A device and method for culturing anaerobic ammonia oxidizing bacteria

Abstract

The present invention discloses a novel device for culturing and proliferating anaerobic ammonia-oxidizing bacteria, including a reactor, a water inlet mechanism, a negative pressure forming mechanism, a low-voltage power supply, an anode, a cathode, a constant temperature mechanism and a water outlet mechanism. The reactor is provided with a reaction tank body and a water bath from the inside to the outside, the water bath is wrapped around the outside of the reaction tank body, a containing chamber is provided in the reaction tank body, the water inlet mechanism is connected to the bottom of the containing chamber, the water outlet mechanism is connected to the upper part of the containing chamber, the negative pressure forming mechanism is installed above the reactor and is connected to the containing chamber, and is used to form a negative pressure environment in the containing chamber, the anode and the cathode are symmetrically installed in the containing chamber, and are respectively electrically connected to the positive and negative poles of the low-voltage power supply, and the constant temperature mechanism is connected to the water bath, and the water in the water bath is circulated and kept warm. The present invention adopts a novel device that combines a specific negative pressure with a low voltage and a specific reaction tank body, and cooperates with a culturing method to achieve rapid recovery of the activity of anaerobic ammonia-oxidizing sludge and promote the proliferation of anaerobic ammonia-oxidizing bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of anaerobic ammonia oxidizing bacteria cultivation, and in particular to a novel device and a cultivation method for culturing and proliferating anaerobic ammonia oxidizing bacteria. Background Art

[0002] A large amount of ammonia nitrogen wastewater is generated in aquaculture, production and life. Excessive ammonia nitrogen wastewater discharged into the water will lead to eutrophication of the water, resulting in a decrease in dissolved oxygen in the water, a large number of aquatic organisms dying, and a foul odor. It not only causes great harm to the environment, but also reduces the ornamental value of the water body. The nitrates and nitrites generated by oxidation will also affect aquatic organisms and even endanger human health. Therefore, it is very necessary to denitrify the wastewater.

[0003] Anaerobic ammonium oxidation is an emerging denitrification process that uses nitric oxide (NO) and hydrazine (N2H4) as intermediates to produce NH4 + As an electron donor, NO2 - As an electron acceptor, it generates N 2 , and N 2 Anaerobic ammonium oxidation has many obvious advantages over traditional denitrification processes. However, the anaerobic ammonium oxidation process is still not widely used because it is restricted by the slow proliferation of anaerobic ammonium oxidizing bacteria and the culture environment is restricted by many factors.

[0004] Patent application number: 201720597197.0, patent name: invention patent for device for enriching and culturing anaerobic ammonia-oxidizing bacteria, discloses a device for enriching and culturing anaerobic ammonia-oxidizing bacteria, including a culture chamber, a deoxygenation mechanism, a filter membrane assembly and an aeration component, the aeration component is located at the bottom of the culture chamber, the deoxygenation mechanism is connected to the aeration component, and is used to pass mixed gas into the culture chamber through the aeration component, and an air outlet is provided at the top of the culture chamber to discharge the gas in the culture chamber. However, this technical solution cannot solve the impact of incoming water on granular sludge, granular sludge is easy to float, sludge loss is obvious, and it has the disadvantages of requiring aeration, long culture cycle, and slow recovery of bacterial activity. Summary of the invention

[0005] The object of the present invention is to provide a novel device and method for culturing the proliferation of anaerobic ammonia-oxidizing bacteria.

[0006] According to one aspect of the present invention, there is provided a novel device for culturing and proliferating anaerobic ammonia-oxidizing bacteria, comprising a reactor, a water inlet mechanism, a negative pressure forming mechanism, a low-voltage power supply, an anode, a cathode, a constant temperature mechanism and a water outlet mechanism. The reactor is provided with a reaction tank body and a water bath from the inside to the outside, the water bath is wrapped around the outside of the reaction tank body, a containing chamber is provided in the reaction tank body, the water inlet mechanism is connected to the bottom of the containing chamber, the water outlet mechanism is connected to the upper part of the containing chamber, the negative pressure forming mechanism is installed above the reactor and is connected to the containing chamber to form a negative pressure environment in the containing chamber, the anode and the cathode are symmetrically installed in the containing chamber and are electrically connected to the positive and negative poles of the low-voltage power supply respectively, the constant temperature mechanism is connected to the water bath to circulate and keep the water in the water bath warm.

[0007] In some embodiments, the negative pressure forming mechanism includes a negative pressure power supply, a power timer, a vacuum pump and a one-way vent valve. The one-way vent valve is installed on the top of the reaction tank body and is connected to the accommodating chamber. The vacuum pump is connected to the one-way vent valve. The negative pressure power supply, the power timer and the vacuum pump are electrically connected in sequence.

[0008] In some embodiments, the water inlet mechanism includes a water inlet tank and a water inlet pump, a water inlet is provided at the bottom of the reaction tank body, the water inlet is connected to the bottom of the accommodating chamber, one end of the water inlet pump is connected to the water inlet pump, and the other end is connected to the water inlet, the water outlet mechanism includes a water outlet pump and a tail water collecting tank, a water outlet is provided at the upper part of the reaction tank body, the water outlet is connected to the accommodating chamber, one end of the water outlet pump is connected to the water outlet, and the other end is connected to the tail water collecting tank.

[0009] In some embodiments, the constant temperature mechanism includes a constant temperature water bath and a circulating pump, a circulating water outlet is provided at the top of the water bath, a circulating water inlet is provided at the bottom, the constant temperature water bath is connected to the circulating water outlet pipe, one end of the circulating pump is connected to the constant temperature water bath, and the other end is connected to the circulating water inlet.

[0010] The present invention also includes a reflux pump. A reflux port is also provided on the upper part of the reaction tank body. The reflux port is connected to the accommodating chamber and is opposite to the water outlet. One end of the reflux pump is connected to the reflux port, and the other end is connected to the water inlet. The reactor is also provided with an insulation layer, which is wrapped around the outside of the water bath.

[0011] In some embodiments, the anode and the cathode are both mesh titanium sheets, the water inlet pump, the water outlet pump and the reflux pump are all peristaltic pumps, and the voltage of the low-voltage power supply is constant at 1V.

[0012] According to another aspect of the present invention, there is provided a method for culturing anaerobic ammonia-oxidizing bacteria, comprising the following steps:

[0013] Step a, pretreatment of bacterial particles: the anaerobic ammonium oxidation sludge to be inoculated is washed with PBS buffer solution, and after the bacterial particles are precipitated, the upper solution is poured out, and the process is repeated three times to wash away the surface residue of the anaerobic ammonium oxidation granular sludge;

[0014] Step b, inoculation: the washed anaerobic ammonium oxidation sludge is inoculated into the containing chamber of the reaction tank. The MLSS value and MLVSS value of the anaerobic ammonium oxidation sludge are 5.12 g·L -1 and 4.74 g·L -1 , the inoculation volume of anaerobic ammonium oxidation sludge is 30% of the volume of the holding chamber;

[0015] Step c, preparing simulated waste liquid: using 5L of waste water as a basic waste liquid, the basic waste liquid contains KH2PO4, CaCl2·2H2O, MgSO4·7H2O and KHCO3, adding trace element composition I solution and trace element composition II solution to the basic waste liquid, then adding ammonium salt and nitrite to the basic waste liquid, and adjusting the pH value to between 7.5 and 8.0 with a hydrochloric acid solution with a molar concentration of 1 mol / L to prepare an initial simulated waste liquid; during the proliferation culture process, the ammonia nitrogen concentration in the initial simulated waste liquid is 20 mg / L and the nitrite nitrogen concentration is 26 mg / L, and new simulated waste liquid is prepared every day, and the ammonia nitrogen concentration in the simulated waste liquid is increased by 20 mg / L every 7 days, and the nitrite nitrogen concentration in the simulated waste liquid is 1.32 times the ammonia nitrogen concentration at that time; when the ammonia nitrogen concentration reaches 180 mg / L and the nitrite nitrogen concentration is 200 mg / L, the ammonia nitrogen concentration and the nitrite nitrogen concentration in the simulated waste liquid no longer increase;

[0016] Step d, water inlet: add the prepared simulated waste liquid into the water inlet tank, and pump it into the reaction tank body through the water inlet pump, the water inlet flow rate is 8 ml / min, the hydraulic retention time is 1 day, and the simulated waste liquid does not need to be treated with nitrogen blowing and deoxygenation;

[0017] Step e, start constant temperature culture: add enough water to the constant temperature water bath, turn on the constant temperature water bath, set the water bath temperature to 32°C, start the circulating water pump to pump the warm water in the constant temperature water bath into the water bath, and the water bath will be automatically drained after it is full, and the water circulation of the constant temperature water bath, the circulating water pump and the water bath is used to achieve the heat preservation of the reaction tank;

[0018] Step f, applying negative pressure and coupling low-voltage direct current: turning on the power timer and the vacuum pump to evacuate the interior of the reaction tank, controlling the air pressure in the accommodating chamber at about -0.04 MPa, and setting the power timer to power the vacuum pump every hour for half an hour each time; starting the low-voltage power supply, setting its voltage intensity to be constant at 1V, and applying low-voltage direct current to the simulated waste liquid in the reaction tank through the anode and cathode titanium plates; when the water inflow volume reaches 5L for the first time, starting the reflux pump for reflux, and the reflux ratio is 2;

[0019] Step g, water discharge: start the water discharge pump to discharge the supernatant in the reaction tank into the tail water collection tank, and the water discharge flow rate is 8 ml / min;

[0020] Step h, detection: sampling and testing of influent water samples and effluent water samples are carried out regularly every day to detect the concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, and the total nitrogen concentration is obtained by the sum of the three. Water samples are taken out every day to determine the dissolved oxygen DO, pH and COD, and the operation status of the reactor is observed.

[0021] In some embodiments, in step c, trace element composition I solution and trace element composition II solution are added to the basic waste liquid at an addition concentration of 1 mL / L, respectively. The trace element composition I solution is an aqueous solution of EDTA and FeSO4·7H2O, wherein the concentrations of EDTA and FeSO4·7H2O are both 5 g / L.

[0022] In some embodiments, the concentration of KH2PO4 in the basic waste liquid of step c is 20 mg / L, the concentration of CaCl2·2H2O is 107 mg / L, the concentration of MgSO4·7H2O is 45 mg / L, the concentration of KHCO3 is 1200 mg / L, the ammonium salt is ammonium sulfate, and the nitrite is sodium nitrite.

[0023] In some embodiments, the trace element composition II solution is an aqueous solution of EDTA, H3BO3, MnCl2·4H2O, CuSO4·5H2O, ZnSO4·7H2O, NiCl2·6H2O, Na2MoO4·2H2O and CoCl2·6H2O, wherein the concentration of EDTA is 15 g / L, the concentration of H3BO3 is 0.014 g / L, the concentration of MnCl2·4H2O is 0.99 g / L, the concentration of CuSO4·5H2O is 0.25 g / L, the concentration of ZnSO4·7H2O is 0.43 g / L, the concentration of NiCl2·6H2O is 0.19 g / L, the concentration of Na2MoO4·2H2O is 0.22 g / L, and the concentration of CoCl2·6H2O is 0.24 g / L.

[0024] The beneficial effects of the present invention are as follows: (1) The present invention develops a device and a culture method for rapidly increasing the growth rate of anaerobic ammonia-oxidizing bacteria and improving the anaerobic ammonia-oxidizing activity by coupling negative pressure with low-voltage direct current. The device is used to apply a negative pressure environment without the need to set up a three-phase separator. During the culture process, a nearly sealed specific negative pressure environment is formed in the reaction tank, thereby increasing the activation rate of the anaerobic ammonia-oxidizing bacteria, greatly reducing the loss of anaerobic ammonia-oxidizing bacterial biomass, and achieving efficient denitrification treatment of waste liquid;

[0025] (2) The new anaerobic ammonium oxidizing bacteria culture device established by the present invention uses a mesh titanium sheet as an electrode, which promotes the growth of anaerobic ammonium oxidizing bacteria without generating excess metal ions to inhibit bacterial growth, and can avoid electrode loss;

[0026] (3) The anaerobic ammonia-oxidizing bacteria culture device established by the present invention can achieve stable mass transfer and has the characteristics of low shear force compared to the SBR reactor, ensuring that the anaerobic ammonia-oxidizing bacteria particles can be stably formed, and the specific negative pressure conditions can effectively reduce the cavity structure inside the bacteria particles and effectively reduce the phenomenon of particle floating;

[0027] (4) The anaerobic ammonium oxidation culture device established by the present invention improves the abundance of anaerobic ammonium oxidizing bacteria while efficiently removing nitrogen, improves the ability of anaerobic ammonium oxidizing bacteria to cope with water quality fluctuations, and reduces the inhibitory effect of high concentrations of nitrite nitrogen on bacteria, while enhancing the stability of the operation of the anaerobic ammonium oxidation system under the impact of water quality changes. Therefore, the present invention adopts a new device that couples low-voltage direct current with specific negative pressure and combines it with a specific reaction tank body, and cooperates with the corresponding culture method to achieve rapid recovery of the activity of anaerobic ammonium oxidation sludge, promote the proliferation of anaerobic ammonium oxidizing bacteria, shorten the proliferation cycle, and increase the system's retained biomass, thereby achieving rapid denitrification of waste liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The schematic diagram is a structural diagram of a novel device for culturing and multiplying anaerobic ammonia-oxidizing bacteria according to the present invention.

[0029] Figure 2 This is a diagram showing the effect of different electrodes on the total nitrogen concentration in the effluent water according to Example 3 of the present invention.

[0030] Figure 3 This is a graph showing the effect of different vacuum intensities on the total nitrogen concentration in the effluent and the nitrogen removal rate in Example 4 of the present invention.

[0031] Figure 4 This is a diagram of the physical state of the anaerobic ammonium oxidation granular sludge of group 1 treated in Example 5 of the present invention.

[0032] Figure 5 This is a diagram of the microbial abundance of treatment group 1 and treatment group 4 in Example 5 of the present invention.

[0033] Figure 6 This is a comparison chart of ammonia nitrogen concentration and specific activity of treatment groups 1-4 and the control group in Example 5 of the present invention. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below in conjunction with the embodiments.

[0035] Example 1

[0036] The novel device for culturing and proliferating anaerobic ammonia-oxidizing bacteria of the present invention comprises a reactor 1, a water inlet mechanism, a negative pressure forming mechanism, a low-voltage power supply 4, an anode 5, a cathode 6, a constant temperature mechanism, a water outlet mechanism and a reflux pump 9, as shown in the attached Figure 1 shown.

[0037] The reactor 1 is provided with a reaction tank body 11, a water bath 12, an insulation layer 13 and a support frame 14. The water bath 12 is wrapped around the outside of the reaction tank body 11, and the insulation layer 13 is wrapped around the outside of the water bath 12. The support frame 14 is located at the bottom of the insulation layer 13, and is used to support and place the entire reactor 14. A accommodating chamber 111 is provided in the reaction tank body 11, and a water inlet is provided at the bottom, and a water outlet and a reflux port are provided at the top, and the reflux port is opposite to the water outlet. The water inlet, the water outlet and the reflux port are all connected to the accommodating chamber 111. The effective height of the accommodating chamber 111 is 47 cm and the effective volume is 5 L. The bottom of the accommodating chamber 111 is inverted cone shape, and the inclination angle of the bottom side wall is 45°. The upper part of the accommodating chamber 111 is cylindrical, and the inner diameter is 14 cm. The accommodating chamber 111 is used to accommodate anaerobic ammonia oxidation granular sludge for cultivation and proliferation, and to purify the waste liquid.

[0038] The water inlet mechanism includes an inlet tank 21 and an inlet pump 22. One end of the inlet pump 22 is connected to the inlet tank 21, and the other end passes through the insulation layer 13 and the water bath 12 in sequence to be connected to the water inlet, so as to pump the simulated waste liquid in the inlet tank 21 into the containing chamber 111, which is used to multiply and culture the anaerobic ammonia oxidation sludge particles in the reaction tank body 11. One end of the reflux pump 9 passes through the insulation layer 13 and the water bath 12 in sequence to be connected to the reflux port, and the other end is connected to the water inlet. The reflux pump 9 pumps out the unreacted liquid in the upper layer of the reaction tank body 11, and mixes it with the inlet water in a ratio of 2:1 as reflux water and enters the containing chamber 111. The reflux ratio is controlled to be 2:1, which is used to dilute the substrate concentration of the inlet water and alleviate the inhibitory effect of the high substrate concentration in the inlet water on the anaerobic ammonia oxidation bacteria. This arrangement eliminates the need to discharge the anaerobic ammonium oxidation granular sludge, retains the biomass in the containing chamber 111 as much as possible, increases the proliferation rate of the anaerobic ammonium oxidation bacteria, and improves the purification effect of the waste liquid.

[0039] The water outlet mechanism includes a water outlet pump 81 and a tail water collection tank 82. One end of the water outlet pump 81 passes through the insulation layer 13 and the water bath 12 in sequence and is connected to the water outlet, and the other end is connected to the tail water collection tank 82. The supernatant after the anaerobic ammonia-oxidizing bacteria treatment is pumped out of the reaction tank body 11 and stored in the tail water collection tank 82 for subsequent use.

[0040] The negative pressure forming mechanism includes a negative pressure power supply 31, a power timer 32, a vacuum pump 33 and a one-way vent valve 34. The one-way vent valve 34 is installed on the top of the reaction tank body 11 and is connected to the accommodating chamber 111. The vacuum pump 33 is connected to the one-way vent valve 34, and is used to evacuate the reaction tank body 11, so as to form a negative pressure environment of -0.04MPa in the reaction tank body 11. The negative pressure power supply 31, the power timer 32 and the vacuum pump 33 are electrically connected in sequence. The negative pressure power supply 31 is used to power the vacuum pump 33, so as to facilitate the normal operation of the vacuum pump 33. The power timer 32 is used to set the negative pressure power supply 31 to be turned on every 1 hour, and each time it is turned on for half an hour, so as to achieve the purpose of maintaining the air pressure intensity in the accommodating chamber 111 at -0.04MPa.

[0041] The anode 5 and the cathode 6 are both mesh titanium sheets, symmetrically installed in the accommodating cavity 111 and extending below the liquid surface. The anode 5 and the cathode 6 are electrically connected to the positive and negative poles of the low-voltage power supply 4 respectively. The voltage of the low-voltage power supply 4 is a constant voltage of 1V, and low-voltage direct current is provided to the culture system in the reaction tank body 11 through the anode 5 and the cathode 6 to shorten the growth cycle of the anaerobic ammonia-oxidizing bacteria and stimulate the cell growth of the anaerobic ammonia-oxidizing bacteria.

[0042] The constant temperature mechanism includes a constant temperature water bath 71 and a circulation pump 72. The upper part of the water bath 12 is provided with a circulating water outlet, and the lower part is provided with a circulating water inlet. The constant temperature water bath 71 is connected to the circulating water outlet pipe, one end of the circulation pump 72 is connected to the constant temperature water bath 71, and the other end passes through the insulation layer 13 and is connected to the circulating water inlet. The water in the water bath 12 continuously flows into the constant temperature water bath 71 for heating to a constant temperature of 32°C, and is then pumped into the water bath 12 by the circulation pump 72 to achieve insulation of the reaction tank body 11, and is used to maintain the internal temperature of the accommodating chamber 111 at a constant temperature of 32°C, so as to facilitate the proliferation of anaerobic ammonia-oxidizing bacteria in the tank and the purification of the waste liquid.

[0043] Example 2

[0044] A method for culturing anaerobic ammonia-oxidizing bacteria of the present invention adopts the novel device for culturing anaerobic ammonia-oxidizing bacteria of Example 1, comprising the following steps:

[0045] Step a, pretreatment of bacterial particles: the anaerobic ammonium oxidation granular sludge to be inoculated is washed with PBS buffer solution, and after the bacterial particles are precipitated, the upper layer solution is poured out, and the process is repeated three times to wash away the surface residue of the anaerobic ammonium oxidation granular sludge;

[0046] Step b, inoculation: the washed anaerobic ammonium oxidation granular sludge is inoculated into the inverted conical bottom of the containing chamber 111 of the reaction tank 11. The MLSS value and MLVSS value of the anaerobic ammonium oxidation granular sludge are 5.12 g·L -1 and 4.74 g·L -1 The inoculation volume of the anaerobic ammonium oxidation granular sludge is 30% of the effective volume in the reaction tank 11;

[0047] Step c, preparing simulated waste liquid: using 5L of waste water as a basic waste liquid, the basic waste liquid contains KH2PO4, CaCl2·2H2O, MgSO4·7H2O and KHCO3, adding trace element composition I solution and trace element composition II solution to the basic waste liquid, then adding ammonium salt and nitrite to the basic waste liquid, and adjusting the pH value to between 7.5 and 8.0 with a hydrochloric acid solution with a molar concentration of 1 mol / L to prepare an initial simulated waste liquid; during the proliferation culture process, the ammonia nitrogen concentration in the initial simulated waste liquid is 20 mg / L and the nitrite nitrogen concentration is 26 mg / L, and new simulated waste liquid is prepared every day, and the ammonia nitrogen concentration in the simulated waste liquid is increased by 20 mg / L every 7 days, and the nitrite nitrogen concentration in the simulated waste liquid is 1.32 times the ammonia nitrogen concentration at that time; when the ammonia nitrogen concentration reaches 180 mg / L and the nitrite nitrogen concentration is 200 mg / L, the ammonia nitrogen concentration and the nitrite nitrogen concentration in the simulated waste liquid no longer increase;

[0048] Step d, water inlet: add the prepared simulated waste liquid into the water inlet tank 21, and pump it into the reaction tank body 11 through the water inlet pump 22, the water inlet flow rate is 8 ml / min, the hydraulic retention time is 1 day, and there is no need to perform nitrogen blowing and deoxygenation treatment on the simulated waste liquid;

[0049] Step e, start constant temperature culture: add enough water to the constant temperature water bath 71, turn on the constant temperature water bath 71, set the water bath temperature to 32°C, start the circulating water pump 72 to pump the warm water in the constant temperature water bath 71 into the water bath 12, and the water bath 12 will be automatically discharged after it is filled and flow into the constant temperature water bath 71. The water circulation of the constant temperature water bath 71, the circulating water pump 72 and the water bath 12 is used to achieve the heat preservation of the reaction tank body 11, and combined with the heat preservation effect of the heat preservation layer 13, the temperature of the anaerobic ammoniation reaction system in the tank is maintained at 32°C;

[0050] Step f, applying negative pressure and coupling low-voltage direct current: turning on the power timer 32 and the vacuum pump 33, evacuating the interior of the reaction tank body 11, controlling the air pressure in the reaction tank body 11 at -0.04MPa, and setting the power timer 32 to supply power to the vacuum pump 33 every hour for half an hour each time; starting the low-voltage power supply 4, setting its voltage intensity to be constant at 1V, applying low-voltage direct current to the simulated waste liquid in the reaction tank body 11 through the two mesh titanium sheet electrodes, the anode 5 and the cathode 6; when the water is inlet for the first time, when the water volume reaches 5L, starting the reflux pump 9 for reflux, and the reflux ratio is 2:1;

[0051] Step g, water discharge: start the water discharge pump 81 to discharge the supernatant in the reaction tank 11 into the tail water collection tank 82, and the water discharge flow rate is 8 ml / min;

[0052] Step h, detection: sampling and testing of influent water samples and effluent water samples are carried out regularly every day to detect the concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, and the total nitrogen concentration is obtained by the sum of the three. Water samples are taken out every day to determine the dissolved oxygen DO, pH and COD, and the operation status of the reactor is observed.

[0053] Example 3 Effects of different electrode materials on the proliferation and cultivation of anaerobic ammonia-oxidizing bacteria

[0054] In the present embodiment, an electrode treatment group (R2 group) and an electrode control group (R1 group) are set. Both the electrode treatment group and the electrode control group adopt the novel device for culturing the proliferation of anaerobic ammonia-oxidizing bacteria of Example 1, wherein the electrode of the electrode treatment group is a mesh titanium sheet electrode, and the electrode of the electrode control group is a stainless steel electrode. Neither the electrode treatment group nor the electrode control group applies a negative pressure environment. The culturing methods of the electrode treatment group and the electrode control group refer to the culturing method of Example 2, except that the part of applying negative pressure in step f is removed. The operating cycle of the electrode treatment group and the electrode control group is 10 days. Samples of the influent and effluent are taken regularly every day to detect the total nitrogen concentration as an evaluation indicator of the impact on the proliferation and cultivation of anaerobic ammonia-oxidizing bacteria. See the attached for specific test results. Figure 2 The total nitrogen concentration is the sum of the concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen.

[0055] According to the attached Figure 2 It can be directly obtained that the electrode control group R1 always maintains a low effluent total nitrogen concentration, and the effluent total nitrogen concentration has a slight downward trend after the fourth day. The electrode treatment group R2 began to show a trend of gradually decreasing effluent total nitrogen concentration in the first four days. Since the anode of the electrode control group electrolyzes some ferrous ions, it has a certain promoting effect on anaerobic ammonia-oxidizing bacteria. Starting from the fourth day, the effluent total nitrogen concentration gradually increased. On the tenth day after the reaction, the effluent total nitrogen concentration was 22 mg / L. The reason is that stainless steel electrodes are used in the electrode control group R1, and the HRT is maintained at 24h, which leads to Fe in the reactor.2+ When Fe 2+ When the concentration exceeds a certain threshold, it will have a certain inhibitory effect on anaerobic ammonia-oxidizing bacteria.

[0056] According to the results of the proliferation culture experiment, when the voltage intensity is lower than 0.5V, the promoting effect on the growth of anaerobic ammonia oxidizing bacteria in the system is not obvious. When the voltage intensity is higher than 1V, the concentration of ferrous ions produced by electrolysis of stainless steel electrodes will be too high, which will inhibit anaerobic ammonia oxidizing bacteria. Therefore, stainless steel electrodes are not suitable for the cultivation method of anaerobic ammonia oxidizing bacteria proliferation in this application. The mesh titanium sheet electrode used in this application does not have the problems of the above-mentioned stainless steel electrode, so this application chooses to use a mesh titanium sheet electrode, which plays a good role in promoting the proliferation and cultivation of anaerobic ammonia oxidizing bacteria.

[0057] Example 4 Effect of different air pressure conditions on the proliferation and cultivation of anaerobic ammonia-oxidizing bacteria

[0058] This embodiment adopts the novel device for culturing the proliferation of anaerobic ammonia-oxidizing bacteria of Example 1, but no electrodes are provided and no low-voltage direct current is applied. Three treatment cycles are provided, namely, air pressure treatment 1, air pressure treatment 2 and air pressure treatment 3. The treatments are operated at air pressures of 0MPa, -0.03-(-0.04MPa) and -0.05-(-0.06MPa) respectively, and each cycle runs for 12 days. The cultivation methods of the three treatment cycles all refer to the cultivation methods of Example 2, except that the negative pressure applied is different. The influent and effluent are sampled regularly every day, and the total nitrogen concentration is detected as an indicator for judging the effect on the proliferation and cultivation of anaerobic ammonia-oxidizing bacteria. See the attached for specific test results. Figure 3 The total nitrogen concentration is the sum of the concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen.

[0059] According to the attached Figure 3 It can be directly obtained that in the first 12 days, during one cycle of air pressure treatment, the air pressure applied in the reactor was 0, and the culture system of anaerobic ammonia-oxidizing bacteria showed a relatively stable effluent total nitrogen removal rate (NRR), and the total nitrogen in the effluent was low; from the 13th to the 25th day, during two cycles of air pressure treatment, the air pressure applied in the reactor was -0.03-(-0.04MPa), and the culture system still showed a relatively stable denitrification rate, and the total nitrogen removal rate (NRR) value on the 25th day was 95.42%. On the 26th day of the reaction, an air pressure intensity of -0.05-(-0.06MPa) was applied to the reactor. At this time, the total nitrogen effluent concentration (TN eff ) began to increase gradually, and the total nitrogen removal rate began to decrease gradually. At the 34th day of reaction, the system was completely destabilized, and at the 36th day, the nitrogen effluent concentration (TN eff) soared to 51.63 mg / L. It can be seen that within the cycle of air pressure treatment 3, the air pressure intensity of -0.05-(-0.06MPa) has a negative effect on the anaerobic ammonia-oxidizing bacteria in the system. Although the air pressure in the containment chamber is 0 and the air pressure is -0.03MPa in a short period of time, which has no direct promoting effect on the denitrification performance, during the cultivation of anaerobic ammonia-oxidizing granular sludge, due to the existence of negative pressure, the exhaust duct of the anaerobic ammonia-oxidizing granular sludge is not easily blocked, and the nitrogen produced by metabolism will not accumulate in the cavity of the granular sludge, and the sludge will not float, which prevents the sludge particles from being discharged from the reactor with water, thereby improving the biomass retention of the anaerobic ammonia-oxidizing granular sludge in the reactor, which is beneficial to the proliferation of anaerobic ammonia-oxidizing granular sludge.

[0060] Example 5 Analysis of the start-up rate, activity recovery, diversity and specific activity of anaerobic ammonia-oxidizing bacteria in reactors with different structures

[0061] 5.1 Experimental Grouping:

[0062] This embodiment sets up treatment group 1, treatment group 2, treatment group 3, treatment group 4 and control group, and the culture period of each group is 60 days, and the anaerobic ammonium oxidizing granular sludge from the same source is used. Among them, treatment group 1: the new device for culturing and proliferating anaerobic ammonium oxidizing bacteria of embodiment 1 is used (without the three-phase separator), and the culture method of embodiment 2 is referred to;

[0063] Treatment Group 2: The novel device for culturing and proliferating anaerobic ammonia-oxidizing bacteria of the electrode treatment group (R1) in Example 3 (without the three-phase separator) was used, and the culturing method of Example 2 was referred to, but the negative pressure environment was not applied;

[0064] Treatment 3: The novel device for culturing and proliferating anaerobic ammonia-oxidizing bacteria of Example 4 (without the three-phase separator) was used, and the culturing method of Example 2 was referred to, but low-voltage direct current was not applied;

[0065] Treatment 4: The novel device for culturing and proliferating anaerobic ammonia-oxidizing bacteria of Example 1 was used (without the three-phase separator), and the anode, cathode and vacuum pump were removed. The culturing method of Example 2 was used with reference to the method, but low-voltage direct current and negative pressure environment were not applied.

[0066] Control group: A UASB reactor with a three-phase separator in the art was used as the reaction tank body of the control group. Other structural settings were the same as the novel device for culturing and proliferating anaerobic ammonia-oxidizing bacteria in Example 1, and the culturing method in Example 2 was referred to.

[0067] 5.2 Measurement indicators

[0068] The influent and effluent of each treatment group and the control group were sampled regularly every day to detect the total nitrogen concentration, which was used as an evaluation index for the effect on the proliferation and cultivation of anaerobic ammonia-oxidizing bacteria. The total nitrogen concentration was the sum of the concentrations of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen. On the 10th day of cultivation, the effluent ammonia nitrogen concentration and specific activity changes of each treatment group and the control group were measured within the initial six hours. On the 30th day of cultivation, the abundance of microorganisms at the genus level of treatment group 1 and treatment group 4 was measured.

[0069] 5.3 Results Analysis

[0070] 5.3.1 Comparison of the status of anaerobic ammonium oxidation granular sludge under different treatments

[0071] On the 1st to 5th day of cultivation, the color status of the anaerobic ammonium oxidation granular sludge in the reaction tank of each treatment group and the control group was observed, as shown in Table 1 below.

[0072] Table 1 Status of anaerobic ammonium oxidation granular sludge under different treatments

[0073]

[0074]

[0075] As can be seen from Table 1, on the fifth day of cultivation, the color of the bacterial particles in the anaerobic ammonium oxidation granular sludge of treatment 1 in this embodiment is reddish brown as a whole, and the proportion of red bacterial particles reaches more than 95%, indicating that more than 95% of the anaerobic ammonium oxidation bacteria have extremely high denitrification performance and activity at this time, proving that the new device of treatment 1 can achieve the purpose of rapid recovery of the activity of anaerobic ammonium oxidation granular sludge in combination with the corresponding cultivation method. During the same cultivation time, the proportion of red bacterial particles in treatments 2, 3, 4 and the control group was less than 95%, indicating that treatments 2, 3, 4 and the control group could not reach the recovery level of the anaerobic ammonium oxidation granular sludge in treatment 1, so the rate of recovery of the activity of anaerobic ammonium oxidation bacteria in treatments 2, 3, 4 and the control group was slower than that in treatment 1.

[0076] On the fifth day of culture, only a very small amount of bacterial particles were suspended on the liquid surface of treatment 1 and treatment 3 in this embodiment, indicating that the new device of the present application can significantly prevent bacterial particles from floating up by setting a negative pressure forming mechanism and improving the airtightness of the reactor, and applying a negative pressure environment of -0.04MPa to the culture system, effectively achieving the retention of the anaerobic ammonia oxidizing bacteria biomass in the reactor and preventing the bacterial particles from being discharged with the effluent. However, no negative pressure environment was applied to treatments 2 and 4, and the control group could not apply a negative pressure environment due to the poor structural sealing of the UASB reactor, resulting in a high proportion of bacterial particles floating up, and it was impossible to avoid bacterial particle loss, resulting in the loss of anaerobic ammonia oxidizing bacteria biomass in the reactor.

[0077] Therefore, the combination of the novel device and the culture method of the present application can quickly restore the activity of the anaerobic ammonium-oxidizing granular sludge, which is beneficial to the proliferation and biomass retention of anaerobic ammonium-oxidizing bacteria, and further improves its denitrification treatment efficiency for wastewater.

[0078] 5.3.2 Comparison of ammonia nitrogen concentration and specific activity under different treatments

[0079] On the 10th day of cultivation, samples were taken every hour for the first six hours to determine the specific activity data of anaerobic ammonia-oxidizing bacteria and ammonia nitrogen concentration in each treatment group and the control group. Figure 5 The specific activity test of anaerobic ammonia-oxidizing bacteria is carried out by taking an appropriate amount of anaerobic ammonia-oxidizing bacteria in the reactor into a 50mL culture bottle, adding simulated waste liquid with an ammonia nitrogen concentration of 50mg / L, passing nitrogen gas to reduce the dissolved oxygen therein, placing the culture bottle in a shaker to maintain a constant temperature of 32°C, and taking samples every hour to measure the ammonia nitrogen concentration to indicate the denitrification efficiency of the colony.

[0080] According to the attached Figure 5 It can be seen that within the first hour of the 10th day of cultivation, the ammonia nitrogen concentration of treatment group 1 decreased from 62.59 mg / L to 46.43 mg / L, a decrease of 16.15 mg / L, and the specific activity was 4.79 mg·N·g·VSS -1 The ammonia nitrogen concentration of treatment group 4 decreased from 62.59 mg / L to 48.49 mg / L, a decrease of 14.1 mg / L, and the specific activity was 4.28 mg·N·g·VSS -1 The ammonia nitrogen concentration of the control group decreased from 63.62 mg / L to 50.79 mg / L, a decrease of 12.83 mg / L, and the specific activity was 3.86 mg·N·g·VSS -1 .

[0081] After six hours of reaction, the ammonia nitrogen concentration of treatment group 1 was 7.21 mg / L, a decrease of 55.38 mg / L, the ammonia nitrogen concentration of treatment group 4 was 18.23 mg / L, a decrease of 44.36 mg / L, and the ammonia nitrogen concentration of the control group was 19.26 mg / L, a decrease of 44.36 mg / L. The specific activity is related to the substrate concentration. The substrate of treatment group 1 was consumed more. After the substrate concentration decreased, the denitrification rate would decrease. Therefore, the specific activity of treatment group 1 decreased significantly after 3.5 hours. Therefore, treatment group 1 has a higher specific activity than treatment group 2 and the control group.

[0082] 5.3.3 Effects of different treatments on microbial diversity during anaerobic ammonium oxidation

[0083] On the 30th day of cultivation, samples were taken from treatment group 1, treatment group 4 and the control group to analyze the microbial diversity in the reaction tank. The abundance diagram of the microbial diversity genus level of each group is attached. Figure 6 .

[0084] According to the attached Figure 6 It can be seen that the abundance of Candidatus Brocadia in treatment group 1 is 13.7%, while the abundance of Candidatus Brocadia in treatment group 4 is only 4.7%. Candidatus Brocadia is a key fungus in the anaerobic ammonium oxidation process. It plays an important role in the proliferation of anaerobic ammonium oxidizing bacteria. They synthesize nitrosamines (hydrazine) through a specific metabolic pathway, and then cooperate with other bacteria to finally generate nitrogen. In this process, Candidatus Brocadia is a key microorganism for organic ammonia oxidation and nitrosamine reduction. Therefore, the culture system of treatment group 1 has a higher anaerobic ammonium oxidation rate than that of treatment group 2.

[0085] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A method for culturing anaerobic ammonia-oxidizing bacteria, characterized in that: The following steps are involved: Step a, pretreatment of bacterial particles: the anaerobic ammonium oxidation sludge to be inoculated is washed with PBS buffer solution, and after the bacterial particles are precipitated, the upper layer solution is poured out, and the process is repeated three times to wash away the surface residue of the anaerobic ammonium oxidation granular sludge; Step b, inoculation: inoculate the washed anaerobic ammonium oxidation sludge into the interior of the reaction tank, the MLSS value and MLVSS value of the anaerobic ammonium oxidation sludge are 5.12 g·L -1 and 4.74 g·L -1 , the inoculation volume of the anaerobic ammonium oxidation sludge is 30% of the effective volume of the reaction tank; Step c, preparing simulated waste liquid: using 5L of wastewater as the basic waste liquid, the basic waste liquid contains KH2PO4, CaCl2·2H2O, MgSO4·7H2O and KHCO3, adding trace element composition I solution and trace element composition II solution to the basic waste liquid at an addition concentration of 1mL / L, respectively, and then adding ammonium salt and nitrite to the basic waste liquid, and adjusting the pH value to between 7.5 and 8.0 with a hydrochloric acid solution with a molar concentration of 1mol / L to prepare an initial simulated waste liquid; during the proliferation culture process, the ammonia nitrogen concentration in the initial simulated waste liquid is 20mg / L and the nitrite nitrogen concentration is 26mg / L, and a new simulated waste liquid is prepared every day, and the ammonia nitrogen concentration in the simulated waste liquid is increased by 20mg / L every 7 days. g / L, the nitrite nitrogen concentration in the simulated waste liquid is 1.32 times the ammonia nitrogen concentration at that time; when the ammonia nitrogen concentration reaches 180 mg / L and the nitrite nitrogen concentration is 200 mg / L, the ammonia nitrogen concentration and the nitrite nitrogen concentration in the simulated waste liquid no longer increase, the trace element composition I solution is an aqueous solution of EDTA and FeSO4·7H2O, wherein the concentrations of EDTA and FeSO4·7H2O are both 5 g / L, and the trace element composition II solution is an aqueous solution of EDTA, H3BO3, MnCl2·4H2O, CuSO4·5H2O, ZnSO4·7H2O, NiCl2·6H2O, Na2MoO4·2H2O and CoCl2·6H2O, wherein the concentration of EDTA is 15 g / L, the concentration of H3BO3 is 0.014 g / L, the concentration of MnCl2·4H2O is 0.99 g / L, the concentration of CuSO4·5H2O is 0.25 g / L, the concentration of ZnSO4·7H2O is 0.43 g / L, the concentration of NiCl2·6H2O is 0.19 g / L, the concentration of Na2MoO4·2H2O is 0.22 g / L, and the concentration of CoCl2·6H2O is 0.24 g / L; Step d, water inlet: add the prepared simulated waste liquid into the water inlet tank, and pump it into the reaction tank body through the water inlet pump, the water inlet flow rate is 8 ml / min, the hydraulic retention time is 1 day, and the simulated waste liquid does not need to be treated with nitrogen blowing and deoxygenation; Step e, start constant temperature culture: add enough water to the constant temperature water bath, turn on the constant temperature water bath, set the water bath temperature to 32°C, start the circulating water pump to pump the warm water in the constant temperature water bath into the water bath, and the water bath will be automatically drained after it is full, and the water circulation of the constant temperature water bath, the circulating water pump and the water bath is used to achieve the heat preservation of the reaction tank; Step f, applying negative pressure and coupling low-voltage direct current: turning on the power timer and the vacuum pump to evacuate the interior of the reaction tank, controlling the air pressure in the reaction tank at -0.04 MPa, and setting the power timer to power the vacuum pump every hour for half an hour each time; starting the low-voltage power supply, setting its voltage intensity to be constant at 1V, and applying low-voltage direct current to the simulated waste liquid in the reaction tank through the anode and cathode titanium plates; when the water inflow volume reaches 5L for the first time, starting the reflux pump for reflux, and the reflux ratio is 2; Step g, water discharge: start the water discharge pump to discharge the supernatant in the reaction tank into the tail water collection tank, and the water discharge flow rate is 8 ml / min; Step h, detection: sampling and testing of influent water samples and effluent water samples are carried out regularly every day to detect the concentrations of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, and the total nitrogen concentration is obtained by the sum of the three. Water samples are taken out every day to determine the dissolved oxygen DO, pH and COD, and the operation status of the reactor is observed.

2. A method for culturing anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: The concentration of KH2PO4 in the basic waste liquid of step c is 20 mg / L, the concentration of CaCl2·2H2O is 107 mg / L, the concentration of MgSO4·7H2O is 45 mg / L, the concentration of KHCO3 is 1200 mg / L, the ammonium salt is ammonium sulfate, and the nitrite is sodium nitrite.

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