A light-controlled extracellular polymer solution, its preparation method and application

Abstract

The present application relates to sewage biological treatment technical field, specifically relates to a kind of light control extracellular polymer solution and its preparation method and application.The present application provides a kind of preparation method of light control extracellular polymer solution, comprising the following steps: (1) using first nitrate wastewater acclimation activated sludge, obtain acclimation activated sludge;(2) using second nitrate wastewater activation the acclimation activated sludge, obtain activated activated sludge;(3) extract the bacteria in the activated activated sludge, then carry out light control metabolic reaction under blue light irradiation in culture solution, after reaction, extract active substance, i.e. the light control extracellular polymer solution is obtained.The present application is irradiated by blue light in light control metabolic reaction to open downstream path or signal transmission, and then open rare metabolic path to generate the light control extracellular polymer solution with biological activity, and the light control extracellular polymer solution can have catalytic effect to improve the denitrification efficiency of denitrifying bacteria.

Description

Technical Field

[0001] This invention relates to the field of wastewater biological treatment technology, specifically to a light-controlled extracellular polymer solution, its preparation method, and its application. Background Technology

[0002] Nitrate pollution caused by excessive fertilization threatens ecological security and human health. On the one hand, nitrate is a common water pollutant; on the other hand, nitrogen in nitrate is a basic component of many biological and chemical products, and the resource utilization methods of converting nitrate into fertilizers, energy, and proteins have attracted attention.

[0003] To achieve efficient nitrate removal, extensive research has been conducted on denitrification biotechnology, including simultaneous nitrification and denitrification, short-cut nitrification and denitrification, and anammox, among which denitrification is a major method for biological nitrate removal. However, biotechnology has consistently faced challenges such as microbial community regulation and process stability, especially under complex water quality conditions such as fluctuating water quality and high-salinity industrial wastewater, where artificial regulation of biological wastewater treatment technologies is extremely difficult. Optical biotechnology, such as photosynthetic cell factories and solar-powered biohybrids, is an economical and environmentally friendly method for synthesizing nitrogen-based products such as ammonia, peptides, and proteins. However, the inhibitory effect of sunlight on microbial communities limits its further application, and currently, the converted products require further processing and purification, posing economic challenges for industrial application.

[0004] In existing technologies, the combined action of extracellular polymer solutions in activated sludge and denitrifying bacteria for nitrate denitrification is a conventional method in wastewater treatment. However, the extracellular polymer solutions in existing technologies have low biological activity and poor catalytic effect on the denitrification reaction of denitrifying bacteria. Therefore, there is an urgent need to develop an extracellular polymer solution with better biological activity. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of low biological activity and poor catalytic effect on denitrification reaction of denitrifying bacteria in the existing extracellular polymer solution, thereby providing a light-controlled extracellular polymer solution, its preparation method and application.

[0006] This invention provides a method for preparing a light-controlled extracellular polymer solution, comprising the following steps:

[0007] (1) The activated sludge was acclimated using the first nitrate wastewater to obtain acclimated activated sludge;

[0008] (2) The acclimatized activated sludge was activated using the second nitrate wastewater to obtain activated activated sludge;

[0009] (3) Extract the bacteria from the activated sludge, and then carry out a photocontrolled metabolic reaction in the culture medium under blue light irradiation. After the reaction, extract the active substances to obtain the photocontrolled extracellular polymer solution.

[0010] Optionally, the acclimated activated sludge is stored at 1-5°C.

[0011] Activated sludge is a collective term for microbial communities and the organic and inorganic substances they adhere to. Its sources can be municipal wastewater treatment plants and industrial wastewater treatment plants. This invention applies to all activated sludge.

[0012] Preferably, the first nitrate wastewater contains 10-11 g / L NaNO3, 6-8 g / L NaAc, 1-6 wt% NaCl, 0.7-0.75 g / L KH2PO4, 0.05-0.15 g / L KHCO3, 0.02-0.04 g / L CaCO3, 0.01-0.03 g / L MgSO4·7H2O, 0.8-1.2 ml / L of a first trace aqueous solution, and 0.8-1.2 ml / L of a second trace aqueous solution.

[0013] The first trace aqueous solution contains 4-6 g / L of EDTA·2Na and 4-6 g / L of FeSO4·7H2O.

[0014] The second trace aqueous solution contains 9-11 g / L EDTA·2Na, 0.012-0.016 g / L H3BO3, 0.4-0.6 g / L ZnSO4·7H2O, 0.2-0.3 g / L CoCl2·6H2O, 0.9-1 g / L MnCl2·4H2O, 0.12-0.2 g / L CuSO4, 0.15-0.2 g / L NiCl2·6H2O, and 0.2-0.3 g / L Na2MoO4·2H2O.

[0015] Preferably, the acclimation of activated sludge is carried out using the sequencing batch activated sludge process;

[0016] Preferably, in the sequencing batch activated sludge process: the influent time is 6-8 min, the anoxic reaction time is 350-400 min, the settling time is 10-20 min, and the effluent time is 3-6 min;

[0017] Preferably, in the sequencing batch activated sludge process, the mass ratio of the first nitrate wastewater to the activated sludge is (7-30):1:.

[0018] Preferably, the second nitrate wastewater contains 50-1000 mg N / L of nitrogen source, carbon source, 10-11 mM NH4Cl2, 17-18 mM KH2PO4, 32-33 mM Na2HPO4·12H2O, 0.4-0.5 mM MgSO4·7H2O, 9-10 mM EDTA·2Na, 8-10 mM FeCl3·6H2O, 0.05-0.2 mM MnCl2·4H2O, 0.5-1.5 mM NaMoO4·2H2O, 0.5-1 mM CuCl2·2H2O, and 2-3 mM ZnCl2;

[0019] The mass ratio of nitrogen in the nitrogen source to carbon in the carbon source is 1-2.

[0020] Preferably, the nitrogen source is selected from NaNO3;

[0021] Preferably, the carbon source is selected from NaAc.

[0022] Preferably, the activation process is carried out at a temperature of 25–37°C, a rotation speed of 100–250 rpm, and a time of 20–27 h.

[0023] Preferably, in step (2), the mass ratio of the second nitrate wastewater to the acclimated activated sludge is (7-20):1.

[0024] Optionally, the activation process is carried out in a constant temperature shaker.

[0025] Preferably, the process of extracting bacteria from the activated sludge includes the following steps: performing a first centrifugal separation on the activated sludge obtained in step (2), washing the product of the first centrifugal separation and then performing a second centrifugal separation, and then adding a first resuspension to resuspend the sludge to obtain a bacterial solution;

[0026] The first suspension contains 10-11 mM NH4Cl2, 17-18 mM KH2PO4, 32-33 mM Na2HPO4·12H2O, and 0.4-0.5 mM MgSO4·7H2O.

[0027] Preferably, the centrifugal force for the first centrifugal separation is 7000-9000g, and the time is 4-6min;

[0028] Preferably, the centrifugal force for the second centrifugal separation is 7000-9000g, and the time is 4-6min;

[0029] Preferably, the cleaning solution used in the cleaning step is PBS buffer;

[0030] Preferably, the volume of the bacterial solution is the same as the volume of the activated sludge before the first centrifugation.

[0031] Optionally, the PBS buffer used in this invention is 0.01–0.1 M PBS;

[0032] More preferably, the PBS buffer concentration used in this invention is 0.01M; wherein, the 0.01M PBS buffer comprises: 8 g·L⁻¹ -1 NaCl, 0.2 g·L -1 KCl, 1.44 g·L -1 Na2HPO4, 0.24 g·L -1 KH2PO4;

[0033] Optionally, the washing step involves washing the first centrifuged product 2-3 times with PBS buffer.

[0034] Preferably, the photocontrolled metabolic reaction is carried out by mixing the bacterial solution with the culture medium and then conducting the photocontrolled metabolic reaction under anaerobic conditions;

[0035] Preferably, the culture medium contains 50-1000 mgN / L of nitrogen source, carbon source, 10-11 mM NH4Cl2, 17-18 mM KH2PO4, 32-33 mM Na2HPO4·12H2O, and 0.4-0.5 mM MgSO4·7H2O;

[0036] The mass ratio of nitrogen in the nitrogen source to carbon in the carbon source is 1-2.

[0037] Preferably, the nitrogen source is selected from NaNO3;

[0038] Preferably, the carbon source is selected from NaAc;

[0039] Preferably, the volume ratio of the culture medium to the bacterial solution is (6-30):1;

[0040] Preferably, the intensity of blue light in the photocontrolled metabolic reaction is 1.0–6.0 mW / cm². 2 The temperature for photocontrolled metabolic reactions is 25-35℃;

[0041] Optionally, the wavelength of the blue light in the photocontrolled metabolic reaction is 400-450 nm;

[0042] Preferably, the concentration of NO3-N in the bacterial culture and culture medium is 100-150 mg NO3-N·L. -1 The photocontrolled metabolic reaction is stopped at that time.

[0043] Optionally, the anaerobic conditions are achieved by aerating argon gas into the mixture of bacterial culture and culture medium;

[0044] Optionally, the argon aeration time is 4-6 minutes.

[0045] Optionally, after argon aeration is completed, the reactants are thoroughly mixed using a nine-piece magnetic stirrer.

[0046] Optionally, the rotation speed of the nine-link magnetic stirrer is 100-300 rpm.

[0047] Optionally, the blue light in the photocontrolled metabolic reaction is generated by a blue LED.

[0048] Preferably, the process of extracting the active substance in step (3) includes the following steps:

[0049] After the product of the photocontrolled metabolic reaction is shaken well, a third centrifugation is performed to collect the first supernatant. The third fraction is then resuspended in a second resuspension. The process is then repeated with a first stirring, a first water bath heating, a second stirring, and a fourth centrifugation. The second supernatant is collected, and the fourth fraction is resuspended in a third resuspension. The process is then repeated with a third stirring, a second water bath heating, a fourth stirring, and a fifth centrifugation. The third supernatant is collected. The first supernatant, the second supernatant, and the third supernatant are mixed to obtain the active substance, namely the photocontrolled extracellular polymer solution.

[0050] Preferably, the volume ratio of the photocontrolled metabolic reaction system, the second suspension, and the third suspension is (25-35):(4-6):(4-6);

[0051] Preferably, the temperature of the third centrifugal separation is 3-6℃, the centrifugal force is 3000-5000g, and the time is 10-20min;

[0052] Preferably, the first stirring is performed by vortex stirring to achieve uniform mixing, and the stirring temperature is 20-25°C;

[0053] Preferably, the temperature of the first water bath is 50-70℃, and the heating time is 2-4 minutes;

[0054] Preferably, the second stirring is performed by vortex stirring to achieve uniform mixing, and the stirring temperature is 20-25°C;

[0055] Preferably, the fourth centrifugal separation is performed at a temperature of 3-6°C, a centrifugal force of 3000-5000g, and a time of 10-20min.

[0056] Preferably, the third stirring is performed by vortex stirring to achieve uniform mixing, and the stirring temperature is 20-25°C;

[0057] Preferably, the temperature of the second water bath is 50-70℃, and the time is 20-40 minutes;

[0058] Preferably, the fourth stirring is performed by using vortex stirring to achieve uniform mixing, and the stirring temperature is 20-25°C.

[0059] Preferably, the fifth centrifugal separation is performed at a temperature of 3-6°C, a centrifugal force of 9000-11000g, and a time of 10-20min.

[0060] Preferably, the second suspension is a 1-6 wt% NaCl aqueous solution;

[0061] Preferably, the third suspension is a 1-6 wt% NaCl aqueous solution;

[0062] Preferably, the volume ratio of the first supernatant, the second supernatant, and the third supernatant in the photocontrolled extracellular polymer solution is (0.9-1.2):(0.9-1.2):(0.9-1.2).

[0063] In this invention, the active ingredient in the first supernatant is mainly soluble extracellular polymer (S-EPS); the active ingredient in the second supernatant is mainly loosely bound extracellular polymer (LB-EPS); and the active ingredient in the third supernatant is mainly tightly bound extracellular polymer (TB-EPS).

[0064] The present invention also provides a light-controlled extracellular polymeric solution, which is prepared by the preparation method described above.

[0065] The present invention also provides a method for denitrification, wherein the light-controlled extracellular polymer solution prepared by the above preparation method is mixed with denitrifying bacteria and the solution to be denitrified, and then a denitrification reaction is carried out.

[0066] Preferably, the concentration of the light-controlled extracellular polymer solution in the solution obtained by mixing the light-controlled extracellular polymer solution, denitrifying bacteria, and the solution to be denitrified is 0.5-5 ml / 100 ml.

[0067] The technical solution of this invention has the following advantages:

[0068] 1. The present invention provides a method for preparing a light-controlled extracellular polymer solution, comprising the following steps:

[0069] (1) The activated sludge is acclimated using the first nitrate wastewater to obtain acclimated activated sludge; (2) The acclimated activated sludge is activated using the second nitrate wastewater to obtain activated activated sludge; (3) The bacteria in the activated activated sludge are extracted, and then a photocontrolled metabolic reaction is carried out in the culture medium under blue light irradiation. After the reaction, the active substances are extracted to obtain the photocontrolled extracellular polymer solution.

[0070] This invention effectively increases the concentration of denitrifying bacteria by acclimating activated sludge with a first nitrate wastewater and activating the activated sludge with a second nitrate wastewater. In the subsequent photocontrolled metabolic reaction, blue light irradiation is used to open downstream pathways or signal transduction, thereby opening rare metabolic pathways to produce a bioactive photocontrolled extracellular polymer solution. This photocontrolled extracellular polymer solution can catalyze and improve the nitrogen removal efficiency of denitrifying bacteria.

[0071] Furthermore, the process of preparing this photocontrolled extracellular polymer solution can consume nitrates in wastewater, enabling the simultaneous removal of nitrates and recovery of active substances.

[0072] 2. The preparation method provided by this invention uses blue light to regulate microbial metabolic denitrification. The method is simple, requires no additional chemical reagents or materials, and does not produce secondary pollution. It is green, efficient, and economical.

[0073] 3. The preparation method provided by this invention can regulate the biological denitrification process by artificially controlling optical parameters. It is highly flexible and universal, and can be applied to the regulation of biological denitrification in different scenarios.

[0074] 4. The intensity of the blue light used in the preparation method provided by this invention is 1.0–6.0 mW / cm². 2 It has a low energy density, does not affect the homeostasis of the microbial community, and is highly practical and low in cost;

[0075] 5. The light-controlled extracellular polymer solution obtained by the preparation method provided by the present invention contains peptides, terpenes, cofactors, etc., and can be combined with other industrial chains, making it a highly promising wastewater resource utilization technology.

[0076] 6. The present invention provides a denitrification method in which a light-controlled extracellular polymer solution can be directly used to regulate and enhance existing processes, reduce sludge production, improve the pollutant removal capacity, artificial controllability and stability of the process, and reduce sludge production. Attached Figure Description

[0077] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0078] Figure 1 This is a comparison chart of the protein content in the first supernatant, second supernatant, and third supernatant prepared in Example 1, Comparative Example 1, and Comparative Example 2 of the present invention.

[0079] Figure 2 This is the metatranscriptome sequencing map of the light-controlled extracellular polymer solution prepared in Example 1 of this invention;

[0080] Figure 3 This is the metatranscriptome sequencing map of the light-controlled extracellular polymer solution prepared in Comparative Example 2 of this invention. Detailed Implementation

[0081] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0082] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0083] The PBS buffer concentration used in the examples and comparative examples was 0.01M; wherein, the 0.01M PBS buffer comprises: 8 g·L⁻¹ -1 NaCl, 0.2 g·L -1 KCl, 1.44 g·L -1 Na2HPO4, 0.24 g·L -1 KH2PO4;

[0084] The denitrifying paracoccus used in the test example ( Paracoccus denitrificans Purchased from Guangdong Microbial Culture Collection Center, model number (ATCC, 19367).

[0085] Example 1

[0086] This embodiment provides a method for preparing a light-controlled extracellular polymer solution, comprising the following steps:

[0087] (1) Place 200g of activated sludge in a sequencing batch reactor and introduce 2000g of first nitrate wastewater over a 7min influent time. Perform an anoxic reaction for 380min, followed by 15min settling and 4min drainage to discharge the wastewater, thereby obtaining acclimated activated sludge. Store the acclimated activated sludge at 4℃ for later use. The first nitrate wastewater contains 10.8g / L NaNO3, 6.6g / L NaAc, 5wt% NaCl, 0.75g / L KH2PO4, 0.1g / L KHCO3, 0.03g / L CaCO3, 0.02g / L MgSO4·7H2O, 1ml / L of first trace aqueous solution, and 1ml / L of second trace aqueous solution.

[0088] The first trace aqueous solution contains 5 g / L EDTA·2Na and 5 g / L FeSO4·7H2O; the second trace aqueous solution contains 10 g / L EDTA·2Na, 0.014 g / L H3BO3, 0.43 g / L ZnSO4·7H2O, 0.24 g / L CoCl2·6H2O, 0.99 g / L MnCl2·4H2O, 0.16 g / L CuSO4, 0.19 g / L NiCl2·6H2O, and 0.22 g / L Na2MoO4·2H2O.

[0089] (2) Place 10g of the acclimatized activated sludge prepared in step (1) and 100g of the second nitrate wastewater in a constant temperature shaker and activate it for 27h at 37℃ and 200rpm to obtain activated activated sludge; wherein, the second nitrate wastewater contains 500mg N·L -1 NaNO3, 1000 mg C·L -1 NaAc, 10.66mM NH4Cl2, 17.93mM KH2PO4, 32.76mM Na2HPO4·12H2O, 0.41mM MgSO4·7H2O, 9.6mM EDTA·2Na, 9.0mM FeCl3·6H2O, 0.1mMnCl2·4H2O, 1.0mM NaMoO4·2H2O, 0.8mM CuCl2·2H2O, 2.5mM ZnCl2, 9.6mM EDTA·2Na, 9.0mM FeCl3·6H2O, 0.1mM MnCl2·4H2O, 1.0mM NaMoO4·2H2O, 0.8mM CuCl2·2H2O, 2.5mM ZnCl2;

[0090] (3) Centrifuge 50 ml of the activated sludge prepared in step (2) at 8000 g for 5 min and discard the supernatant. Wash the centrifuged product 3 times with PBS buffer. Then centrifuge at 8000 g for 5 min. After washing the centrifuged product with PBS buffer, resuspend the centrifuged product in 50 ml with the first resuspension to obtain bacterial solution.

[0091] (4) Place 3 ml of the bacterial culture obtained in step (3) and 27 ml of the culture medium into a quartz reactor. Seal the reactor with a rubber gasket cover and wrap it with tin foil to create a dark environment. Then, aerate the reactor with argon gas for 5 minutes to create an anaerobic environment. Then, use a nine-link magnetic stirrer to thoroughly mix the reactants in the quartz reactor. The magnetic stirring speed is 200 rpm. After the reactants are thoroughly mixed, turn on the LED blue light (wavelength 400-450 nm) and irradiate the reactants with a light intensity of 5.0 mw / cm2 to carry out the photocontrolled metabolic reaction. Detect the NO3-N concentration in the reactants when it reaches 142.83 mg NO3-N·L. -1 The photocontrolled metabolic reaction was stopped. The product was then thoroughly shaken and centrifuged at 4000g for 15 min at 4°C, and the first supernatant was collected. The centrifuged product was resuspended in 5 ml of 5wt% NaCl aqueous solution and stirred evenly using a vortex mixer at 24°C. The mixture was then heated in a water bath at 60°C for 3 min, and stirred again using a vortex mixer at 24°C. The mixture was then centrifuged at 4000g for 15 min at 4°C, and the second supernatant was collected. The product was resuspended in 5 ml of 5wt% NaCl aqueous solution and stirred evenly using a vortex mixer at 24°C. The mixture was then heated in a water bath at 60°C for 30 min, and stirred again using a vortex mixer at 24°C. The mixture was then centrifuged at 10000g for 15 min at 4°C, and the third supernatant was collected. Equal volumes of the first, second, and third supernatants were mixed to obtain the photocontrolled extracellular polymer solution.

[0092] The culture medium contains 500 mg N·L -1 NaNO3, 1000 mg C·L -1 NaAc, 10.66mMNH4Cl2, 17.93mM KH2PO4, 32.76mM Na2HPO4·12H2O, 0.41mM MgSO4·7H2O, 9.6mM EDTA·2Na, 9.0mM FeCl3·6H2O, 0.1mM MnCl2·4H2O, 1.0mM NaMoO4·2H2O, 0.8mM CuCl2·2H2O, 2.5mMZnCl2.

[0093] Example 2

[0094] This embodiment provides a method for preparing a light-controlled extracellular polymer solution, comprising the following steps:

[0095] (1) Place 200g of activated sludge in a sequencing batch reactor and introduce 2000g of first nitrate wastewater over a 7min influent time. Perform an anoxic reaction for 380min, followed by 15min settling and 4min drainage to discharge the wastewater, thereby obtaining acclimated activated sludge. Store the acclimated activated sludge at 4℃ for later use. The first nitrate wastewater contains 10.8g / L NaNO3, 6.6g / L NaAc, 5wt% NaCl, 0.75g / L KH2PO4, 0.1g / L KHCO3, 0.03g / L CaCO3, 0.02g / L MgSO4·7H2O, 1ml / L first trace aqueous solution, and 1ml / L second trace aqueous solution.

[0096] The first trace aqueous solution contains 5 g / L EDTA·2Na and 5 g / L FeSO4·7H2O; the second trace aqueous solution contains 10 g / L EDTA·2Na, 0.014 g / L H3BO3, 0.43 g / L ZnSO4·7H2O, 0.24 g / L CoCl2·6H2O, 0.99 g / L MnCl2·4H2O, 0.16 g / L CuSO4, 0.19 g / L NiCl2·6H2O, and 0.22 g / L Na2MoO4·2H2O.

[0097] (2) Place 6g of the acclimatized activated sludge prepared in step (1) and 100g of the second nitrate wastewater in a constant temperature shaker and activate it for 27h at 37℃ and 200rpm to obtain activated activated sludge; wherein, the second nitrate wastewater contains 500mg N·L -1 NaNO3, 1000 mg C·L -1 NaAc, 10.66mM NH4Cl2, 17.93mM KH2PO4, 32.76mM Na2HPO4·12H2O, 0.41mM MgSO4·7H2O, 9.6mM EDTA·2Na, 9.0mM FeCl3·6H2O, 0.1mMnCl2·4H2O, 1.0mM NaMoO4·2H2O, 0.8mM CuCl2·2H2O, 2.5mM ZnCl2, 9.6mM EDTA·2Na, 9.0mM FeCl3·6H2O, 0.1mM MnCl2·4H2O, 1.0mM NaMoO4·2H2O, 0.8mM CuCl2·2H2O, 2.5mMZnCl2;

[0098] (3) Centrifuge 40 ml of the activated sludge prepared in step (2) at 8000 g for 5 min and discard the supernatant. Wash the centrifuged product 3 times with PBS buffer. Then centrifuge at 8000 g for 5 min. After washing the centrifuged product with PBS buffer, resuspend the centrifuged product in the first resuspension solution to 40 ml to obtain bacterial solution.

[0099] (4) Place 2 ml of the bacterial culture obtained in step (3) and 28 ml of culture medium into a quartz reactor. Seal the reactor with a rubber gasket cover and wrap it with tin foil to create a dark environment. Then, aerate the reactor with argon gas for 5 minutes to create an anaerobic environment. Then, use a nine-link magnetic stirrer to thoroughly mix the reactants in the quartz reactor. The magnetic stirring speed is 200 rpm. After the reactants are thoroughly mixed, turn on the LED blue light (wavelength 400-450 nm) to irradiate the reactants with a light intensity of 4.0 mw / cm2 to carry out the photo-controlled metabolic reaction. Detect the NO3-N concentration in the reactants when it reaches 142.83 mg NO3-N·L. -1 The photocontrolled metabolic reaction was stopped. The product was then thoroughly shaken and centrifuged at 4000g for 15 min at 4°C, and the first supernatant was collected. The centrifuged product was resuspended in 5 ml of 5wt% NaCl aqueous solution and stirred evenly using a vortex mixer at 24°C. The mixture was then heated in a water bath at 60°C for 3 min, and stirred again using a vortex mixer at 24°C. The mixture was then centrifuged at 4000g for 15 min at 4°C, and the second supernatant was collected. The product was resuspended in 5 ml of 5wt% NaCl aqueous solution and stirred evenly using a vortex mixer at 24°C. The mixture was then heated in a water bath at 60°C for 30 min, and stirred again using a vortex mixer at 24°C. The mixture was then centrifuged at 10000g for 15 min at 4°C, and the third supernatant was collected. Equal volumes of the first, second, and third supernatants were mixed to obtain the photocontrolled extracellular polymer solution.

[0100] The culture medium contains 500 mg N·L -1 NaNO3, 1000 mg C·L -1 NaAc, 10.66mMNH4Cl2, 17.93mM KH2PO4, 32.76mM Na2HPO4·12H2O, 0.41mM MgSO4·7H2O, 9.6mM EDTA·2Na, 9.0mM FeCl3·6H2O, 0.1mM MnCl2·4H2O, 1.0mM NaMoO4·2H2O, 0.8mM CuCl2·2H2O, 2.5mM ZnCl2.

[0101] Example 3

[0102] This embodiment provides a method for preparing a light-controlled extracellular polymer solution, comprising the following steps:

[0103] (1) Place 200g of activated sludge in a sequencing batch reactor and introduce 5000g of first nitrate wastewater over a 7min influent time. Perform an anoxic reaction for 380min, followed by 15min settling and 4min drainage to discharge the wastewater, thereby obtaining acclimated activated sludge. Store the acclimated activated sludge at 4℃ for later use. The first nitrate wastewater contains 5wt% NaCl, 150g / L KH2PO4, 50g / L KHCO3, 30g / L CaCO3, 20g / L MgSO4·7H2O, 1ml / L of first trace aqueous solution, and 1ml / L of second trace aqueous solution.

[0104] The first trace aqueous solution contains 5 g / L EDTA·2Na and 5 g / L FeSO4·7H2O; the second trace aqueous solution contains 10 g / L EDTA·2Na, 0.014 g / L H3BO3, 0.43 g / L ZnSO4·7H2O, 0.24 g / L CoCl2·6H2O, 0.99 g / L MnCl2·4H2O, 0.16 g / L CuSO4, 0.19 g / L NiCl2·6H2O, and 0.22 g / L Na2MoO4·2H2O.

[0105] (2) Place 5g of the acclimatized activated sludge prepared in step (1) and 100g of the second nitrate wastewater in a constant temperature shaker and activate it for 27h at 37℃ and 200rpm to obtain activated activated sludge; wherein, the second nitrate wastewater contains 500mg N·L -1 NaNO3, 1000 mg C·L -1NaAc, 10.66mM NH4Cl2, 17.93mM KH2PO4, 32.76mM Na2HPO4·12H2O, 0.41mM MgSO4·7H2O, 9.6mM EDTA·2Na, 9.0mM FeCl3·6H2O, 0.1mMnCl2·4H2O, 1.0mM NaMoO4·2H2O, 0.8mM CuCl2·2H2O, 2.5mM ZnCl2, 1.0mM NaMoO4·2H2O, 0.8mM CuCl2·2H2O, 2.5mM ZnCl2, 9.6mM EDTA·2Na, 9.0mM FeCl3·6H2O, 0.1mM MnCl2·4H2O, 1.0mM NaMoO4·2H2O、0.8mM CuCl2·2H2O, 2.5mM ZnCl2;

[0106] (3) Centrifuge 60ml of the activated sludge prepared in step (2) at 8000g for 5min and discard the supernatant. Wash the centrifuged product 3 times with PBS buffer. Then centrifuge at 8000g for 5min. After washing the centrifuged product with PBS buffer, resuspend the centrifuged product in the first resuspension solution to 60ml to obtain bacterial solution.

[0107] (4) Place 4 ml of the bacterial culture obtained in step (3) and 24 ml of the culture medium into a quartz reactor. Seal the reactor with a rubber gasket cover and wrap it with tin foil to create a dark environment. Then, aerate the reactor with argon gas for 5 minutes to create an anaerobic environment. Next, use a nine-link magnetic stirrer to thoroughly mix the reactants in the quartz reactor. The magnetic stirring speed is 200 rpm. After the reactants are thoroughly mixed, turn on the LED blue light (wavelength 400-450 nm) at 6 mw / cm². 2 The reactants were irradiated with light of high intensity to carry out a photocontrolled metabolic reaction. The concentration of NO3-N in the reactants was measured to be 142.83 mg NO3-N·L. -1The photocontrolled metabolic reaction was stopped. The product was then thoroughly shaken and centrifuged at 4000g for 15 min at 4°C, and the first supernatant was collected. The centrifuged product was resuspended in 5 ml of 5wt% NaCl aqueous solution and stirred evenly using a vortex mixer at 20°C. The mixture was then heated in a water bath at 60°C for 3 min, and stirred again using a vortex mixer at 20°C. The mixture was then centrifuged at 4000g for 15 min at 4°C, and the second supernatant was collected. The product was resuspended in 5 ml of 5wt% NaCl aqueous solution and stirred evenly using a vortex mixer at 20°C. The mixture was then heated in a water bath at 60°C for 30 min, and stirred again using a vortex mixer at 20°C. The mixture was then centrifuged at 10000g for 15 min at 4°C, and the third supernatant was collected. Equal volumes of the first, second, and third supernatants were mixed to obtain the photocontrolled extracellular polymer solution.

[0108] The culture medium contains 500 mg N·L -1 NaNO3, 1000 mg C·L -1 NaAc, 10.66mMNH4Cl2, 17.93mM KH2PO4, 32.76mM Na2HPO4·12H2O, 0.41mM MgSO4·7H2O, 9.6mM EDTA·2Na, 9.0mM FeCl3·6H2O, 0.1mM MnCl2·4H2O, 1.0mM NaMoO4·2H2O, 0.8mM CuCl2·2H2O, 2.5mMZnCl2.

[0109] Comparative Example 1

[0110] This comparative example provides a method for preparing an extracellular polymer solution, which is similar to the preparation method in Example 1, except that there is no blue light irradiation in step (4).

[0111] Comparative Example 2

[0112] This comparative example provides a method for preparing a light-controlled extracellular polymer solution, which is similar to the preparation method in Example 1, except that the blue light irradiation in step (4) is replaced with yellow light irradiation of equal intensity, and the wavelength of the yellow light is 589 nm.

[0113] Test case

[0114] 1. The protein concentration in the first, second, and third supernatants prepared in Example 1, Comparative Example 1, and Comparative Example 2 was tested using a BCA protein concentration assay kit. The results are as follows: Figure 1 As shown in the figure, the bar chart from top to bottom represents the protein concentrations in the first supernatant, the second supernatant, and the third supernatant.

[0115] 2. Catalytic denitrification test

[0116] The denitrification efficiency of the extracellular polymer solutions prepared in Example 1, Comparative Examples 1 and 2 was tested using the following steps:

[0117] (1) 5g of Paracoccus denitrificans and 100g of secondary nitrate wastewater were placed in a constant temperature shaker and activated for 27h at 37℃ and 200rpm to obtain activated sludge; wherein, the secondary nitrate wastewater contained 500mg N·L -1 NaNO3, 1000 mg C·L -1 NaAc, 10.66mM NH4Cl2, 17.93mM KH2PO4, 32.76mM Na2HPO4·12H2O, 0.41mM MgSO4·7H2O, 9.6mM EDTA·2Na, 9.0mM FeCl3·6H2O, 0.1mM MnCl2·4H2O, 1.0mM NaMoO4·2H2O, 0.8mM CuCl2·2H2O, 2.5mM ZnCl2; 9.6mM EDTA·2Na, 9.0mM FeCl3·6H2O, 0.1mM MnCl2·4H2O, 1.0mM NaMoO4·2H2O, 0.8mM CuCl2·2H2O, 2.5mM ZnCl2;

[0118] (2) Centrifuge 40 ml of the denitrified paracoccus prepared in step (1) at 8000 g for 5 min and discard the supernatant. Wash the centrifuged product 3 times with PBS buffer. Then centrifuge at 8000 g for 5 min. After washing the centrifuged product with PBS buffer, resuspend the centrifuged product in the first resuspension solution to 25 ml to obtain bacterial solution.

[0119] (3) Place 1 ml of the bacterial culture prepared in step (2), 1 ml of the extracellular polymer solution and 28 ml of the culture medium into a quartz reactor. Seal the reactor with a rubber gasket cover and wrap it with tin foil to create a dark environment. Then, argon gas is introduced through a needle for 5 min to create an anaerobic environment. Then, the reactants in the quartz reactor are thoroughly mixed with a nine-link magnetic stirrer. The magnetic stirring speed is 200 rpm. After the reactants are thoroughly mixed, the denitrification reaction is carried out in the dark for 30 h. Samples are taken with a syringe at 0 h, 22 h and 24 h after the start of the reaction. The samples are filtered through a 22 μm filter membrane and the NO3-N concentration is determined by ion chromatography and converted into the removal rate (removal rate = 1 - (NO3-N concentration at the time of test / original NO3-N concentration)). The removal rate results are shown in Table 1.

[0120] Table 1

[0121]

[0122]

[0123] 3. Metatranscriptome sequencing

[0124] The light-controlled extracellular polymer solutions prepared in Example 1 and Comparative Example 2 were tested by metatranscriptome sequencing, and the results are as follows: Figure 2 , 3 As shown, where Figure 2 The terms Streptomycin biosynthesis, RIG-like receptor signaling pathway, Galactosine metabolism, Apoptosis-fly, Folate biosynthesis, Carbapenem biosynthesis, Pyrimidine metabolism, Enzymes with EC numbers, Porphyrin metabolism, Ecopolysaccharide biosynthesis, Sulfur relay system, Terpenoid backbone biosynthesis, Secretion system, and Energy metabolism refer to streptomycin biosynthesis, RIG-like receptor signaling pathway, galactose metabolism, apoptosis-fly, folic acid biosynthesis, carbapenem biosynthesis, pyrimidine metabolism, enzymes with EC numbers, porphyrin metabolism, ecopolysaccharide biosynthesis, secretion system, and energy metabolism, respectively. Figure 3The following pathways are identified: sulfur metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, cardiac muscle contraction, chlorroalkane and chloroalkene degradation, limonene and pinenedearadation, biosynthesis of ansamycins, ascorbate and aldarate metabolism, glycocaproic acid and pinenedearadation, and retrograde endocannabinoid signaling.

[0125] Metatranscriptomics revealed changes in metabolic pathway expression induced by yellow light irradiation. Figure 3 As shown in the diagram, the average expression level of the yellow light signaling pathway in the signaling gene panel is low, about 1 / 5 of that of the blue light pathway. The enrichment level is low, and the metabolic pathways related to cofactors and other active substances are not major pathways. Therefore, they cannot play a biocatalytic role with the extracellular polymers generated by blue light irradiation in Example 1.

[0126] Analysis of the catalytic denitrification tests and metatranscriptome results in Example 1 and Comparative Examples 1 and 2 reveals that different light irradiations can directionally regulate and redirect the metabolic flux of the microbial community. Blue light can allocate the metabolic flux of the microbial community to the pathways for synthesizing bioactive substances, thereby converting nitrate into high-value bioactive substances, achieving simultaneous nitrate removal and bioactive substance production, and directly enhancing biological denitrification using bioactive substances. Yellow light, on the other hand, allocates the metabolic flux to central metabolites, reducing the allocation of metabolic flux to bioactive substances; therefore, the extracellular polymers produced under yellow light are unlikely to exert effective biocatalytic activity.

[0127] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a photo-controllable extracellular polymer solution, characterized by, The method comprises the following steps: (1) domesticating activated sludge by using first nitrate wastewater to obtain domesticated activated sludge; (2) activating the domesticated activated sludge by using second nitrate wastewater to obtain activated activated sludge; (3) extracting bacteria from the activated activated sludge, and then performing light-controlled metabolic reaction under blue light irradiation in a culture solution, and extracting active substances after the reaction to obtain the light-controlled extracellular polymer solution.

2. The production method according to claim 1, characterized by, The first nitrate wastewater comprises 10-11 g / L NaNO3, 6-8 g / L NaAc, 1-6 wt% NaCl, 0.7-0.75 g / L KH2PO4, 0.05-0.15 g / L KHCO3, 0.02-0.04 g / L CaCO3, 0.01-0.03 g / L MgSO4·7H2O, 0.8-1.2 ml / L first trace aqueous solution, and 0.8-1.2 ml / L second trace aqueous solution. The first trace aqueous solution comprises 4-6 g / L EDTA·2Na and 4-6 g / L FeSO4·7H2O. The second trace aqueous solution comprises 9-11 g / L EDTA·2Na, 0.012-0.016 g / L H3BO3, 0.4-0.6 g / L ZnSO4·7H2O, 0.2-0.3 g / L CoCl2·6H2O, 0.9-1 g / L MnCl2·4H2O, 0.12-0.2 g / L CuSO4, 0.15-0.2 g / L NiCl2·6H2O, and 0.2-0.3 g / L Na2MoO4·2H2O.

3. The preparation method according to claim 1, characterized in that, The domesticated activated sludge is obtained by using a sequencing batch activated sludge process.

4. The production method according to claim 3, characterized by, In the sequencing batch activated sludge process, the water inlet time is 6-8 min, the anoxic reaction time is 350-400 min, the sedimentation time is 10-20 min, and the water discharge time is 3-6 min.

5. The method of claim 1, wherein, The second nitrate wastewater comprises 50-1000 mg N / L nitrogen source, carbon source, 10-11 mM NH4Cl, 17-18 mM KH2PO4, 32-33 mM Na2HPO4·12H2O, 0.4-0.5 mM MgSO4·7H2O, 9-10 mM EDTA·2Na, 8-10 mM FeCl3·6H2O, 0.05-0.2 mM MnCl2·4H2O, 0.5-1.5 mM NaMoO4·2H2O, 0.5-1 mM CuCl2·2H2O, and 2-3 mM ZnCl2.

6. The preparation method according to claim 5, characterized in that, The nitrogen source is selected from NaNO3.

7. The preparation method according to claim 5, characterized in that, The carbon source is selected from NaAc.

8. The method of claim 1, wherein, The temperature of the activation process is 25-37°C, the rotation speed is 100-250 rpm, and the time is 20-27 h.

9. The method of claim 1, wherein, The process of extracting the bacteria from the activated activated sludge comprises the following steps: the activated activated sludge obtained in step (2) is subjected to first centrifugal separation, and the first centrifugal separation product is washed and then subjected to second centrifugal separation, and then resuspended by adding a first resuspension solution to obtain a bacterial solution; The first resuspension solution comprises 10-11 mM NH4Cl, 17-18 mM KH2PO4, 32-33 mM Na2HPO4·12H2O, and 0.4-0.5 mM MgSO4·7H2O.

10. The method of claim 9, wherein, The first centrifugal separation is performed at a centrifugal force of 7000-9000g for 4-6 min.

11. The preparation method according to claim 9, characterized in that, The second centrifugal separation is performed at a centrifugal force of 7000-9000g for 4-6 min.

12. The method of claim 9, wherein, The washing solution used in the washing step is a PBS buffer solution.

13. The preparation method according to claim 9, characterized in that, The volume of the bacterial solution is the same as that of the activated activated sludge before the first centrifugal separation.

14. The method of claim 1, wherein, The light-controlled metabolic reaction is performed by mixing the bacterial solution with a culture solution under anaerobic conditions.

15. The method of claim 1, wherein, The culture solution comprises 50-1000 mg N / L of a nitrogen source, a carbon source, 10-11 mM NH4Cl, 17-18 mM KH2PO4, 32-33 mM Na2HPO4·12H2O, and 0.4-0.5 mM MgSO4·7H2O.

16. The method of claim 15, wherein, The nitrogen source is selected from NaNO3.

17. The preparation method according to claim 15, characterized in that, The carbon source is selected from NaAc.

18. The method of claim 14, wherein, The volume ratio of the culture solution to the bacterial solution is (6-30):

1.

19. The method of claim 1, wherein, The light intensity of blue light in the light-controlled metabolic reaction is 1.0-6.0 mw / cm 2 The temperature of the light-controlled metabolic reaction is 25-35℃.

20. The method of claim 14, wherein, When the NO3-N concentration in the culture solution was 100-150 mg NO3-N•L -1 , the light-controlled metabolic reaction was stopped.

21. The method of claim 1, wherein, The process of extracting the active substance in step (3) comprises the following steps: The product after the light-controlled metabolic reaction is shaken and then subjected to third centrifugal separation, the first supernatant is collected, the third separation product is resuspended using a second resuspension solution, and then subjected to first stirring, first water bath heating, second stirring, fourth centrifugal separation, the second supernatant is collected, the fourth separation product is resuspended using a third resuspension solution, and then subjected to third stirring, second water bath heating, fourth stirring, and fifth centrifugal separation, the third supernatant is collected, and the first supernatant, the second supernatant, and the third supernatant are mixed to obtain the active substance solution, i.e., the light-controlled extracellular polymer solution.

22. The method of claim 21, wherein, The volume ratio of the light-controlled metabolic reaction system, the second resuspension solution, and the third resuspension solution is (25-35):(4-6):(4-6).

23. The preparation method according to claim 21, characterized in that, The third centrifugal separation is performed at a temperature of 3-6℃, a centrifugal force of 3000-5000g, and for 10-20 min.

24. The method of claim 21, wherein, The first stirring is uniform stirring using vortex stirring, and the stirring temperature is 20-25℃.

25. The method of claim 21, wherein, The first water bath heating is performed at a temperature of 50-70℃ for 2-4 min.

26. The method of claim 21, wherein, The second stirring is uniform stirring using vortex stirring, and the stirring temperature is 20-25℃.

27. The method of claim 21, wherein, The fourth centrifugal separation is performed at a temperature of 3-6℃, a centrifugal force of 3000-5000g, and for 10-20 min.

28. The method of claim 21, wherein, The third stirring is uniform stirring using vortex stirring, and the stirring temperature is 20-25℃.

29. The method of claim 21, wherein, The second water bath heating is performed at a temperature of 50-70℃ for 20-40 min.

30. The method of claim 21, wherein, The fourth stirring is uniform stirring using vortex stirring, and the stirring temperature is 20-25℃.

31. The method of claim 21, wherein, The temperature of the fifth centrifugal separation is 3-6℃, the centrifugal force is 9000-11000g, and the time is 10-20min.

32. The method of claim 21, wherein, The second resuspension is a 1-6wt% NaCl aqueous solution.

33. The method of claim 21, wherein the method is carried out at a temperature of about 20°C to about 30°C. The third resuspension is a 1-6wt% NaCl aqueous solution.

34. The method of claim 21, wherein, The volume ratio of the first supernatant, the second supernatant, and the third supernatant in the light-controlled extracellular polymer solution is (0.9-1.2):(0.9-1.2):(0.9-1.2).

35. A photo-controllable extracellular polymer solution, characterized by, The light-controlled extracellular polymer solution is prepared by the preparation method of any one of claims 1-34.

36. A method of denitrification, characterized by, The light-controlled extracellular polymer solution prepared by the preparation method of any one of claims 1-34 is mixed with denitrifying bacteria and a solution to be denitrified, and then a denitrification reaction is performed.

37. The method of claim 36, wherein, The concentration of the light-controlled extracellular polymer solution in the solution after the light-controlled extracellular polymer solution prepared by the preparation method of any one of claims 1-34 is mixed with denitrifying bacteria and a solution to be denitrified is 0.5-5 ml / 100ml.

Images