g - C3N4 - CQDs to construct the S. oneidensis - g - C3N4 - CQDs system; after incubating in the dark for 6 - 18 h, wash the cells three times with 0.9% NaCl, collect by centrifugation and resuspend in 5 mL of 0.9% NaCl, with a centrifugation speed of 5000 - 10000 rpm and a centrifugation time of 5 - 10 minutes.

[0009] Preferably, a method for establishing a catalyst / microorganism coupling system, wherein the Luria Bertani medium is 10 g / L NaCl, 10 g / L tryptone, and 5 g / L yeast extract; the temperature is maintained at 30 °C; the oscillation speed is 150 r / min; until the logarithmic growth phase OD 600 reaches 0.2; then 10 mg of the prepared g-C3N4-CQDs is added to construct the S. oneidensis-g-C3N4-CQDs system; after incubating in the dark for 12 h, the cells are washed three times with 0.9% NaCl, centrifuged for 6 minutes, collected, and resuspended in 0.9% NaCl; the centrifugation speed is 8000 rpm.

[0010] Preferably, a method for establishing a catalyst / microorganism coupling system, and a preparation method of the above g-C3N4-CQDs material: The carbon quantum dots CQD are synthesized by the microwave synthesis method. 2-6 grams of urea and citric acid are added to a beaker, and 15 ml of deionized water is added to form a clear solution; the obtained clear solution is heated in a microwave oven at 750 W for 5-15 minutes; during the heating process, the mixture gradually reacts and evolves from a transparent solution into a dark brown solid; the dark brown solid CQD is dispersed in deionized water and centrifuged at a speed of 2000-15000 rpm for 5-20 minutes, and repeated 3-5 times to remove the agglomerated CQD. The obtained CQD liquid is dried in an oven at 60 °C to obtain a powder;

[0011] In the reactor, the CQD solution at 1 g / L is mixed with ammonia water at a volume ratio of 1:1-3, and hydrothermal treatment is carried out in a reaction kettle at 120 °C-180 °C for 4-12 hours; it is opened at room temperature to remove the excess ammonia until the pH value reaches 7; using melamine as a precursor, it is heated at 550 °C for 2 hours to synthesize the g-C3N4 photocatalyst. 0, 3, 5, 7, or 9 ml of the ammonia-treated carbon quantum dot solution is mixed with the melamine aqueous solution, evaporated at 80 °C, and then continuously heated in a muffle furnace at 550 °C for 2 hours to synthesize g-C3N4-CQD3, g-C3N4-CQD5, g-C3N4-CQD7, or g-C3N4-CQD9 respectively.

[0012] Preferably, a method for establishing a catalyst / microorganism coupling system, and the preparation method of the above g-C3N4-CQDs material: The carbon quantum dots CQD are synthesized by microwave synthesis method. Add 4 grams of urea and 1-4 grams of citric acid into a beaker, add 15 milliliters of deionized water to form a clear solution, and heat the obtained clear solution in a microwave oven at 750W for 10 minutes; during the heating process, the mixture gradually reacts and evolves from a transparent solution into a dark brown solid. Then, disperse the dark brown solid CQD in deionized water and centrifuge it at a speed of 10,000 rpm for 10 minutes, repeat 5 times to remove the agglomerated CQD, dry the obtained CQD liquid in an oven at 60°C to obtain a powder; mix the 1 g / L CQD solution with ammonia water in a reactor at a volume ratio of 1:1, and perform hydrothermal treatment in a reaction kettle at 150°C for 7 hours; open it at room temperature to remove the excess ammonia gas until the pH value reaches 7; use melamine as a precursor and heat it at 550°C for 2 hours to synthesize the g-C3N4 photocatalyst; mix 5 milliliters of ammonia-treated carbon quantum dot solution with an aqueous melamine solution, evaporate it at 80°C, and then continuously heat it in a muffle furnace at 550°C for 2 hours to synthesize g-C3N4-CQD5 as a light-driven material.

[0013] Preferably, in the establishment of the catalyst / microorganism coupling system, the aqueous melamine solution is 1 gram of melamine dissolved in 40 milliliters of deionized water.

[0014] Preferably, a method for applying a catalyst / microorganism coupling system, mix the S.oneidensis-g-C3N4-CQDs system suspension with 95 mL of sterilized carbon-free nitrate medium and place it in a sealed reaction bottle, purge with argon for 10-20 minutes, and the reaction is carried out in a photoreactor under the irradiation of a xenon lamp; control the temperature at 20-35°C, take samples from the bottle at regular intervals, filter them with a syringe filter with a pore size of 0.22 μm, and use ultraviolet spectrophotometry, n-1-naphthylethylenediamine dihydrochloride spectrophotometry and Nessler's reagent colorimetry to measure the concentrations of nitrate nitrogen, nitrite nitrogen and ammonia nitrogen respectively.

[0015] Preferably, a method for applying a catalyst / microorganism coupling system, mix the S.oneidensis-g-C3N4-CQDs system suspension with 95 mL of sterilized carbon-free nitrate medium and place it in a sealed reaction bottle, purge with argon for 15 minutes, and the reaction is carried out in a photoreactor under the irradiation of a 250W xenon lamp; control the temperature at 30°C, take samples from the bottle at regular intervals, filter them with a syringe filter with a pore size of 0.22 μm, and use ultraviolet spectrophotometry at 220-275 nm, n-1-naphthylethylenediamine dihydrochloride spectrophotometry at 540 nm and Nessler's reagent colorimetry at 420 nm to measure the concentrations of nitrate nitrogen, nitrite nitrogen and ammonia nitrogen respectively.

[0016] Preferably, an application method of a catalyst / microorganism coupling system, wherein the composition of the carbon-free nitrate medium includes, by mass percentage: 1000 mg / L of NaHCO3, 500 mg / L of KH2PO4, 500 mg / L of Na2HPO4, 200 mg / L of MgSO4·7H2O, and 5000 mg / L of NaCl; a trace element solution is added at 2 mL / L, and the trace element solution is, by mass percentage: 57100 mg / L of EDTA-2Na, 3900 mg / L of ZnSO4·7H2O, 7000 mg / L of CaCl2·2H2O, 1000 mg / L of MnCl2·4H2O, 5000 mg / L of FeSO4·7H2O, 1100 mg / L of (NH4)6MO 24 -4H2O, 1600 mg / L of CuSO4·5H2O, and 1600 mg / L of CoCl2·6H2O; adjust the pH to 5-9, the nitrate nitrogen concentration in the medium is 5-30 mg / L, and the reaction time is 12-60 h.

[0017] Preferably, when preparing the carbon-free nitrate medium, it is necessary to adjust the pH to 7; the nitrate nitrogen concentration in the medium is 15 mg / L; the reaction time is 48 h.

[0018] Beneficial effects

[0019] Through the synergistic effect of light driving and microbial enzyme transformation, the present invention has successfully developed an innovative method for efficiently reducing nitrate to ammonia by visible light under carbon-free conditions. Its beneficial effects are significantly reflected in the following aspects: The present invention adopts a wide-spectrum response catalyst and microorganism synergistic system, directly uses visible light to excite and generate photogenerated electrons, and directionally reduces nitrate to ammonia by optimizing the electron transfer path. At the same time, it completely gets rid of the dependence on organic carbon sources and external electric energy, providing a resource integration solution for the treatment of nitrate-containing sewage and wastewater and the green synthesis of ammonia, with both environmental benefits and economic value. Description of the drawings

[0020] Figure 1 Scanning electron microscope images of the g-C3N4-CQDs, Shewanella oneidensis MR-1, and S.oneidensis-g-C3N4-CQDs systems.

[0021] Figure 2 Concentration changes of nitrate nitrogen and ammonia nitrogen in Example 1, Example 2, Example 3, Example 4, Example 5, and the control example. Specific implementation schemes

[0022] The present invention will be described in detail below in conjunction with embodiments, so that those skilled in the art can better understand the present invention. However, the present invention is not limited to the following embodiments.

[0023] A method for constructing a coupling system of g-C3N4-CQDs catalyst and Shewanella oneidensis MR-1: Shewanella is first grown in Luria Bertani (LB) medium (10 g / L NaCl, 10 g / L tryptone and 5 g / L yeast extract), the temperature is maintained at 20-35 °C, preferably 30 °C, and the oscillation speed is 100-200 r / min, preferably 150 r / min, until the logarithmic growth phase (OD 600 reaches 0.2-0.5, preferably 0.2). Then the prepared g-C3N4-CQDs (5 mg-20 mg, preferably 10 mg) is added to construct the S. oneidensis-g-C3N4-CQDs system. After incubating in the dark for 6-18 h, preferably 12 h, the cells are washed three times with 0.9% NaCl and collected by centrifugation (rotation speed 5000-10000 rpm, preferably 8000 rpm, time 5-10 minutes, preferably 6 minutes) and resuspended in 0.9% NaCl (5 mL).

[0024] The method for preparing the g-C3N4-CQDs material is as follows: Carbon quantum dots (CQDs) are synthesized by microwave synthesis. Add 2 - 6 g, preferably 4 g of urea and 1 - 4 g, preferably 2 g of citric acid into a beaker. Add 15 mL of deionized water to form a clear solution. Heat the obtained clear solution in a microwave oven at 750 W for 5 - 15 minutes, preferably 10 minutes. During the heating process, the mixture gradually reacts and evolves from a transparent solution into a dark brown solid. Then, disperse the dark brown solid CQD in deionized water and centrifuge it at a speed of 2000 - 15000 rpm, preferably 10,000 rpm for 5 - 20 minutes, preferably 10 minutes, and repeat 3 - 5 times, preferably 5 times, to remove the agglomerated CQD. The obtained CQD liquid is dried in an oven at 60 °C to obtain a powder. Mix the CQD solution (1 g / L) with ammonia water in a reactor at a volume ratio of 1:1 - 3, preferably 1:1, and perform hydrothermal treatment in a reaction kettle at 120 °C - 180 °C, preferably 150 °C for 4 - 12 hours, preferably 7 hours. Then, open it at room temperature to remove the excess ammonia gas until the pH value reaches 7. Using melamine as a precursor, heat it at 550 °C for 2 hours to synthesize the g-C3N4 photocatalyst. Mix different volumes (0, 3, 5, 7, and 9 mL) of ammonia-treated carbon quantum dot solutions with an aqueous melamine solution (1 g of melamine dissolved in 40 mL of deionized water), evaporate it at 80 °C, and then continuously heat it in a muffle furnace at 550 °C for 2 hours to synthesize g-C3N4-CQD3, g-C3N4-CQD5, g-C3N4-CQD7, and g-C3N4-CQD9 respectively. Preferably, g-C3N4-CQD5 is used as the light-driven material.

[0025] An application method of a g-C3N4-CQDs catalyst and Shewanella oneidensis MR-1 coupling system: Mix the S.oneidensis-g-C3N4-CQDs system suspension with 95 mL of sterilized carbon-free nitrate medium and place it in a sealed reaction bottle, and purge it with argon for 10 - 20 minutes, preferably 15 minutes. The reaction is carried out in a photoreactor under the irradiation of a xenon lamp (250 W). The temperature is controlled at 20 - 35 °C, preferably 30 °C. Take samples from the bottle at regular intervals, filter them with a syringe filter with a pore size of 0.22 μm, and measure the concentrations of nitrate nitrogen, nitrite nitrogen, and ammonia nitrogen by ultraviolet spectrophotometry (220 - 275) nm, n-1-naphthylethylenediamine dihydrochloride spectrophotometry (540 nm), and Nessler's reagent colorimetry (420 nm) respectively.

[0026] The composition of the carbon-free nitrate medium (mg / L): NaHCO3, 1000; KH2PO4, 500; Na2HPO4, 500; MgSO4·7H2O, 200 and NaCl, 5000. And a trace element solution is added at 2 mL / L, where the trace element solution is (mg / L): EDTA-2Na, 57100; ZnSO4·7H2O, 3900; CaCl2·2H2O, 7000; MnCl2·4H2O, 1000; FeSO4·7H2O, 5000; (NH4)6MO7O 24 -4H2O, 1100; CuSO4·5H2O, 1600 and CoCl2·6H2O, 1600. Adjust the pH to 5 - 9, preferably 7. The nitrate nitrogen concentration in the medium is 5 - 30 mg / L, preferably 15 mg / L. The reaction time is 12 - 60 h, preferably 48 h.

[0027] Figure 1 are the scanning electron microscope images of the g-C3N4-CQDs, Shewanella oneidensis MR-1 and S.oneidensis-g-C3N4-CQDs systems.

[0028] Example 1

[0029] Shewanella bacteria were first grown in Luria Bertani (LB) medium at a temperature maintained at 30 °C and an oscillation speed of 150 r / min until the logarithmic growth phase (OD 600 reached 0.2). Then 10 mg of the prepared g-C3N4-CQD3 was added to construct the S.oneidensis-g-C3N4-CQD3 system. After incubation in the dark for 12 h, the cells were washed three times with 0.9% NaCl, collected by centrifugation (8000 rpm, 6 minutes) and resuspended in 0.9% NaCl (5 mL). The suspension of the S.oneidensis-g-C3N4-CQD3 system was mixed with 95 mL of sterilized carbon-free nitrate medium (nitrate concentration controlled at 15 mg / L) and placed in a sealed reaction bottle, and purged with argon for preferably 15 minutes. The reaction was carried out in a photoreactor with a 250 W xenon lamp. The temperature was controlled at 30 °C. After 48 h of illumination, samples were taken from the bottle, filtered through a syringe filter with a pore size of 0.22 μm, and the nitrate nitrogen concentration was measured to be 3 mg / L by ultraviolet spectrophotometry (220 - 275), the nitrite nitrogen concentration was measured to be 5 mg / L by N-(1-naphthyl)ethylenediamine dihydrochloride spectrophotometry (540 nm), and the ammonia nitrogen concentration was measured to be 7 mg / L by Nessler's reagent colorimetry (420 nm).

[0030] Example 2

[0031] Shewanella was first grown in Luria Bertani (LB) medium at a temperature of 30 °C and an oscillation speed of 150 r / min until the logarithmic growth phase was reached (OD 600 reached 0.2). Then, 10 mg of the prepared g-C3N4-CQD5 was added to construct the S. oneidensis-g-C3N4-CQD5 system. After incubation in the dark for 12 h, the cells were washed three times with 0.9% NaCl, collected by centrifugation (8000 rpm, 6 minutes), and resuspended in 0.9% NaCl (15 mL). The suspension of the S. oneidensis-g-C3N4-CQD5 system was mixed with 95 mL of sterilized carbon-free nitrate medium (nitrate concentration controlled at 15 mg / L) and placed in a sealed reaction bottle, and purged with argon for preferably 15 minutes. The reaction was carried out under a photoreactor with a 250 W xenon lamp. The temperature was controlled at 30 °C. After 48 h of illumination, samples were taken from the bottle, filtered through a syringe filter with a pore size of 0.22 μm, and the nitrate nitrogen concentration was measured to be 0.5 mg / L by ultraviolet spectrophotometry (220 - 275), the nitrite nitrogen concentration was measured to be 2 mg / L by N-(1-naphthyl)ethylenediamine dihydrochloride spectrophotometry (540 nm), and the ammonia nitrogen concentration was measured to be 13.5 mg / L by Nessler's reagent colorimetry (420 nm).

[0032] Example 3

[0033] Shewanella was first grown in Luria Bertani (LB) medium at a temperature of 30 °C and an oscillation speed of 150 r / min until the logarithmic growth phase was reached (OD 600 reached 0.2). Then, 10 mg of the prepared g-C3N4-CQD7 was added to construct the S. oneidensis-g-C3N4-CQD7 system. After incubation in the dark for 12 h, the cells were washed three times with 0.9% NaCl, collected by centrifugation (8000 rpm, 6 minutes), and resuspended in 0.9% NaCl (15 mL). The suspension of the S. oneidensis-g-C3N4-CQD7 system was mixed with 95 mL of sterilized carbon-free nitrate medium (nitrate concentration controlled at 15 mg / L) and placed in a sealed reaction bottle, and purged with argon for preferably 15 minutes. The reaction was carried out under a photoreactor with a 250 W xenon lamp. The temperature was controlled at 30 °C. After 48 h of illumination, samples were taken from the bottle, filtered through a syringe filter with a pore size of 0.22 μm, and the nitrate nitrogen concentration was measured to be 1.5 mg / L by ultraviolet spectrophotometry (220 - 275), the nitrite nitrogen concentration was measured to be 5.5 mg / L by N-(1-naphthyl)ethylenediamine dihydrochloride spectrophotometry (540 nm), and the ammonia nitrogen concentration was measured to be 8 mg / L by Nessler's reagent colorimetry (420 nm).

[0034] Example 4

[0035] Shewanella was first grown in Luria Bertani (LB) medium at a temperature of 30 °C and an oscillation speed of 150 r / min until the logarithmic growth phase (OD 600 reached 0.2). Then, 910 mg of the prepared g-C3N4-CQD was added to construct the S. oneidensis-g-C3N4-CQD9 system. After incubation in the dark for 12 h, the cells were washed three times with 0.9% NaCl, collected by centrifugation (8000 rpm, 6 minutes), and resuspended in 0.9% NaCl (5 mL). The suspension of the S. oneidensis-g-C3N4-CQD9 system was mixed with 95 mL of sterilized carbon-free nitrate medium (nitrate concentration controlled at 15 mg / L) and placed in a sealed reaction bottle, and purged with argon for preferably 15 minutes. The reaction was carried out under a photoreactor with a 250 W xenon lamp. The temperature was controlled at 30 °C. After 48 h of illumination, samples were taken from the bottle, filtered through a syringe filter with a pore size of 0.22 μm, and the nitrate nitrogen concentration was measured to be 2 mg / L by ultraviolet spectrophotometry (220 - 275), the nitrite nitrogen concentration was measured to be 5 mg / L by N-(1-naphthyl)ethylenediamine dihydrochloride spectrophotometry (540 nm), and the ammonia nitrogen concentration was measured to be 7.5 mg / L by Nessler's reagent colorimetry (420 nm).

[0036] Example 5

[0037] Shewanella was first grown in Luria Bertani (LB) medium at a temperature of 20 °C and an oscillation speed of 150 r / min until the logarithmic growth phase (OD 600 reached 0.2). Then, 510 mg of the prepared g-C3N4-CQD was added to construct the S. oneidensis-g-C3N4-CQD5 system. After incubation in the dark for 12 h, the cells were washed three times with 0.9% NaCl, collected by centrifugation (8000 rpm, 6 minutes), and resuspended in 0.9% NaCl (5 mL). The suspension of the S. oneidensis-g-C3N4-CQD5 system was mixed with 95 mL of sterilized carbon-free nitrate medium (nitrate concentration controlled at 15 mg / L) and placed in a sealed reaction bottle, and purged with argon for preferably 15 minutes. The reaction was carried out under a photoreactor with a 250 W xenon lamp. The temperature was controlled at 30 °C. After 48 h of illumination, samples were taken from the bottle, filtered through a syringe filter with a pore size of 0.22 μm, and the nitrate nitrogen concentration was measured to be 4 mg / L by ultraviolet spectrophotometry (220 - 275), the nitrite nitrogen concentration was measured to be 6 mg / L by N-(1-naphthyl)ethylenediamine dihydrochloride spectrophotometry (540 nm), and the ammonia nitrogen concentration was measured to be 5 mg / L by Nessler's reagent colorimetry (420 nm).

[0038] Example 6

[0039] Shewanella was first grown in Luria Bertani (LB) medium at a temperature of 30 °C and an oscillation speed of 150 r / min until the logarithmic growth phase (OD 600 reached 0.2). Then, 10 mg of the prepared g-C3N4-CQD5 was added to construct the S. oneidensis-g-C3N4-CQD5 system. After incubation in the dark for 12 h, the cells were washed three times with 0.9% NaCl, collected by centrifugation (8000 rpm, 6 minutes), and resuspended in 0.9% NaCl (5 mL). The suspension of the S. oneidensis-g-C3N4-CQD5 system was mixed with 95 mL of sterilized carbon-free nitrate medium (nitrate concentration controlled at 30 mg / L) and placed in a sealed reaction bottle, and purged with argon for preferably 15 minutes. The reaction was carried out under a photoreactor with a 250 W xenon lamp. The temperature was controlled at 30 °C. After 48 h of illumination, samples were taken from the bottle, filtered through a syringe filter with a pore size of 0.22 μm, and the nitrate nitrogen concentration was measured to be 4 mg / L by ultraviolet spectrophotometry (220 - 275), the nitrite nitrogen concentration was measured to be 10 mg / L by N-(1-naphthyl)ethylenediamine dihydrochloride spectrophotometry (540 nm), and the ammonia nitrogen concentration was measured to be 15 mg / L by Nessler's reagent colorimetry (420 nm).

[0040] Control Example

[0041] Shewanella was first grown in Luria Bertani (LB) medium at a temperature of 30 °C and an oscillation speed of 150 r / min until the logarithmic growth phase (OD 600 reached 0.2). Then, 10 mg of the prepared g-C3N4-CQD5 was added to construct the S. oneidensis-g-C3N4-CQD5 system. After incubation in the dark for 12 h, the cells were washed three times with 0.9% NaCl, collected by centrifugation (8000 rpm, 6 minutes), and resuspended in 0.9% NaCl (5 mL). The suspension of the S. oneidensis-g-C3N4-CQD5 system was mixed with 95 mL of sterilized carbon-free nitrate medium (nitrate concentration controlled at 15 mg / L) and placed in a sealed reaction bottle, and purged with argon for preferably 15 minutes. The reaction was carried out under dark conditions. The temperature was controlled at 30 °C. After 48 h of illumination, samples were taken from the bottle, filtered through a syringe filter with a pore size of 0.22 μm, and the nitrate nitrogen concentration was measured to be 15 mg / L by ultraviolet spectrophotometry (220 - 275), the nitrite nitrogen concentration was measured to be 0 mg / L by N-(1-naphthyl)ethylenediamine dihydrochloride spectrophotometry (540 nm), and the ammonia nitrogen concentration was measured to be 0 mg / L by Nessler's reagent colorimetry (420 nm).

[0042] Figure 2 Concentration changes of nitrate nitrogen and ammonia nitrogen in Example 1, Example 2, Example 3, Example 4, Example 5 and the control example. It can be seen from this: According to the control example, there are no obvious changes in nitrate nitrogen and ammonia nitrogen in the dark environment, proving that light is a necessary condition for driving the reduction of nitrate to synthesize ammonia in the coupled system. According to the concentration changes of nitrate nitrogen and ammonia nitrogen in Examples 1-4, it is proved that the S.oneidensis-g-C3N4-CQD5 system has the optimal light-driven reduction effect of nitrate to synthesize ammonia. According to the concentration changes in Example 2, Example 4 and Example 6, it is proved that 30 °C is the optimal temperature for the reaction.

[0043] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made by using the specification of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of the present invention.

Claims

1. A method for establishing a catalyst / microorganism coupling system, characterized in that Shewanella bacteria were first grown in Luria Bertani medium at a temperature maintained at 20 - 35 °C and an oscillation speed of 100 - 200 r / min until the optical density (OD) reached 0.2 - 0.5 in the logarithmic growth phase. 600 Then, 5 mg - 20 mg of the prepared g-C3N4-CQDs was added to construct the S.oneidensis-g-C3N4-CQDs system. After incubation in the dark for 6 - 18 h, the cells were washed three times with 0.9% NaCl, collected by centrifugation, and resuspended in 5 mL of 0.9% NaCl at a centrifugation speed of 5000 - 10,000 rpm for 5 - 10 minutes.

2. The method for establishing a catalyst / microorganism coupling system according to claim 1, wherein The Luria Bertani medium was 10 g / L NaCl, 10 g / L tryptone, and 5 g / L yeast extract; the temperature was maintained at 30 °C; the oscillation speed was 150 r / min; until the logarithmic growth phase OD 600 was 0.2; then 10 mg of the prepared g-C3N4-CQDs was added to construct the S. oneidensis-g-C3N4-CQDs system; after incubation in the dark for 12 h, the cells were washed three times with 0.9% NaCl, collected after centrifugation for 6 minutes and resuspended in 0.9% NaCl; the centrifugation speed was 8000 rpm.

3. The method for establishing a catalyst / microorganism coupling system according to claim 1, characterized in that Preparation method of the above-mentioned g-C3N4-CQDs material: Carbon quantum dots CQD are synthesized by microwave synthesis method. Add 2-6 grams of urea and citric acid into a beaker, and add 15 ml of deionized water to form a clear solution. Heat the obtained clear solution in a microwave oven at 750 W for 5-15 minutes. During the heating process, the mixture gradually reacts and evolves from a transparent solution into a dark brown solid. Disperse the dark brown solid CQD in deionized water and centrifuge it at a speed of 2000-15000 rpm for 5-20 minutes, repeat 3-5 times to remove the agglomerated CQD, and dry the obtained CQD liquid in an oven at 60 °C to obtain a powder. In a reactor, mix a 1 g / L CQD solution with ammonia water at a volume ratio of 1:1-3, and carry out hydrothermal treatment in a reaction kettle at 120 °C-180 °C for 4-12 hours. Open it at room temperature to remove the excess ammonia gas until the pH value reaches 7. Using melamine as a precursor, heat it at 550 °C for 2 hours to synthesize the g-C3N4 photocatalyst. Mix 0, 3, 5, 7 or 9 ml of the ammonia-treated carbon quantum dot solution with the melamine aqueous solution, evaporate it at 80 °C, and then continuously heat it in a muffle furnace at 550 °C for 2 hours to synthesize g-C3N4-CQD3, g-C3N4-CQD5, g-C3N4-CQD7 or g-C3N4-CQD9 respectively.

4. The method for establishing a catalyst / microorganism coupling system according to claim 1, characterized in that: Preparation method of the above-mentioned g-C3N4-CQDs material: Carbon quantum dots CQD are synthesized by microwave synthesis method. Add 4 grams of urea and 1-4 grams of citric acid into a beaker, and add 15 ml of deionized water to form a clear solution. Heat the obtained clear solution in a microwave oven at 750 W for 10 minutes. During the heating process, the mixture gradually reacts and evolves from a transparent solution into a dark brown solid. Then, disperse the dark brown solid CQD in deionized water and centrifuge it at a speed of 10,000 rpm for 10 minutes, repeat 5 times to remove the agglomerated CQD, and dry the obtained CQD liquid in an oven at 60 °C to obtain a powder. In a reactor, mix a 1 g / L CQD solution with ammonia water at a volume ratio of 1:1, and carry out hydrothermal treatment in a reaction kettle at 150 °C for 7 hours. Open it at room temperature to remove the excess ammonia gas until the pH value reaches 7. Using melamine as a precursor, heat it at 550 °C for 2 hours to synthesize the g-C3N4 photocatalyst. Mix 5 ml of the ammonia-treated carbon quantum dot solution with the melamine aqueous solution, evaporate it at 80 °C, and then continuously heat it in a muffle furnace at 550 °C for 2 hours to synthesize g-C3N4-CQD5 as a light-driven material.

5. The method for establishing a catalyst / microorganism coupling system according to claim 4, wherein: The melamine aqueous solution is 1 gram of melamine dissolved in 40 ml of deionized water.

6. An application method of the catalyst / microorganism coupling system according to claim 1, characterized in that: Mix the S.oneidensis-g-C3N4-CQDs system suspension with 95 mL of sterilized carbon-free nitrate medium and place it in a sealed reaction bottle. Purge with argon for 10 - 20 minutes. The reaction is carried out in a photoreactor under the irradiation of a xenon lamp; the temperature is controlled at 20 - 35 °C. Take samples from the bottle at regular intervals, filter them with a syringe filter with a pore size of 0.22 μm, and measure the concentrations of nitrate nitrogen, nitrite nitrogen, and ammonia nitrogen by ultraviolet spectrophotometry, n-1-naphthylethylenediamine dihydrochloride spectrophotometry, and Nessler's reagent colorimetry, respectively.

7. The application method of a catalyst / microorganism coupling system according to claim 6, characterized in that: Mix the S.oneidensis-g-C3N4-CQDs system suspension with 95 mL of sterilized carbon-free nitrate medium and place it in a sealed reaction bottle. Purge with argon for 15 minutes. The reaction is carried out in a photoreactor under the irradiation of a 250W xenon lamp; the temperature is controlled at 30 °C. Take samples from the bottle at regular intervals, filter them with a syringe filter with a pore size of 0.22 μm, and measure the concentrations of nitrate nitrogen, nitrite nitrogen, and ammonia nitrogen by ultraviolet spectrophotometry at 220 - 275 nm, n-1-naphthylethylenediamine dihydrochloride spectrophotometry at 540 nm, and Nessler's reagent colorimetry at 420 nm, respectively.

8. The application method of a catalyst / microorganism coupling system according to claim 6, characterized in that: The composition of the carbon-free nitrate medium includes, by mass percentage: 1000 mg / L of NaHCO3, 500 mg / L of KH2PO4, 500 mg / L of Na2HPO4, 200 mg / L of MgSO4·7H2O, and 5000 mg / L of NaCl; a trace element solution is added at 2 mL / L, and the trace element solution is, by mass percentage: 57100 mg / L of EDTA-2Na, 3900 mg / L of ZnSO4·7H2O, 7000 mg / L of CaCl2·2H2O, 1000 mg / L of MnCl2·4H2O, 5000 mg / L of FeSO4·7H2O, 1100 mg / L of (NH4)6MO7O 24 -4H2O, 1600 mg / L of CuSO4·5H2O, and 1600 mg / L of CoCl2·6H2O; adjust the pH to 5 - 9, the nitrate nitrogen concentration in the medium is 5 - 30 mg / L, and the reaction time is 12 - 60 h.

9. The application method of a catalyst / microorganism coupling system according to claim 8, wherein: Adjust the pH to 7; the concentration of nitrate nitrogen in the medium is 15 mg / L; the reaction time is 48 h.

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