Method for in-situ efficient enrichment of anaerobic ammonia oxidation bacteria

Abstract

A method for in-situ efficient enrichment of anaerobic ammonia oxidation bacteria, comprising: using anaerobic digestion sludge as inoculation sludge and using nitrate wastewater as an influent substrate, the inoculation sludge and the influent substrate reacting with each other under a light-proof, constant-temperature and closed condition so as to enrich anaerobic ammonia oxidation bacteria. After the anaerobic ammonia oxidation bacteria are enriched about 30 days by using the anaerobic digestion sludge as the inoculation sludge and the nitrate wastewater as the influent substrate, the number of functional genes HzsB of the anaerobic ammonia oxidation bacteria is increased by about 100 times than that in a control group (using a liquid mixture of ammonia nitrogen and nitrite nitrogen as an influent substrate). Thus, on one hand, there is no need to add extra ammonia nitrogen and nitrite nitrogen, and on the other hand, there is no need to regulate and control complex process parameters. Therefore, in-situ rapid and sustainable enrichment of anaerobic ammonia oxidation bacteria can be implemented in wastewater treatment plants simply by means of an economical and simple operation mode.

Description

A method for efficiently enriching anaerobic ammonia-oxidizing bacteria in situ Technical Field

[0001] The present invention relates to the technical field of sewage treatment, and in particular to a method for efficiently enriching anaerobic ammonia-oxidizing bacteria in situ. Background Art

[0002] Anaerobic ammonium oxidation technology refers to a biological process in which anaerobic ammonium-oxidizing bacteria (AnAOB) use ammonia nitrogen as an electron donor and nitrite nitrogen as an electron acceptor to convert nitrogen gas in an anaerobic environment (oxygen less than 0.2 mg / L). Compared with traditional nitrification and denitrification biological denitrification processes, anaerobic ammonium oxidation technology does not require the addition of an organic carbon source and can reduce carbon dioxide emissions by 90%, aeration volume by 60%, and floor space by 50%. Anaerobic ammonium oxidation technology has been maturely applied in the treatment of wastewater such as high-ammonia nitrogen sludge digestate from side streams of sewage treatment plants, but the total amount of this type of wastewater is small, accounting for only about 1-2% of the total water volume of the sewage treatment plant. The mainstream water volume of sewage treatment plants is large and the nitrogen load is high. The use of anaerobic ammonium oxidation technology to denitrify mainstream wastewater has huge potential for energy saving and consumption reduction.

[0003] However, anaerobic ammonium oxidizing bacteria (ANAMMOX) have a low growth rate (0.063 to 0.069 mmol / day) and are sensitive to environmental conditions. Conventional methods (providing ammonia and nitrite nitrogen substrates) often use activated sludge from sewage treatment plants as inoculum, and successful enrichment typically requires at least 2-3 months. Furthermore, compared to sidestream wastewater, mainstream sewage treatment plant wastewater has low temperatures and low ammonia nitrogen levels (usually below 50 mgN / L). In situ enrichment of ANAMMOX bacteria from mainstream wastewater takes longer, or may even be impossible.

[0004] Patent CN110615525A discloses a method for enriching anaerobic ammonium oxidizing bacteria and its application. By controlling the concentration of ammonia nitrogen and / or nitrite nitrogen in simulated wastewater and different hydraulic mixing conditions, the enrichment culture of anaerobic ammonium oxidizing bacteria is optimized in stages, which significantly improves the enrichment culture of anaerobic ammonium oxidizing bacteria and the startup process of the anaerobic ammonium oxidation process. The process startup can usually be completed within about 3 to 4 months.

[0005] The traditional method of starting the anaerobic ammonium oxidation process with ammonia nitrogen and nitrite nitrogen as the influent matrix, the starting effect of activated sludge or mixed sludge containing mature anaerobic ammonium oxidation sludge is better than that of anaerobic digestion sludge. This may be because the activity of anaerobic ammonium oxidizing bacteria in anaerobic digestion sludge is usually lower than that in activated sludge, and the anaerobic digestion sludge is mainly composed of reduced pollutants, which have a certain inhibitory effect on anaerobic ammonium oxidizing bacteria. At the same time, the denitrification-related microorganisms lack sufficient oxidizing electron acceptors to react. In the biochemical reaction stage of the sewage treatment plant, increasing aeration is a common method used in sewage treatment plants, and a series of processes are also developed and applied based on the principle of anaerobic-anoxic-aerobic. However, oxygen has an inhibitory effect on anaerobic ammonium oxidizing bacteria, so it is difficult to achieve stable and sustainable enrichment of anaerobic ammonium oxidizing bacteria in the biochemical treatment stage of the sewage treatment plant.

[0006] Therefore, there is an urgent need to find a method for in situ enrichment of anaerobic ammonium-oxidizing bacteria suitable for anaerobic digestion sludge in sewage treatment plants to promote the widespread application of anaerobic ammonium oxidation technology in mainstream wastewater.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to overcome the limitations of the existing technology and provide a method for in situ efficient enrichment of anaerobic ammonia oxidizing bacteria, aiming to use anaerobic digestion sludge from urban sewage treatment plants as inoculated sludge and nitrate wastewater from the mainstream wastewater treatment plant as the influent matrix, thereby achieving rapid enrichment of anaerobic ammonia oxidizing bacteria.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] The technical solution of the present invention is to provide a method for efficiently enriching anaerobic ammonia-oxidizing bacteria in situ, which is:

[0011] Anaerobic digestion sludge is used as inoculated sludge, and nitrate wastewater is used as influent matrix. The inoculated sludge and influent matrix are reacted under light-proof, constant temperature and closed conditions to enrich anaerobic ammonia-oxidizing bacteria.

[0012] In some specific embodiments, the anaerobic digestion sludge is selected from sludge containing any one or more of ammonia nitrogen, carbon source, and sulfide.

[0013] In some specific embodiments, the concentration of ammonia nitrogen in the anaerobic digestion sludge is greater than or equal to 0.5 g / kg, the concentration of the carbon source is greater than or equal to 20 g / L, and the concentration of sulfide is greater than or equal to 4 g / kg.

[0014] In some specific embodiments, the anaerobic sludge is pretreated before the reaction. The specific process of the pretreatment is: removing the sludge portion that is oxidized and discolored on the surface of the anaerobic sludge.

[0015] In some specific embodiments, the influent matrix is ​​pretreated before the reaction. The specific process of the pretreatment is: nitrogen blowing to remove oxygen in the influent matrix so that the dissolved oxygen concentration in the influent matrix is ​​lower than 0.2 mg / L.

[0016] In some embodiments, the nitrate wastewater is selected from the group consisting of - -N, NO2 - -N、NH4 + -N any one or more nitrate wastewater.

[0017] In some embodiments, the nitrate wastewater contains NO3 - -N concentration is 60-70mg / L, NO2 - -N concentration is 0.5-1.0mg / L, NH4 + -N concentration is 5-8 mg / L.

[0018] In some embodiments, the volume ratio of the inoculum sludge to the influent substrate is 1:9 to 1:1.

[0019] In some embodiments, the constant temperature is 33-37°C.

[0020] In some specific embodiments, the reaction time is 30 cycles, each cycle is 24 hours, including 0.5 hours of water addition, 22 hours of stirring reaction, 1 hour of standing, and 0.5 hours of water discharge.

[0021] The mechanism of the in-situ enrichment of anaerobic ammonia-oxidizing bacteria of the present invention is as follows: since heterotrophic bacteria are more competitive for electron acceptors than autotrophic bacteria, when the nitrate concentration of the influent is appropriate, in a short period of time, the activity of heterotrophic denitrifying bacteria in the anaerobic digestion sludge will be promoted first. As the carbon-nitrogen ratio gradually decreases, the lack of endogenous carbon will cause a short-term denitrification process to generate nitrite nitrogen; subsequently, the sulfur autotrophic denitrification activity will gradually increase. As the sulfur-nitrogen ratio gradually decreases, the lack of endogenous sulfur will also cause a partial denitrification process to generate nitrite nitrogen. Specifically, the entire enrichment process can be roughly divided into two stages. In the first stage, under the conditions of suitable carbon-nitrogen ratio and sulfur-nitrogen ratio, high concentrations of organic matter and sulfide are rapidly reduced in a short period of time, which creates a favorable habitat for the occurrence of the anaerobic ammonia-oxidizing process; in the second stage, as the carbon-nitrogen ratio and sulfur-nitrogen ratio gradually decrease, the anaerobic ammonia-oxidizing bacteria can utilize the nitrite nitrogen produced by partial denitrification and the background ammonia nitrogen to react, and the activity of the anaerobic ammonia-oxidizing bacteria is improved.

[0022] The traditional startup strategy using ammonia nitrogen and nitrite nitrogen as the influent matrix has three disadvantages: (1) the startup process can only adopt the method of gradually increasing the load from a low matrix, because the initial influent matrix is ​​too high, which will inhibit the anaerobic ammonia oxidizing bacteria; (2) the low concentration of the influent matrix at the initial startup cannot effectively improve the inhibitory effect of the high carbon and high sulfur environment of the inoculated sludge on the anaerobic ammonia oxidizing bacteria; (3) the low concentration of the influent matrix will cause the inoculated sludge microbial community, such as denitrifying bacteria, to form a competitive relationship with the anaerobic ammonia oxidizing bacteria, which is manifested as competing for nitrite nitrogen as an electron acceptor, resulting in the anaerobic ammonia oxidizing bacteria being unable to obtain sufficient matrix at the initial startup.

[0023] The startup strategy of the present invention using nitrate as the influent matrix has the following advantages: (1) the influent nitrate concentration level usually does not inhibit anaerobic ammonia-oxidizing bacteria; (2) nitrate is more oxidizing than nitrite, so a higher influent nitrate concentration (higher than the sum of ammonia nitrogen and nitrite nitrogen concentrations in the traditional startup strategy) can effectively improve the inhibitory effect of the high carbon and high sulfur environment of the seed mud on anaerobic ammonia-oxidizing bacteria, creating a good habitat for the occurrence of the anaerobic ammonia oxidation process; (3) the standard electrode potential (E) of nitrate reduction to nitrogen gas under the same conditions is higher than that of nitrite. 0 ) is about +1.24V, and the E of nitrite reduction to nitrogen gas 0 The value is about 0.96V, indicating that in an environment containing both nitrate and nitrite, denitrifying bacteria will give priority to using nitrate. When the ratio of endogenous electron donors to electron acceptors is low, short-range denitrification will occur to produce nitrite, which can form a synergistic relationship between denitrifying bacteria and anaerobic ammonia-oxidizing bacteria.

[0024] The uniqueness of the mechanism for in situ enrichment of anaerobic ammonia-oxidizing bacteria provided by the present invention lies in: using anaerobic digestion sludge as inoculum sludge, utilizing the high concentration of reduced sulfur and bioavailable carbon source contained in the sludge itself as electron donors, without the need for external carbon and sulfur sources, using nitrate wastewater as influent, and determining the optimal volume ratio of influent to inoculum sludge to maximize the activity of anaerobic ammonia-oxidizing bacteria and achieve in situ enrichment of anaerobic ammonia-oxidizing bacteria.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This method uses anaerobic digestion sludge as inoculum and nitrate wastewater as the influent matrix to enrich anaerobic ammonium oxidizing bacteria. After approximately 30 days of operation, the number of anaerobic ammonium oxidizing bacteria increased by approximately three times compared to the original inoculum sludge. This method eliminates the need for additional ammonia and nitrite nitrogen additions and complex process parameter control, achieving rapid and sustainable in-situ enrichment of anaerobic ammonium oxidizing bacteria in sewage treatment plants through an economical and simple operation method. DETAILED DESCRIPTION

[0027] The present invention is described in detail below with reference to specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0028] In the following examples and comparative examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0029] Anaerobic digestion sludge: taken from a sewage treatment plant, containing an ammonia nitrogen concentration of 929.3 mg / L, a carbon source concentration of 23614.5 mg TCOD / L, and a sulfide concentration of 7880.2 mg / kg.

[0030] Nitrate wastewater: taken from the effluent of the anaerobic ammonium oxidation reactor, containing NO3 - -N concentration is 65mg / L, containing NO2 - -N concentration is 0.7mg / L, containing NH4 + -N concentration is 5.3 mg / L.

[0031] Example 1:

[0032] A method for efficiently enriching anaerobic ammonia-oxidizing bacteria in situ comprises the following steps:

[0033] (1) Remove the surface oxidized and discolored parts of the collected anaerobic digestion sludge.

[0034] (2) Using an anaerobic serum bottle with a volume of 100 mL, inoculate 10 mL of the anaerobic digested sludge treated in step (1) above.

[0035] (3) Nitrogen is blown through the nitrate wastewater to remove oxygen, so that the dissolved oxygen concentration in the nitrate wastewater is reduced to below 0.2 mg / L.

[0036] (4) Add 90 mL of nitrate wastewater to the anaerobic serum bottle loaded with anaerobic digestion sludge in step (2) daily for reaction. The anaerobic serum bottle is placed in a constant temperature shaker for incubation, the temperature is set at 35°C, the speed is 180 rpm, the operation cycle is 24 hours (including 0.5 hours of water inlet, 22 hours of stirring reaction, 1 hour of standing, and 0.5 hours of water outlet), and the operation is carried out in the dark. The water change operation is to use a syringe to first suck out the original nitrate wastewater in the anaerobic serum bottle, and then inject the nitrate wastewater after nitrogen blowing into the anaerobic serum bottle. The water change process is carried out in an anaerobic operation box.

[0037] The first stage of the enrichment process in this example occurs in 1-5 days, showing the nitric nitrogen (NO3 - -N) is removed, but nitrite (NO2 --N) has no obvious accumulation phenomenon; the second stage occurs in 6-30 days, in which nitric nitrogen (NO3 - -N) removal rate and nitrite nitrogen (NO2 - -N) accumulation rate increases simultaneously, and nitrate nitrogen (NO3 - -N) removal rate is stable and there is continuous nitrite (NO2 - -N) accumulation phenomenon. On the 7th day of operation, obvious nitrite nitrogen accumulation (about 1.4 mg / L) appeared in the anaerobic serum bottle, and the average accumulation concentration of nitrite nitrogen was about 2.2 mg / L thereafter. After the 16th day, the ammonia nitrogen removal rate was basically stable and greater than 0. On the 29th day, the ammonia nitrogen removal rate reached a maximum of 52.1%. The removal rates of total organic carbon (TOC) and acid volatile sulfur (AVS) of the inoculated sludge were 91.8% and 93.3%, respectively. The results of fluorescent quantitative PCR showed that the number of anaerobic ammonia oxidizing bacteria functional gene HzsB (6.90E+04) increased by about 100 times compared with the control example 1 (6.73E+06).

[0038] Comparative Example 1:

[0039] Compared with Example 1, other conditions are the same as those of Example 1, except that the influent matrix is ​​replaced by "nitrate wastewater" with a mixture of ammonia nitrogen and nitrite nitrogen, with concentrations of 14 mg / L and 18.5 mg / L respectively. No ammonia nitrogen removal phenomenon was observed within 30 cycles, and nitrite nitrogen (NO2 - -N) removal rate was less than 50%. No significant removal of total organic carbon (TOC) and acid volatile sulfur (AVS) was observed in the inoculated sludge. Fluorescence quantitative PCR results showed that the number of HzsB functional gene of anaerobic ammonium oxidizing bacteria was basically the same as that in the original inoculated sludge.

[0040] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for efficiently enriching anaerobic ammonia-oxidizing bacteria in situ, characterized in that: for: The anaerobic digestion sludge is used as inoculated sludge, and the nitrate wastewater is used as the influent matrix. The inoculated sludge and the influent matrix are reacted under light-proof, constant temperature and closed conditions to enrich the anaerobic ammonia-oxidizing bacteria.

2. The method for in-situ efficient enrichment of anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: The anaerobic digestion sludge is selected from sludge containing any one or more of ammonia nitrogen, carbon source and sulfide.

3. The method for in-situ efficient enrichment of anaerobic ammonia-oxidizing bacteria according to claim 2, characterized in that: The concentration of ammonia nitrogen in the anaerobic digestion sludge is greater than or equal to 0.5 g / kg, the concentration of carbon source is greater than or equal to 20 g / L, and the concentration of sulfide is greater than or equal to 4 g / kg.

4. The method for in-situ efficient enrichment of anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: The anaerobic sludge is pretreated before the reaction. The specific process of the pretreatment is: removing the oxidized and discolored sludge part on the surface of the anaerobic sludge.

5. The method for in-situ efficient enrichment of anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: The influent matrix is ​​pretreated before the reaction. The specific process of the pretreatment is: nitrogen blowing to remove oxygen in the influent matrix so that the dissolved oxygen concentration in the influent matrix is ​​lower than 0.2 mg / L.

6. The method for in-situ efficient enrichment of anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: The nitrate wastewater is selected from the group consisting of NO3 - -N, NO2 - -N、NH4 + -N any one or more nitrate wastewater.

7. The method for in-situ efficient enrichment of anaerobic ammonia-oxidizing bacteria according to claim 6, characterized in that: The nitrate wastewater NO3 - -N concentration is 60-70mg / L, NO2 - -N concentration is 0.5-1.0mg / L, NH4 + -N concentration is 5-8mg / L.

8. The method for in-situ efficient enrichment of anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: The volume ratio of inoculated sludge to influent substrate is 1:9 to 1:

1.

9. The method for in-situ efficient enrichment of anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: The constant temperature is 33-37℃.

10. The method for in-situ efficient enrichment of anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: The reaction time is 30 cycles, each cycle is 24 hours, including 0.5 hours of water inlet, 22 hours of stirring reaction, 1 hour of standing, and 0.5 hours of water outlet.