CN115520962B - With NO3-Method for domesticating and culturing electroactive anaerobic ammonia oxidizing microorganism serving as single nitrogen source - Google Patents

Abstract

The present invention relates to the field of sewage treatment, and provides a method for taming and cultivating electroactive anaerobic ammonium oxidizing microorganisms with NO ₃ ^‑ as a single nitrogen source. First, electroactive anaerobic ammonium oxidizing microorganisms are tamed by means of an external power supply, and then the dissimilatory nitrate reduction metabolic pathway of the electroactive anaerobic ammonium oxidizing microorganisms is stimulated by substrate taming, so as to realize autotrophic denitrification of anaerobic ammonium oxidation with NO ₃ ^‑ as a single nitrogen source and using electric field cathode electrons as electron donors. The present invention realizes the anaerobic ammonium oxidation reaction under a single NO ₃ ^‑nitrogen source mode, and achieves the purpose of autotrophic removal of NO ₃ ^‑ in a water body. The tamed electroactive anaerobic ammonium oxidizing microorganisms with NO ₃ ^‑ as a single nitrogen source can achieve a direct removal rate of NO ₃ ^‑ in a water body of more than 75%, and the specific anaerobic ammonium oxidation activity can reach more than 0.3 g‑N/(g ^‑1 ‑VSS·d ^‑1 ).

Description

Method for acclimatizing and cultivating electroactive anaerobic ammonium-oxidizing microorganisms using NO3- as the sole nitrogen source

Technical Field

The invention belongs to the technical field of sewage treatment and relates to a method for acclimatizing and cultivating electroactive anaerobic ammonia oxidizing microorganisms using NO ₃ ^- as a single nitrogen source.

Background technique

Eutrophication of water bodies is still one of the most serious environmental problems in the ^world . Anaerobic ammonium oxidation is the most valuable biological denitrification technology for sewage. Traditional anaerobic ammonium oxidation microorganisms use nitrite nitrogen _NO2- as an electron acceptor to oxidize ammonia nitrogen into _N2 under anaerobic conditions, thereby achieving autotrophic removal of nitrogen in water bodies and avoiding the problems of consuming organic carbon sources and causing greenhouse gas _N2O emissions in traditional denitrification. However, it is still impossible to directly and effectively remove nitrate nitrogen _NO3- ^that exists stably in water bodies.

Summary of the invention

The electrons provided by the electric field cathode in the tamed electroactive anaerobic ammonium oxidizing microorganisms of the present invention are used to reduce NO ₃ ^- and accumulate NO ₂ ^- , and the cathode electrons are used to replace the position of NH ₄ ⁺ electron donor, thereby realizing the anaerobic ammonium oxidation reaction under the single NO ₃ ^- nitrogen source mode, thereby achieving autotrophic removal of NO ₃ ^- in the water body and solving the problem of eutrophication of the water body.

The technical solution of the present invention is:

The method for acclimatizing and cultivating electroactive anaerobic ammonium oxidizing microorganisms using NO ₃ ^- as a single nitrogen source comprises the following steps:

① Inoculum sludge pretreatment: The anaerobic ammonium oxidation sludge granules are used as inoculum. After starvation pretreatment, ultrasound is used to promote the disintegration and flocculation of the granules. After sedimentation and concentration, the flocculent sludge is inoculated into the cathode reaction chamber of the double-chamber electrolytic cell for acclimatization and cultivation.

Furthermore, the particle size of the granular sludge is 1.0-3.0 mm, and after 5-15 days of starvation pretreatment, ultrasound is applied for 1 hour to promote the disintegration and flocculation of the particles;

② Operating condition control of electroactive anaerobic ammonium oxidizing microorganism acclimation culture: the concentration of flocculent sludge in the cathode reaction chamber is controlled at 2500-4000 mg/L, 0.5% penicillin is added to inhibit the growth of heterotrophic bacteria, Ar and _CO2 are introduced to create an anaerobic environment, the initial _NH4 ⁺ and _NO2- concentrations are 100 mg-N/L and 120 mg-N/L, respectively, and the ^system is operated in a continuous mode with a hydraulic retention time (HRT) of 12 h. _NaHCO3 and trace elements are supplemented every 2 days to supply the growth of anaerobic ammonium oxidizing microorganisms, the load is increased by 25% every 10 days as a cycle, and the system is stirred with a stirring paddle to ensure uniform mass transfer in the system.

Furthermore, the addition ratio of Ar to CO ₂ was 95%:5%, and the stirring paddle was stirred for 10 min every 60 min to ensure uniform mass transfer in the system.

When the concentrations of NH ₄ ⁺ and NO ₂ ^- in the cathode reaction chamber influent reach 300 mg-N/L and 360 mg-N/L respectively, and the total nitrogen removal rate is stabilized at above 75%, the proportion of NH ₄ ⁺ is gradually reduced by 25% until it reaches 0, thereby constructing a reaction pathway for reducing NO ₂ ^- to generate N ₂ by cathode electrons and domesticating electrically active anaerobic ammonia-oxidizing microorganisms.

③ Control of the working conditions of stimulating cathode electron supply to electroactive anaerobic ammonium oxidizing microorganisms to reduce nitrate (NO ₃ ^- ) and accumulate nitrite (NO ₂ ^- ): in the cathode reaction chamber where electroactive microorganisms are successfully domesticated, the substrate composition is changed to use NO ₃ ^- as the nitrogen source, and 1% EDTA disodium iron is added to promote the stimulation of the DNRA pathway;

The initial NO ₃ ^- concentration was 120 mg-N/L, and the system was operated in a continuous mode with a hydraulic retention time (HRT) of 12 h. NaHCO ₃ and trace elements were supplemented every 2 days to supply the growth of anaerobic ammonium oxidizing microorganisms. The load was increased by 25% every 10 days as a cycle until the influent NO ₃ ^- concentration of the system reached 360 mg-N/L. When the NO ₃ ^- removal rate was stabilized at above 75%, it indicated that the pathway of electroactive anaerobic ammonium oxidizing microorganisms to reduce nitrate (NO ₃ ^- ) and accumulate nitrite (NO ₂ ^- ) was stimulated, and further cathode electrons were used to reduce NO ₂ ^- to generate N ₂ , thereby achieving effective removal of NO ₃ ^- in water by electroactive anaerobic ammonium oxidizing microorganisms. That is, electroactive anaerobic ammonium oxidizing microorganisms using NO ₃ ^- as the single nitrogen source were successfully domesticated, and their specific anaerobic ammonium oxidizing activity could reach above 0.3 gN/(g ^-1 -VSS·d ^-1 ).

The electroactive anaerobic ammonium oxidizing microorganisms described above use a double-chamber electrolytic cell as a reaction device, a DC regulated power supply controls a voltage range of 0.8-1.2 V, and the electroactive anaerobic ammonium oxidizing microorganisms are domesticated and enriched in a cathode reaction cell.

Further, the double-chamber electrolytic cell comprises a cathode reaction chamber, an anode chamber, a DC regulated power supply, and a salt bridge, the two ends of the salt bridge are respectively inserted into the cathode reaction chamber and the anode chamber, the electric field cathode and the reference electrode of the DC regulated power supply are arranged in the cathode reaction chamber, the electric field anode is arranged in the anode chamber, the anode chamber is filled with a KCl buffer solution, the cathode reaction chamber is used for the acclimation and cultivation of electroactive anaerobic ammonia oxidizing microorganisms, and further, a stirrer is arranged in the cathode reaction chamber;

The above-mentioned electroactive anaerobic ammonium oxidizing microorganisms are domesticated and cultivated with _NO3- ^as the single nitrogen source and are applied to the removal of nitrate nitrogen in sewage. The steps are as follows: first, the electroactive anaerobic ammonium oxidizing microorganisms are domesticated and cultivated, and then the dissimilatory nitrate reduction to ammonia (DNRA) metabolic pathway of the electroactive anaerobic ammonium oxidizing microorganisms is stimulated through substrate domestication to achieve anaerobic ammonium oxidizing autotrophic nitrogen removal with electric field cathode electrons as electron donors and _NO3- ^as the single nitrogen source.

Beneficial effects of the present invention:

The electrically active anaerobic ammonium oxidizing microorganisms domesticated in the present invention utilize electrons provided by the electric field cathode to reduce NO ₃ ^{- and} accumulate NO ₂ ^- , and utilize cathode electrons to replace the position of NH ₄ ⁺ electron donor, thereby realizing anaerobic ammonium oxidation reaction under a single NO ₃ ^- nitrogen source mode, thereby achieving autotrophic removal of NO ₃ ^- in water.

The present invention uses electroactive anaerobic ammonium oxidizing microorganisms with nitrate nitrogen (NO ₃ ^- ) as a single nitrogen source to remove NO ₃ ^- in water, avoiding the problems of consuming organic carbon sources and causing greenhouse gas N ₂ O emissions in traditional denitrification. The present invention has positive significance for solving the problem of eutrophication of water bodies.

A double-chamber electrolytic cell is used as a reactor, and an external direct current voltage of 0.8-1.2 V is applied. The domestication and cultivation method of the electroactive anaerobic ammonium oxidizing microorganism of the present invention includes three steps: inoculated sludge pretreatment, working condition control of the domestication and cultivation of the electroactive anaerobic ammonium oxidizing microorganism, and working condition control of the pathway of stimulating cathode electrons to supply the electroactive anaerobic ammonium oxidizing microorganism with nitrate reduction (NO ₃ ^- ) and nitrite accumulation (NO ₂ ^- ). By coordinating various parameters, the electroactive anaerobic ammonium oxidizing microorganism successfully domesticated with NO ₃ ^- as a single nitrogen source under synergistic effect can achieve a removal rate of 75% for NO ₃ ^- in water, and its specific anaerobic ammonium oxidizing activity can reach more than 0.3 gN/(g ^-1 -VSS·d ^-1 ).

BRIEF DESCRIPTION OF THE DRAWINGS

Fig.1 Influent and effluent indicators and total nitrogen removal rate during the acclimation stage

Figure 2 Removal effect of single NO ₃ - by electroactive anaerobic ammonium oxidizing microorganisms

Detailed ways

Example 1, a method for acclimatizing and culturing electroactive anaerobic ammonium oxidizing microorganisms using NO ₃ ^- as a single nitrogen source, comprising the following steps:

① Inoculum sludge pretreatment: anaerobic ammonia oxidation sludge granular sludge was used as inoculum, and the particle size of the granular sludge was 1.0-3.0 mm. After 15 days of starvation pretreatment, ultrasound was used for 1 hour to promote the disintegration and flocculation of the particles. After sedimentation and concentration, 400 ml of flocculent sludge (MLSS of about 3000-3500 mg/L) was inoculated and placed in the cathode reaction chamber of a 1 L double-chamber electrolytic cell for acclimation and cultivation. The DC regulated power supply of the double-chamber electrolytic cell controlled the voltage in the range of 0.8-1.2 V.

② Control of operating conditions for the acclimatization and cultivation of electroactive anaerobic ammonium-oxidizing microorganisms: The concentration of flocculent sludge in the cathode reaction chamber was controlled at 2500-4000 mg/L, 0.5% penicillin was added to inhibit the growth of heterotrophic bacteria, Ar and _CO2 were introduced to create an anaerobic environment, the addition ratio of Ar to _CO2 was 95%:5%, the initial concentrations of _NH4 ⁺ and _NO2- were 100 mg-N/L and 120 mg-N/L, respectively, ^and the system was operated in a continuous mode with a hydraulic retention time (HRT) of 12 h. _NaHCO3 and trace elements were supplemented every 2 d to supply the growth of anaerobic ammonium-oxidizing microorganisms, the load was increased by 25% every 10 d as a cycle, and the agitator was stirred for 10 min every 60 min to ensure uniform mass transfer in the system.

As shown in Figure 1, when the reactor was operated to the 40th day, the concentrations of NH ₄ ⁺ and NO ₂ ^- in the influent reached 300 mg-N/L and 360 mg-N/L, respectively, and the total nitrogen removal rate was stabilized at more than 75%, and then it was stably operated for 20 days. From the 60th day, the proportion of NH ₄ ⁺ was gradually reduced by 25% until it reached 0 while maintaining the conservation of matrix nitrogen mass. At this time, the total nitrogen removal rate was still more than 75%. The above phenomenon shows that the reaction pathway of reducing NO ₂ ^- to generate N ₂ by cathode electrons was constructed, and electroactive anaerobic ammonium oxidizing microorganisms were domesticated.

③ Control of the operating conditions of the pathway for stimulating cathode electron supply to electroactive anaerobic ammonium oxidizing microorganisms to reduce nitrate (NO ₃ ^- ) and accumulate nitrite (NO ₂ ^- ): In the cathode reaction chamber where electroactive microorganisms have been successfully domesticated, the substrate composition is changed to use NO ₃ ^- as the nitrogen source, and 1% disodium iron EDTA is added to promote the stimulation of the DNRA pathway.

As shown in Figure 2, the initial influent NO ₃ ^- concentration was 120 mg-N/L, and the system was operated in a continuous mode with a hydraulic retention time (HRT) of 12 h. NaHCO ₃ and trace elements were supplemented every 2 days to supply the growth of anaerobic ammonium oxidizing microorganisms. The load was increased by 25% every 10 days as a cycle. From the 50th day, the influent NO ₃ ^- concentration of the system reached 360 mg-N/L, and the NO ₃ ^- removal rate was stable at more than 75%. The above phenomenon indicates that the pathway of electroactive anaerobic ammonium oxidizing microorganisms to reduce nitrate (NO ₃ ^- ) and accumulate nitrite (NO ₂ ^- ) is stimulated, and NO ₂ ^- can be successfully reduced to N ₂ with cathode electrons, thereby achieving the effective removal of NO ₃ ^- in water by electroactive anaerobic ammonium oxidizing microorganisms. That is, electroactive anaerobic ammonium oxidizing microorganisms using NO ₃ ^- as the single nitrogen source were successfully domesticated, and their specific anaerobic ammonium oxidation activity can reach more than 0.3 gN/(g ^-1 -VSSd ^-1 ).

The above experimental process shows that there are electroactive anaerobic ammonium oxidizing microorganisms that can achieve extracellular electron transfer through an external electric field, and when there are sufficient electron donors, the metabolic pathway of dissimilatory nitrate reduction to ammonia (DNRA) of anaerobic ammonium oxidizing microorganisms can be stimulated to reduce NO ₃ ^- to accumulate NO ₂ ^- , thereby achieving the removal of NO ₃ ^- in water bodies. Therefore, the domestication and cultivation of electroactive anaerobic ammonium oxidizing microorganisms with NO ₃ ^- as the sole nitrogen source can effectively achieve the removal of NO ₃ ^- in water bodies, which is of positive significance for solving the problem of eutrophication of water bodies, especially for the treatment of sewage with a low carbon-nitrogen ratio, without the need for an external carbon source, and the treatment effect is better.

The electrically active anaerobic ammonium oxidizing microorganisms domesticated in the present invention utilize electrons provided by the electric field cathode to reduce NO ₃ ^{- and} accumulate NO ₂ ^- , and utilize cathode electrons to replace the position of NH ₄ ⁺ electron donor, thereby realizing anaerobic ammonium oxidation reaction under a single NO ₃ ^- nitrogen source mode, thereby achieving autotrophic removal of NO ₃ ^- in water.

The above is a detailed description of an embodiment of the present invention, but the content is only a preferred embodiment of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims (4)

1. An electro-active anaerobic ammonia oxidation microorganism domestication culture method taking NO ₃ ^- as a single nitrogen source is characterized in that: the method comprises the following steps:

① Pretreatment of inoculated sludge: anaerobic ammonia oxidation sludge granular sludge is used as an inoculum, after starvation pretreatment, ultrasonic promotion of particle disintegration and flocculation is carried out, flocculent sludge is inoculated into a cathode reaction chamber of a double-cavity electrolytic cell after sedimentation and concentration, the double-cavity electrolytic cell comprises a cathode reaction chamber, an anode chamber, a direct-current stabilized power supply and a salt bridge, two ends of the salt bridge are respectively inserted into the cathode reaction chamber and the anode chamber, an electric field cathode and a reference electrode of the direct-current stabilized power supply are arranged in the cathode reaction chamber, an electric field anode is arranged in the anode chamber, KCl buffer solution is filled in the anode chamber, and the cathode reaction chamber is used for domestication culture of electroactive anaerobic ammonia oxidation microorganisms;

② Domestication culture of electroactive anaerobic ammonia oxidizing microorganisms: controlling the concentration of flocculent sludge in the cathode reaction chamber to 2500-4000 mg/L, adding 0.5% penicillin to inhibit the growth of heterotrophic bacteria, introducing Ar and CO ₂ to create anaerobic environment, enabling the initial concentration of NH ₄ ⁺ and NO ₂ ^- to be 100 mg-N/L and 120 mg-N/L respectively, running in a continuous mode, enabling the hydraulic retention time to be 12 h, supplementing NaHCO ₃ and microelements to every 2d to supply anaerobic ammonia oxidizing microorganisms for growth, and lifting the load by 25% every 10 d as a period, and stirring by a stirring paddle to ensure uniform mass transfer in the system;

Gradually reducing the NH ₄ ⁺ ratio to 0 in a range of 25% on the premise of keeping the mass conservation of matrix nitrogen when the concentrations of the inlet water NH ₄ ⁺ and NO ₂ ^- in the cathode reaction chamber reach 300 mg-N/L and 360 mg-N/L respectively and the total nitrogen removal rate is more than 75%, so as to construct an N ₂ reaction path generated by cathode electron reduction of NO ₂ ^-, and domesticating anaerobic ammonia oxidizing microorganisms with electric activity;

③ Excitation of cathodic electrons supply the electroactive anaerobic ammonia oxidizing microorganism to reduce nitrate (NO ₃ ^-) accumulation nitrite (NO ₂ ^-) pathway: in the cathode reaction chamber for successfully domesticating electroactive microorganisms, changing substrate components, taking NO ₃ ^- as a nitrogen source, and simultaneously adding 1% of EDTA disodium iron to promote excitation of DNRA pathway;

The initial concentration of NO ₃ ^- is 120 mg-N/L, the operation is continuous, the Hydraulic Retention Time (HRT) is 12 h, naHCO ₃ and microelements are supplemented every 2d and supplied to the growth of the anaerobic ammonia oxidation microorganism, the load is lifted by 25% every 10 d as a period, when the concentration of the inflow water NO ₃ ^- of the system reaches 360 mg-N/L, the removal rate of NO ₃ ^- is stabilized to be more than 75%, the way that the electro-active anaerobic ammonia oxidation microorganism reduces nitrate (NO ₃ ^-) to accumulate nitrite (NO ₂ ^-) is excited, and the cathode electron is further utilized to reduce NO ₂ ^- to generate N ₂, so that the electro-active anaerobic ammonia oxidation microorganism in water body NO ₃ ^- is effectively removed, namely the electro-active anaerobic ammonia oxidation microorganism which takes NO ₃ ^- as a single nitrogen source is successfully domesticated.

2. The method for domesticating and culturing an electroactive anaerobic ammonium oxidation microorganism using NO ₃ ^- as a single nitrogen source according to claim 1, wherein the method comprises the steps of: in the step ①, the particle size of the granular sludge is 1.0-3.0 mm, and after starvation pretreatment of 5-15 and d, the granular sludge is subjected to ultrasonic treatment for 1h to promote disintegration and flocculation of the granules;

In step ②, the addition ratio of Ar to CO ₂ is 95%:5, stirring paddles stir 10min every 60min to ensure uniform mass transfer in the system.

3. The method for domesticating and culturing an electroactive anaerobic ammonium oxidation microorganism using NO ₃ ^- as a single nitrogen source according to claim 1 or 2, wherein: the control voltage range of the direct current stabilized power supply in the double-cavity electrolytic cell is 0.8-1.2V.

4. Use of a microorganism obtained by the method for domesticating and culturing an electroactive anaerobic ammonia oxidizing microorganism using NO ₃ ^- as a single nitrogen source according to any one of claims 1 to 3 for treating sewage with a low carbon nitrogen ratio.