Simultaneous treatment of raw and aerated wastewater using bioelectrochemical system

Abstract

To provide an advanced livestock wastewater treatment system, apparatus and method for the simultaneous removal of nitrate ions (nitrite ions) from treated wastewater in a cathodic tank and organic matter, suspended solids and odor (generated by volatile fatty acids) from raw wastewater in an anodic tank, using an anaerobic bio-electrochemical system (BES).SOLUTION: To provide an apparatus having at least one anode tank with at least one anode therein and at least one cathode tank with at least one cathode therein, in which the anode tank is attached to the cathode tank via a separator for transporting anions or cations between the anode tank and the cathode tank.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority to a Japanese patent application (Patent Application No. 2021-198900) filed on 7 December 2021, the entirety of which is incorporated herein by reference.

[0002] This application relates to an advanced swine wastewater treatment system using an anaerobic bioelectrochemical system (BES) for the simultaneous removal of nitrate ions (nitrite ions) from treated wastewater in the cathode tank, particularly from aerated wastewater, and organic matter, suspended solids, and odors (generated by volatile fatty acids) from raw wastewater in the anode tank. [Background technology]

[0003] Sustainable wastewater treatment aims not only to reuse water and minimize pollution, but also to maximize the recovery of valuable resources, such as energy and nutrients (Verstraete et al., 2009). Agricultural wastewater is rich in recyclable nutrients. Wastewater treatment is a pressing issue in areas like the Okinawa Islands of Japan, where intensive pig farming significantly increases wastewater volume and leads to the accumulation of undesirable products that contribute to environmental pollution. This ammonium-rich wastewater from livestock farms is typically treated by aeration systems (Rosso et al., 2008). Nitrate ions are toxic and abundant inorganic pollutants commonly found in aeration tanks used to remove ammonia-containing wastewater discharged from livestock farms. Nitrate ion contamination of wastewater is of great concern due to its toxicity to human health and the environment (Powlson et al., 2008). When a person ingests nitrate ions, they are converted to nitrite ions, which then bind to hemoglobin in the body, forming methemoglobin, which cannot carry oxygen. Therefore, excessive levels of nitrate ions in drinking water can cause methemoglobinemia (also known as blue baby syndrome) (Majumdar and Gupta, 2000).

[0004] Biological denitrification is the reduction of nitrogen oxides, such as nitrate or nitrite ions, to nitrogen gas, and has traditionally been achieved by heterotrophic facultative anaerobic microorganisms (Schmidt et al., 2003). Although biological denitrification is a well-established technique, competition for available organic matter often occurs between aerobic and denitrifying microorganisms. This competition can lead to insufficient substrate (organic matter) supply, resulting in improper denitrification. Therefore, in many cases, additional carbon sources are required to achieve complete denitrification in wastewater containing low concentrations of organic matter.

[0005] Bioelectrochemical systems (BES), a cutting-edge environmental technology, have the potential to address the limitations of anaerobic digestion and complement aeration approaches. BES combine the oxidation of electron donors at the anode with the reduction of electron acceptors at the cathode, utilizing bacteria that catalyze one or both of these reactions (Clauwaert et al., 2007). Typically, in the anode of a BES, electrogenic bacteria oxidize organic matter, releasing electrons to the anode. In the cathode of a BES, nitrogen-containing electron acceptors, such as nitrate ions (NO3), are used. - ), nitrite ion (NO2 - Furthermore, nitrous oxide (N2O) can be reduced to nitrogen gas by electrotrophic denitrifying bacteria. Compared to traditional biological nutrient removal techniques, denitrifying BES achieves high nitrogen removal efficiency even at low C / N ratios due to the abundant bacterial biofilm on the cathode (Zhang and He, 2012; Tian and Yu, 2020). Understanding the behavior of microbial communities in BES has been a focus of many recent studies. The genus Geobacter has been found to be able to directly use the graphite cathode as an electron donor to reduce nitrate ions to nitrite ions in a half-cell mode applied by a potentiostat (Gregory et al., 2004). Another study using a similar system showed that nitrate ions were completely reduced to nitrogen gas by electrotrophic microorganisms that directly consume electrons from the cathode (Park et al., 2005). These electrotrophic denitrifying bacteria are autotrophic bacteria that can use electrodes as electron donors and inorganic carbon (e.g., carbon dioxide and carbonate) as a carbon source. Therefore, the biocathode serves as a safe and inexhaustible electron source. Furthermore, such microbial communities can easily adapt to electrically stimulated environments and accumulate after an adaptation period.

[0006] Recent advances in the development of biocathode denitrification in BES have utilized synthetic wastewater (Park et al., 2017; N Pous et al., 2015). Therefore, a particular interest of this study was to verify whether autotrophic denitrification by cathode could be achieved with pig wastewater and to identify the optimal conditions for stabilizing such a system. To our knowledge, this study is the first to achieve simultaneous treatment of undiluted pig wastewater in the anode tank and aerated water in the cathode tank. The removal capacity and efficiency of organic matter and odor compounds in the anode tank were evaluated along with the nitrate ion removal performance in the cathode tank. Furthermore, the system was operated over a long period.

[0007] The bacterial groups involved in autotrophic denitrification have been identified to date (Van Doan et al., 2013; Vilar-Sanz et al., 2013). However, the long-term survival of these bacterial groups in livestock wastewater remains largely unknown.

[0008] [Prior art document] [Non-patent literature] [Table 1] TIFF2023084705000002.tif241169 TIFF2023084705000003.tif242169 TIFF2023084705000004.tif213169 [Overview of the project] [Problems that the invention aims to solve]

[0009] Nitrate ions in wastewater are a cause for concern because they have adverse effects on human and environmental health. In Japan, the livestock industry uses 500 mg NO3 - -N / L (as of December 2021) is below the provisional wastewater standard, which corresponds to 400 mg NO3 -The level was lowered to -N / L (July 2022). This level corresponds to 300mg NO3 - -N / L or 200mg NO3 - - It was further reduced to N / L, and ultimately, it met the same general wastewater discharge standards as other industries (100mg NO3 - It is expected to drop to -N / L). Nitrate ions are designated as hazardous substances. Furthermore, phosphorus levels in livestock wastewater are below the current provisional wastewater standard (22 mg / L) and are expected to be lowered to the general wastewater standard (16 mg / L). Conventional activated sludge wastewater treatment systems, widely used in wastewater treatment, generally remove ammonia but not nitrate ions or phosphorus. To remove nitrate ions, conventional wastewater systems require maintaining a C / N ratio of 3-5 or higher and low dissolved oxygen (DO), both of which are difficult to control. Conventional swine farm wastewater treatment methods utilize aeration (activated sludge method) that does not remove phosphorus, which is a cause of eutrophication and a scarce resource. In conventional pig farming wastewater treatment (activated sludge method), excess sludge is generated during COD removal, which must be removed periodically or even daily. This, along with the electricity costs for aeration, is one of the main operating costs. [Means for solving the problem]

[0010] The present invention provides an advanced wastewater treatment system, system, and method for livestock farming, which uses an anaerobic bioelectrochemical system (BES) to simultaneously remove nitrate ions (nitrite ions) from aerated wastewater (hereinafter also called aerated wastewater or secondary treated wastewater) in a cathode tank and organic matter, suspended solids, and odors (volatile fatty acids) from raw wastewater in an anode tank. This advanced wastewater treatment system comprises at least two separate tanks. In the anode tank, BOD is oxidized to remove suspended solids (SS), odors, and pathogens from raw pig farm wastewater, while simultaneously, in the cathode tank, nitrate ions are reduced to nitrogen gas from aerated wastewater. Raw wastewater from livestock barns can be fed into the anode tank for treatment, reducing the organic load on the aeration tank, thereby extending the lifespan of the aeration tank and reducing the cost of removing excess sludge associated with aeration. Subsequently, the wastewater treated in the anode tank enters the existing aeration tank, where residual BOD can be removed and ammonium can be nitrified to nitrate ions. The water treated in the aeration tank can then be used as aerated wastewater.

[0011] The present invention includes the following embodiments. (1) A device for simultaneously performing denitrification of aerated wastewater and treatment of raw wastewater, It comprises at least one anode inside, and at least one anode tank for treating raw wastewater, It comprises at least one cathode inside and at least one cathode tank for denitrifying aerated wastewater, Here, the anode tank is attached to the cathode tank via a separator for transporting anions and / or cations between the anode tank and the cathode tank. (2) The apparatus according to (1), wherein the cathode tank and / or anode tank further comprises a reference electrode inside. (3) The apparatus according to (1), wherein the anode and / or cathode are conductive electrodes. (4) The apparatus according to (3), wherein the conductive electrode comprises carbon fiber or stainless steel. (5) The apparatus according to (1), wherein at least one of the anode tank and the cathode tank is equipped with means for continuously or periodically stirring the inside of the tank. (6) The apparatus according to (1), wherein the apparatus is connected to an aeration tank, which is preferably an existing aeration tank already installed on site and located in front of the apparatus of the Disclosure, thereby enabling the farmer to comply with new regulations regarding wastewater standards for nitrate ions, nitrite ions and / or ammonium, and the apparatus can be connected to the aeration tank directly or via one or more intermediate tanks or containers, such as a sedimentation tank.

[0012] (1A) A system for simultaneously performing anaerobic removal of organic compounds, such as organic matter, suspended solids and volatile fatty acids and pathogens, from raw wastewater containing organic compounds, and removal of nitrate ions and / or nitrite ions from aerated wastewater containing nitrate ions and / or nitrite ions, 1) The apparatus described in (1) or (2), 2) Means for adjusting the potential between the cathode and the anode, or between the cathode or anode and a reference electrode, Here, this means is connected to the anode and cathode, or to the anode, cathode and reference electrode, In the anode tank, the power-generating bacteria decompose organic compounds in the raw wastewater and supply electrons through the anode; and In the cathode tank, denitrifying bacteria receive electrons through the cathode and reduce nitrate ions and / or nitrite ions in the aerated wastewater to N2 gas. system. (2A) The system according to (1A), wherein the means for adjusting the potential is a potentiostat, an external resistor, or an open-circuit potential (OCP) mode. (3A) The system according to (1A), wherein the power-generating bacteria include at least one bacterium selected from the group consisting of the species Geobacter, Desulfovibrio, Syntrophobacter, Clostridium, Alicycliphilus, Thauera, Acidovorax, Xanthomonas, Bacteroides, Rhodopseudomonas, Thiomonas, Acinetobacter, Stenotrophomonas, Dechloromonas, Pseudomonas, Azoarcus, and Ralstonia. (4A) The system according to (1A), wherein the denitrifying bacteria include at least one bacterium selected from the group consisting of the species Syderoxidans, Gallionela, Thiobacillus, Thauera, Mycobacterium, Alicycliphilus, Azoarcus, Acidovorax, Psudomonas, Dechloromonas, Methylibium, Burkholderia, Leptothrix, Ralstonia, Aromatoleum, Cupriavidus, Delfia, Nitrosomonas, Methylococcus, and Maribacter.

[0013] (1B) At least one anode tank having at least one anode inside, It includes at least one cathode tank having at least one cathode inside, Here, the anode tank is used by using a device attached to the cathode tank via a separator to transport anions and / or cations between the anode tank and the cathode tank, A method for simultaneously removing organic compounds, such as organic matter, suspended solids, and volatile fatty acids, as well as pathogens, from raw wastewater containing organic compounds via anaerobic means, and removing nitrate ions and / or nitrite ions from aerated wastewater containing nitrate ions and / or nitrite ions, 1) Adding raw wastewater to the anode tank and adding aerated wastewater to the cathode tank, and then, 2) The process includes the step of adjusting the potential between the anode and the cathode, Here, in the anode tank, the power-generating bacteria decompose organic compounds, thereby supplying electrons through the anode; and in the cathode tank, the denitrifying bacteria receive electrons through the cathode and reduce nitrate ions and / or nitrite ions to N2 gas. method. (2B) At least one anode tank having at least one anode inside, A cathode tank having at least one cathode inside, Includes a reference electrode in a cathode tank or anode tank, Here, the anode tank is attached to the cathode tank via a separator to transport anions and / or cations between the anode tank and the cathode tank, by using the apparatus. A method for simultaneously removing organic compounds, such as organic matter, suspended solids, and volatile fatty acids, as well as pathogens, from raw wastewater containing organic compounds via anaerobic means, and removing nitrate ions and / or nitrite ions from aerated wastewater containing nitrate ions and / or nitrite ions, 1) Adding raw wastewater to the anode tank and adding aerated wastewater to the cathode tank, and then, 2) The process includes the step of adjusting the potential of either the cathode or anode relative to the reference electrode, Here, in the anode tank, the power-generating bacteria decompose organic compounds, thereby supplying electrons through the anode; and in the cathode tank, the denitrifying bacteria receive electrons through the cathode and reduce nitrate ions and / or nitrite ions to N2 gas. method. (3B) The method according to (1B) or (2B), wherein the power-generating bacteria comprises at least one bacterium selected from the group consisting of the species Geobacter, Desulfovibrio, Syntrophobacter, Clostridium, Alicycliphilus, Thauera, Acidovorax, Xanthomonas, Bacteroides, Rhodopseudomonas, Thiomonas, Acinetobacter, Stenotrophomonas, Dechloromonas, Pseudomonas, Azoarcus and Ralstonia. (4B) The method according to (1B) or (2B), wherein the denitrifying bacteria comprises at least one bacterium selected from the group consisting of the species Syderoxidans, Gallionela, Thiobacillus, Thauera, Mycobacterium, Alicycliphilus, Azoarcus, Acidovorax, Psudomonas, Dechloromonas, Methylibium, Burkholderia, Leptothrix, Ralstonia, Aromatoleum, Cupriavidus, Delfia, Nitrosomonas, Methylococcus and Maribacter. (5B) The method according to (1B) or (2B), wherein the raw wastewater is livestock wastewater or its supernatant. (6B) The method according to (1B) or (2B), wherein the raw wastewater is swine wastewater or its supernatant. (7B) The method according to (1B) or (2B), wherein the aerated wastewater is aerated livestock wastewater or its supernatant containing low levels of organic compounds. (8B) The method according to (1B) or (2B), wherein the aerated wastewater is aerated swine wastewater or its supernatant containing low levels of organic compounds. (9B) The method of (2B), wherein in step 2), a potential of -0.2 to -0.8 V relative to the reference electrode (Ag / AgCl) is applied to and adjusted at the cathode. (10B) In step (2), the method according to (2B), wherein the potential is applied and adjusted to the cathode at -0.4 to -0.6 V with respect to the reference electrode (Ag / AgCl). (11B) (0) The method according to (1B) or (2B), further comprising the step of inoculating the anode tank and / or the cathode tank with activated sludge at a volume of 0% to 60% thereof. (12B) The method according to (11B), wherein step (0) is the step of inoculating the anode tank and / or the cathode tank with activated sludge at a volume of 20% to 25% thereof. (13B) The aerated wastewater after step (2) contains NO3 with a total nitrogen equivalent of 100 mg / L or less - and NO2 - The method according to (1B) or (2B).

[0014] (1C) At least one anode tank having at least one anode therein, and at least one cathode tank having at least one cathode therein, wherein the anode tank is attached to the cathode tank via a separator for transporting anions and / or cations between the anode tank and the cathode tank, A method for simultaneously performing anaerobic removal of organic compounds, such as organic matter, suspended substances, volatile fatty acids, and pathogens, from raw wastewater containing electricity-generating bacteria and organic compounds, and removal of nitrate ions and / or nitrite ions and phosphorus from aerated wastewater containing denitrifying bacteria and nitrate ions and / or nitrite ions and phosphorus, 1) A step of adding raw wastewater to the anode tank and adding aerated wastewater to the cathode tank, and then, 2) A step of adjusting the potential between the anode and the cathode, wherein in the anode tank, the electricity-generating bacteria decompose the organic compounds, thereby supplying electrons through the anode; and in the cathode tank, the denitrifying bacteria receive electrons through the cathode, reduce nitrate ions and / or nitrite ions to N2 gas, and the phosphate precipitates in the cathode tank. Method. (2C) At least one anode tank having at least one anode inside, A cathode tank having at least one cathode inside, Includes a reference electrode in a cathode tank or anode tank, Here, the anode tank is used by using a device attached to the cathode tank via a separator to transport anions and / or cations between the anode tank and the cathode tank, A method for simultaneously performing anaerobic removal of organic compounds, such as organic matter, suspended solids, and volatile fatty acids, and pathogens from raw wastewater containing power-generating bacteria and organic compounds, and removal of nitrate ions and / or nitrite ions from aerated wastewater containing denitrifying bacteria and nitrate ions and / or nitrite ions, 1) Add raw wastewater to the anode tank and aerated wastewater to the cathode tank, and then 2) The process includes the step of adjusting the potential of either the cathode or anode relative to the reference electrode, Here, in the anode tank, the power-generating bacteria decompose organic compounds, thereby supplying electrons through the anode; and in the cathode tank, the denitrifying bacteria receive electrons through the cathode, reducing nitrate ions and / or nitrite ions to N2 gas, and phosphate precipitates in the cathode tank. method. (3C) The method according to claim 24 or 25, wherein by weight of phosphorus, more than 30% of the phosphate phosphorus present in the aerated wastewater is removed by step 2).

[0015] (A1) At least one anode tank having at least one anode inside, It comprises at least one cathode tank having at least one cathode inside, Here, the anode tank is attached to the cathode tank via an anion exchange membrane or a cation exchange membrane to transport anions or cations that can move between the anode tank and the cathode tank; and The anode tank contains power-generating bacteria internally, preferably on the surface of the anode, and the cathode tank contains denitrifying bacteria internally, preferably on the surface of the cathode. Device. (A2) The apparatus according to (A1), wherein the cathode chamber further comprises a reference electrode inside. (A3) The apparatus according to (A1) or (A2), wherein the anode and / or cathode are carbon electrodes. (A4) The apparatus according to any one of (A1) to (A3), wherein the power-generating bacteria include at least one bacterium selected from the group consisting of the species Geobacter, Desulfovibrio, Syntrophobacter, Clostridium, Alicycliphilus, Thauera, Acidovorax, Xanthomonas, Bacteroides, Rhodopseudomonas, Thiomonas, Acinetobacter, Stenotrophomonas, Dechloromonas, Pseudomonas, Azoarcus, and Ralstonia. (5) The apparatus according to any one of (A1) to (A4), wherein the denitrifying bacteria include at least one bacterium selected from the group consisting of the species Syderoxidans, Gallionela, Thiobacillus, Thauera, Mycobacterium, Alicycliphilus, Azoarcus, Acidovorax, Psudomonas, Dechloromonas, Methylibium, Burkholderia, Leptothrix, Ralstonia, Aromatoleum, Cupriavidus, Delfia, Nitrosomonas, Methylococcus, Methylobacter, Nitrosococcus, Mesorhizobium, and Maribacter.

[0016] (A1A) 1) The apparatus described in (A2), 2) A potentiostat for applying a potential to the cathode electrode relative to the reference electrode, comprising an external resistance mode or an open-circuit mode, system. (A1B) A method for simultaneously removing organic compounds, such as organic matter, suspended solids and volatile fatty acids, from raw wastewater containing organic compounds, and removing nitrate ions and / or nitrite ions from aerated wastewater containing nitrate ions and / or nitrite ions, 1) Add raw wastewater to the anode tank of the apparatus described in (A2), and add aerated wastewater to the cathode tank of the apparatus, and then, 2) The process includes the step of applying a potential to the cathode, preferably using a reference electrode (both connected to a potentiostat), Here, in the anode tank, the power-generating bacteria decompose organic compounds, thereby supplying electrons to the anode connected to the potentiostat; and in the cathode tank, the denitrifying bacteria receive electrons through the cathode and reduce nitrate ions and / or nitrite ions by electrons, preferably to NO, N2O and / or N2. method. (A2B) The method according to (A1B), wherein the raw wastewater is livestock wastewater or its supernatant, preferably pig farming wastewater or its supernatant. (A3B) The method according to (A1B), wherein the aerated wastewater is aerated livestock wastewater or its supernatant containing low levels of organic compounds, preferably aerated swine wastewater or its supernatant. (A4B) Raw wastewater contains organic compounds and NH4 + The aerated wastewater contains, and in step 2) organic compounds are removed, and then NH4 + The method described in (A1B), wherein the raw wastewater is obtained after the nitrates are converted to nitrate ions and / or nitrite ions by nitrifying bacteria under aeration. (A5B) The method described in (A1B), wherein in step 2), the cathode is set to -0.2 to -0.8 V (Ag / AgCl), preferably -0.4 to -0.6 V (Ag / AgCl). (A6B) The method described in (A1B), wherein at least step 2) is carried out under anaerobic conditions. [Effects of the Invention]

[0017] One embodiment of the present invention can exhibit the following effects. 1. Tertiary treatment of aerated wastewater (inside the cathode tank) A potential advantage of this system is that, by using electrodes as electron donors, it allows autotrophic denitrifying bacteria to reduce nitrate ions to nitrogen gas without the labor-intensive adjustment of organic matter or dissolved oxygen (DO) levels and the addition of additional compounds required by conventional methods. Nitrate-containing wastewater with small amounts of organic matter, such as aerated nitrification wastewater, nitrate-contaminated groundwater, or aquaculture water, is suitable for this system (low C / N wastewater), but is not limited to these. In addition, this system may enable conventional heterotrophic denitrification. Nitrate nitrogen can be removed to below the general wastewater standard (100 mg / L).

[0018] 2. Secondary treatment of raw wastewater (in the anode tank) One embodiment of the present invention can function as a secondary wastewater treatment that reduces the organic load from raw wastewater (by more than 80%), thereby reducing the burden on existing aeration tanks and saving aeration costs and overall operating costs.

[0019] In addition, this system can remove suspended solids (80%), odors (80%), and Escherichia coli (a pathogenicity indicator). This system can be added to existing wastewater treatment as an advanced treatment system (Figures 10 and 11). The system consists of a continuous sedimentation tank for removing large particles from wastewater and a BES bioreactor. The overall advantage of this system is that it reduces operating costs compared to conventional treatment systems, including the removal of excess sludge, the addition of chemicals, and electricity usage, and thus reduces CO2 emissions. [Brief explanation of the drawing]

[0020] [Figure 1]Biocathode denitrification performance in short-term experiments. A) Removal of NO3--N at an initial concentration of 300 mg L-1; B) Cathode current generation curves under applied potentials of -0.6 V vs. Ag / AgCl, -0.4 V vs. Ag / AgCl, and -0.2 V vs. Ag / AgCl; C) NO2--N concentration; D) NH4-+-N concentration. [Figure 2] Biocathode denitrification performance with cation exchange membrane (CEM) versus anion exchange membrane (AEM). A) Removal of NO3-N at an initial concentration of 300 mg L-1. Here, BES with AEM was supplemented with an additional 300 mg L-1 of nitrate nitrogen for its complete removal on day 6; B) Cathode current generation curve. [Figure 3] Average cumulative removal amount of NO3-N in long-term experiments. The BES was operated in fed-batch mode for 45 cycles with a 3-day HRT. In each odd-numbered cycle, the anode tank was filled with untreated wastewater and the cathode tank with aerated wastewater. In each even-numbered cycle, only the cathode tank was filled with aerated wastewater from a new batch. [Figure 4] A) Cumulative amount of current output and nitrate ion reduction on the final day of the operating cycle for 0-15 days; B) 15-35 days; C) 145-170 days. Blue diamond: Cumulative amount of NO3-N removed on day 3. [Figure 5] Taxonomic classification of microbial communities on the cathode. A) Relative occupancy at the genus level and B) at the species level (heatmap of the top 30 most matching species. Color intensity indicates the relative occupancy value after applying a binary logarithmic transformation). CEM: Cation exchange membrane (short-term experiment), AEM: Anion exchange membrane (long-term experiment). [Figure 6] The occupancy rate of the most cohesive dominant species involved in denitrification. The composition of denitrifying microorganisms exhibiting a occupancy rate higher than 3% in samples from Stage I under an applied potential of -0.6V and OCP. [Figure 7]Schematic diagram of one embodiment of the present application. Figure 7A shows one embodiment of an apparatus having one anode tank and one cathode tank. 7A-i. Application of potential by a potentiostat, ii. Adjustment of potential by an external resistor, and iii. Open-circuit potential. Figure 7B shows one embodiment of the method cascade in the present application. Microbial communities containing denitrifying bacteria were confirmed to be localized on the cathode surface by SEM. Figure 7C shows the conversion cascade of NO3- to N2 by denitrifying bacteria. Figure 7D shows that the removal of NO3- was enhanced by applying a potential to the cathode in the cathode tank. Figure 7E Removal of nitrate ions by applying a potential (-0.4V) to the cathode and using an external resistor (500Ω). Figure 7F Cathode potential, anode potential, and current in the external resistor experiment (R=500Ω). [Figure 8] A demonstration reactor system having multiple anode tanks and multiple cathode tanks. [Figure 9] Schematic diagram of the demonstration reactor system. Two anode tanks and four cathode tanks; cathode compartment: 36L; anode compartment: 18L; carbon brushes / fibers as electrodes; anion exchange membrane; tray volume approximately 65L; -0.4V vs Ag / AgCl (at the cathode). The raw wastewater is biologically treated in the anode tanks, including with power-generating bacteria. The treated raw wastewater is then transferred to the aeration tank, where the remaining organic matter is treated by aeration and by nitrifying bacteria that can convert NH4+ to NO3-. The aerated wastewater is then transferred to the cathode tanks as aerated wastewater. [Figure 10] Schematic diagram of the system installation. [Figure 11] Installation on existing wastewater treatment facilities. [Figure 12] Removal of organic compounds in the anode tank. Treatment in the anode tank reduces the COD in the raw wastewater. Figure 12A shows the change in COD over time. Figure 12B shows the COD value, and Figure 12C shows the turbidity. [Figure 13]Removal of NO3- in the cathode tank. Figure 13A shows the change in NO3- over time. Figure 13B shows the concentration of NO3-. Figure 13C shows the change in NO2- over time, which indicates that NO3- is reduced and converted by denitrifying bacteria. Figure 13D shows the relationship between acquired current and NO3- removal. [Figure 14] Overview of the water quality after treatment in this system.

[0021] (Intentionally left blank) [Modes for carrying out the invention]

[0022] Unless otherwise specified, all terms in this invention have the same meaning as those generally understood by those skilled in the art in the field to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural references unless otherwise specified. Similarly, the word “or” is intended to include “and” unless otherwise specified. The term “a few” in this description means two to three numbers. The term “several” in this description means two to six numbers. In case of any conflict, this specification, including the definitions of terms, shall prevail. In addition, materials, methods, and examples are illustrative and not intended to limit the scope.

[0023] In one embodiment, the present application includes an apparatus comprising at least one anode tank having at least one anode inside, and at least one cathode tank having at least one cathode inside. The anode tank may have at least one inlet for adding raw wastewater to the anode tank and at least one outlet for recovering the treated raw wastewater from the anode tank. In the anode tank, the inlet may be the same as the outlet. The cathode tank may have at least one inlet for adding aerated wastewater to the cathode tank and at least one outlet for recovering the treated aerated wastewater from the cathode tank. In the cathode tank, the inlet may be the same as the outlet. The volume and number of the tanks are not limited. If the apparatus comprises multiple anode tanks and multiple cathode tanks, the anode tanks may be directly connected to each other so as to allow drainage to move between the anode tanks, and the cathode tanks may be directly connected to each other so as to allow drainage to move between the cathode tanks. The anode tank may be connected to the cathode tank so that ions, particularly anions, can move between the anode and cathode tanks. In one embodiment, the anode tank may be connected to the cathode tank via a separator, for example, an ion exchange membrane (but not other), preferably via an anion exchange membrane. The anion exchange membrane allows anions to permeate between the tanks, while the anion exchange membrane allows cations, for example, NH4 + Do not allow it to pass through. The anode tank can be used to treat raw wastewater. Activated sludge or raw wastewater can be inoculated into the anode tank as an inoculum containing electrogenic bacteria. As a result, the anode tank can preferably contain electrogenic bacteria on the surface of the anode. Electrogenic bacteria, or electron-producing bacteria (exoelectrogens), are a group of microorganisms that, under anaerobic or microaerophilic conditions, can transfer electrons to or from electron acceptors, including electrodes, oxidized minerals, and other bacteria, through the cell envelope to the extracellular space. Electrogenic bacteria can decompose organic compounds in the raw wastewater to produce CO2 and electrons, which can then be supplied to the anode. Electricity-generating bacteria may be autotrophic and include, but are not limited to, species of Geobacter, Desulfovibrio, Syntrophobacter, Clostridium, Alicycliphilus, Thauera, Acidovorax, Xanthomonas, Bacteroides, Rhodopseudomonas, Thiomonas, Acinetobacter, Stenotrophomonas, Dechloromonas, Pseudomonas, Azoarcus, and Ralstonia. Further examples of electricity-generating bacteria include Escherichia, Methanospirillum, Rhodobacter, and Stenotrophomonas. A cathode tank can be used for denitrification. Activated sludge or aerated wastewater can be inoculated into the cathode tank as an inoculant containing denitrifying bacteria. As a result, the cathode tank can preferably contain denitrifying bacteria on the surface of the cathode. The denitrifying bacteria, preferably produced by electrogenic bacteria, and using the anode, means for applying and / or adjusting the potential, and electrons supplied through the cathode, perform denitrification as part of the nitrogen cycle, metabolizing nitrogen compounds using various enzymes, producing nitrate ions and nitrite ions (NO3). - NO2 - ) can be converted back to nitrogen gas (N2) or nitric oxide, nitrous oxide (NO, N2O). Preferably, denitrifying bacteria can convert nitrate ions (NO3 - ) and nitrite ions (NO2 -They reduce ) to nitrogen gas (N2). Denitrifying bacteria may be autotrophic and include, but are not limited to, the species Syderoxidans, Gallionela, Thiobacillus, Thauera, Mycobacterium, Alicycliphilus, Azoarcus, Acidovorax, Psudomonas, Dechloromonas, Methylibium, Burkholderia, Leptothrix, Ralstonia, Aromatoleum, Cupriavidus, Delfia, Nitrosomonas, Methylococcus, and Maribacter. Further examples of denitrifying bacteria include Janthinobacterium, Hyphomicrobium, Mesorhizobium, Methylobacillus, and Rhodobacter and Rhodopseudomonas.

[0024] In one embodiment, the cathode and / or anode chamber may further include a reference electrode. When a potential is applied to either the cathode or the anode, the potential is preferably adjusted using the reference electrode.

[0025] In one embodiment, the anode and / or cathode are preferably resistant to corrosion caused by drainage. The anode and / or cathode can be conductive electrodes, preferably carbon fiber electrodes or stainless steel.

[0026] In one embodiment, the cathode tank and / or anode tank may further include means for continuously or periodically agitating the contents of the tank. These means may include, but are not limited to, a stirring pump or a bubbling machine. These means may be the shape or structure of the tank itself.

[0027] In one embodiment, the present application includes a system comprising the aforementioned apparatus and means for adjusting the potential. The potential can be adjusted to either the cathode or anode relative to a reference electrode, or between the anode and cathode. This means may have the function of applying the potential. This means includes, but is not limited to, a potentiostat, an external resistor, and an open-circuit potential. In some embodiments, the external resistor may have a resistance of 100Ω to 1000Ω. The term “open-circuit potential” corresponds to the use of a resistor with infinite or near-infinite resistance, for example, when the termination of the circuit is removed or when there is no external load between the electrodes.

[0028] In one embodiment, the present application includes a method for removing organic compounds, such as organic matter, suspended solids, and volatile fatty acids, from raw wastewater containing organic compounds; a method for removing nitrate ions and / or nitrite ions from aerated wastewater containing nitrate ions and / or nitrite ions; a method for removing phosphorus from aerated wastewater containing phosphorus; a method for simultaneously removing organic compounds, such as organic matter, suspended solids, and volatile fatty acids, from raw wastewater containing organic compounds, and removing nitrate ions and / or nitrite ions from aerated wastewater containing nitrate ions and / or nitrite ions; and a method for simultaneously performing anaerobic removal of organic compounds, such as organic matter, suspended solids, and volatile fatty acids, and pathogens, from raw wastewater containing power-generating bacteria and organic compounds, and removing nitrate ions and / or nitrite ions, and phosphorus, from aerated wastewater containing denitrifying bacteria and nitrate ions and / or nitrite ions.

[0029] These methods 1) The process of adding raw wastewater to the anode tank of the aforementioned apparatus and adding aerated wastewater to the cathode tank of the apparatus, and then, 2) The process may include the step of adjusting the potential to either the cathode or the anode (preferably relative to a reference electrode), where “adjusting the potential” may include “applying a potential.” In the anode tank, the power-generating bacteria can supply electrons via an anode connected to means for decomposing organic compounds and applying and / or adjusting the potential. In the cathode tank, denitrifying bacteria can receive electrons through the cathode and use these electrons to reduce nitrate ions and / or nitrite ions, preferably converting them into NO, N2O, and / or N2 gases. If the wastewater contains phosphorus, phosphates, such as calcium phosphate, can be precipitated in the cathode tank.

[0030] In one embodiment, the raw wastewater added to the anode tank may be livestock wastewater or its supernatant, preferably swine wastewater or its supernatant. In particular, the raw wastewater may preferably be wastewater obtained by applying any sedimentation treatment and removing any precipitates and / or solids, yet still containing abundant organic compounds (e.g., COD values ​​of 1000 mg / L to 10000 mg / L, more preferably 1000 mg / L to 3000 mg / L). The raw wastewater may contain live power-generating bacteria.

[0031] Aerated wastewater may be aerated livestock wastewater or its supernatant, preferably aerated pig wastewater or its supernatant. Aerated wastewater may be aerated wastewater obtained by applying any sedimentation treatment and removing any sediment and / or solids. In particular, aerated wastewater may be rich in, for example, 100 mg / L or more (NO3 - -N), 200mg / L or more (NO3 - -N) or 100-400 mg / L (NO3 - The aerated wastewater may contain nitrate ions and / or nitrite ions (-N). Furthermore, the aerated wastewater may contain fewer organic compounds (e.g., a BOD value of 5 to 30, but not limited to this). Therefore, the BOD / N ratio of the aerated wastewater may be 3 or less, 2 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less. The aerated wastewater may contain live denitrifying bacteria.

[0032] In another embodiment, the raw wastewater treated in the anode tank can be used as a source for aerated wastewater. In this case, the raw wastewater preferably contains organic compounds and NH4 + This may include: The raw wastewater treated in the anode tank is recovered and applied to aeration treatment. In this case, NH4 + This is due to nitrifying bacteria, NO3 - and / or NO2 - It is converted to aeration. If necessary, the aerated wastewater can be further applied to any sedimentation treatment to remove any sediment and / or solids. Treated wastewater derived from the raw wastewater may be available as aerated wastewater to be added to the cathode tank. Thus, the apparatus used may be connected to the aeration tank via an inlet. This connection makes it possible to introduce the raw wastewater treated in the anode tank into the aeration tank, where it is aerated, converting the treated raw wastewater into aerated wastewater, and then introducing the aerated wastewater into the cathode tank. Nitrifying bacteria obtain energy by oxidizing inorganic nitrogen compounds. Nitrifying bacteria may be autotrophic bacteria and include, but are not limited to, species of the genera Nitrosomonas, Nitrosococcus, Nitrobacter, Nitrospina, Nitrospira, and Nitrococcus.

[0033] When a potentiostat is used as a means to adjust the potential, the cathode can be balanced in step 2 at approximately or on average -0.1 to -1V, preferably -0.2 to -0.8V, and more preferably -0.4 to -0.6V pairs (Ag / AgCl). This potential can increase the accumulation of denitrifying bacteria on the cathode.

[0034] If the bacteria in the anode and cathode tanks are anaerobic bacteria, at least step 2) can be carried out under anaerobic conditions. Furthermore, this method can be carried out at ambient temperature (i.e., 10 to 35°C, preferably 20 to 30°C or more preferably 22 to 28°C or about 25°C).

[0035] In addition to the raw wastewater, activated sludge containing live bacteria (including, but not limited to, power-generating bacteria, nitrifying bacteria, and denitrifying bacteria) can be added to the anode tank in an amount of 0% to 60% of the anode tank's capacity, preferably 20% to 25%. In addition to the aerated wastewater, activated sludge containing live bacteria (including, but not limited to, power-generating bacteria, nitrifying bacteria, and denitrifying bacteria) can be added to the cathode tank in an amount of 0% to 60% of the cathode tank's capacity, preferably 20% to 25%. Activated sludge can be added before step 2). Activated sludge can be added before adding wastewater, at the same time as adding wastewater, or after adding wastewater.

[0036] The above method uses NO3 with a total nitrogen equivalent of 100 mg / L or less, 50 mg / L or less, or 10 mg or less. - and NO2 - This allows for the provision of aerated wastewater after step 2, including the aerated wastewater. The above method can provide raw wastewater after step 2 containing organic compounds removed to a COD value of 100 mg / L to 1000 mg / L or 1000 mg / L or higher.

[0037] The above method can be carried out such that, by weight of phosphorus, more than 30%, more than 40%, more than 50%, or more than 60% of the phosphate phosphorus present in the aerated wastewater is removed by step 2). [Examples]

[0038] The present invention will be described in more detail in the following examples. However, the present invention is not limited to these examples. In these examples herein, experiments using commercially available kits and reagents were performed according to the accompanying protocols unless otherwise specified. The present invention will be demonstrated by the following non-limiting examples.

[0039] Example 1 Materials and methods 1. Design and construction of BES A two-chamber BES was fabricated using a transparent polyacrylic sheet. To provide a large surface area for bacterial growth, two carbon brush electrodes containing ZOLTEK Panex 35 carbon fiber with a density of 800K chips per 2.5 cm, each containing two fragments of 3.5 mm diameter, 10 cm long stainless steel wire (Hengshui Chiehwang Industry and Trade Co, China), were used for both the anode and cathode. Before first use, the brushes were immersed in acetone overnight, heated in a muffle furnace at 450°C for 30 minutes (Feng et al., 2010), and washed three times with distilled water. The distance between the anode and cathode electrodes was set to 2 cm. Nafion® 117 (Dupont, USA) membrane was used as the cation exchange membrane (CEM) between the anode and cathode chambers, and AMI-7001 (Membranes International Inc, USA) was used as the anion exchange membrane (AEM). The frame of the two membranes is 48cm 2 The system was installed with the specified surface area. The electrodes were connected to a potentiostat (Uniscan PG580RM) using titanium wire. All experiments were performed with a 3-electrode setup or open-circuit potential. The cathode and anode compartments were each 1L. This system was operated at a controlled temperature of 25°C.

[0040] 2. Vaccination, collection, and system operation Both swine wastewater (raw and aerated wastewater) and activated sludge from the aeration tank were obtained from the Okinawa Prefectural Livestock Research Center (Japan). Activated sludge was inoculated into both the anode and cathode tanks at an initial ratio of 1:3 to the wastewater flow. After inoculation, the anode tank was filled with undiluted (full-strength) raw swine wastewater, while the cathode tank was filled with the wastewater after aeration treatment. Table 1 shows the chemical composition of the two types of wastewater compared to the same wastewater after treatment in the BES. Before supplying to the BES, the wastewater was passed through a mesh with a pore size of 1 mm to remove any remaining sludge particles. The average initial pH of the wastewater used in the anode tank was 6.86 ± 0.26, and the conductivity was 263 ± 28 μScm. -1The average initial pH of the aerated wastewater used in the cathode tank was 7.96 ± 0.34, and the conductivity was 315.5 ± 8.5 μScm. -1 The nitrate nitrogen level of the wastewater used in the cathode tank was reduced to 300 mg L using sodium nitrate. -1 The settings were adjusted accordingly. Throughout all experiments, both tanks were maintained under anaerobic conditions and operated in fed-batch mode.

[0041] 3. BES operation All experiments were performed in a three-electrode setup or open-circuit potential. In this case, the cathode was used as the working electrode controlled by chronoamperometry, and the Ag / AgCl electrode was used as the reference electrode (Radiometer XR300 Reference Electrode, Hach, 0.197V versus a standard hydrogen electrode). After inoculation, the BES was pre-incubated under open-circuit potential (OCP) to allow the bacterial biofilm to adapt to the environment. Table 2 shows an overview of the three stages of the experimental operation tested in the examples. In Stage I, nitrate ion removal and the resulting final product were evaluated under the following conditions: different applied cathode potentials (-0.2V, -0.4V, and -0.6V against Ag / AgCl reference electrodes), open-circuit potential (OCP), a reactor with no inoculation and an applied potential of -0.6V, and a reactor without electrodes. In Stages II and III, the BES was operated under an applied potential of -0.6V and in OCP mode. All BESs were operated in duplicate. For the experiments in Stage III, the BES was operated for 180 days to examine its performance and analyze how the microbial community adapted over time. Cell voltage in open-circuit (OCP) mode was monitored with a data logger (GRAPHTEC Midi Logger GL240). Coulomb efficiency was given by the following equation:

number

[0042] 4.Chemical analysis Using the HACH test kit (USA), chemical oxygen demand (COD), volatile fatty acids (VFA), and ammonium (NH4) were measured. + -N), nitrate ion (NO3 - -N) and nitrite ions (NO2 - The concentration of -N) was analyzed. All samples (except COD) were filtered through a 0.45 μm filter before measurement. pH and conductivity were measured using a pH meter (LAQUAtwin-pH-33, Horiba Scientific, Japan) and an EC meter (LAQUAtwin-EC-33, Horiba Scientific, Japan). N2O in the liquid phase was analyzed using gas chromatography-mass spectrometry (PEGASUS 4D GCxGC-TOFMS, LECO, MI, USA) equipped with a PLOT column particle trap (0.25 mm × 2.5 m, GL Science, Tokyo, Japan) and an RT-Q-BOND separation column (0.25 mm × 30 m, 8 μm, RESTEK, PA, USA). Suspended solids (SS) were measured in accordance with the environmental standards in Appendix 9 of the Water Pollution Control Act (Japan).

[0043] 5. Microbiological analysis Biofilm samples were collected from both cathode electrodes at 7 days after Stage I and at 180 days after cycle #45 in Stage III, when the microbial community had stabilized. Genomic DNA was extracted from solid samples using the Maxwell RSC DNA Kit (Promega, USA). RNA was extracted using the Maxwell RSC RNA Kit (Promega, USA). The quality of the extracted DNA and RNA was analyzed using a 4200 TapeStation (Agilent, USA). For DNA shotgun sequencing, the NEBNext Ultra® II FS DNA Library Prep Kit for Illumina was used, and sequencing was performed on a NovaSeq6000 (Illumina). For ribosomal RNA removal, the Ribo-Zero rRNA removal kit (Bacteria) was used. Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, and sequencing was performed on NovaSeq6000 and HiSeq2500 Rapid (Illumina). Coliform bacteria were counted according to the method of Japanese Industrial Standard K350-20-10:2001. For scanning electron microscopy (SEM) analysis, small pieces of the cathode electrode containing the biofilm were taken from the BES and immersed in 2.5% (w / v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The samples were then washed and sequentially dehydrated with an ethanol series. The fixed samples were dried in a critical point dryer and sputtered with a gold layer. The coated samples were examined using a 15kV SEM (JEOL JSM-7900F), and images were digitally captured.

[0044] 6. Sequencing and Data Analysis The combined taxonomic domain information analysis was performed using the MG-RAST (Meta Genome Rapid Annotation using Subsystem Technology) server under the following conditions: the taxonomic domain filter was set to bacteria and archaea, with % identity set to 90% and length set to 50; all other parameters were set to their default values. Bar plots and heatmaps showing genome abundance were generated using the ggplot2 package (Wickham, 2016) in R (R Core Team, 2013). The plot data was exported as a TSV file using the RefSeq database in MG-RAST.

[0045] [Table 2]

[0046] [Table 3]

[0047] [Table 4]

[0048] result 1. Overall performance of denitrification BES for simultaneous processing 1.1 Biocathode denitrification in BES under different electrochemical conditions (Stage I) A 2L biocathode BES was constructed to treat undiluted raw wastewater containing high levels of organic matter and volatile fatty acids in the anode tank, and treated aerated wastewater in the cathode tank. In the cathode tank, the BOD was 10 mg L. -1 Even when the levels were below a certain threshold, nitrate ions were reduced to nitrite ions, nitrous oxide, and nitrogen gas by denitrification via the microbial community in the cathode. From Stage I, the importance of the applied potential at the cathode for the nitrate ion removal rate was demonstrated. The applied potential was used in fed-batch mode during the 7-day experimental period for NO3 - The effect on N removal is shown in Figure 1A. 300 mg L -1 Initial NO.3 - -N concentration was set based on the highest value observed at local farms. This initial concentration was almost completely removed after 7 days in a BES under an applied potential of -0.6V, and the removed NO3 - -The amount of N is 282±12 mg L -1 The applied cathode potential of -0.6V showed the best removal compared to -0.4V and -0.2V (226±21 mg L under applied potentials of -0.4V and -0.2V, respectively). -1 NO3 - -N and 213±19 mg L -1 NO3 - -N). Furthermore, the amount of nitrate ions removed under OCP was significantly lower compared to the applied potential condition (145 ± 13 mg L). -1 NO3 - -N). Other control conditions, including electrode-free BES and inoculum-free BES, also showed low removal capacity (82±7 mg L each). -1 NO3 - -N and 87±11mg L -1 NO3 - -N). These results indicate the importance of applied potential and the presence of inoculum for faster denitrification. This is likely due to the accumulation of microbial communities under these conditions. Figure 1B shows the current response over time in the reactor under different applied potentials (-0.6V, -0.4V, and -0.2V). The decrease in cathode current indicates an active electrochemical reduction process, which, as previously reported (Chen et al., 2017), allows denitrification to proceed with the help of electrotrophic denitrifying bacteria. Figures 1A and 1B show a clear correlation between nitrate ion removal and cathode potential, with lower cathode potentials resulting in higher nitrate ion removal rates. This correlation is consistent with a previous report (Yu et al., 2015). As more nitrate ions are removed, the cathode current decreases rapidly, reflecting the consumption of electrons donated for denitrification by the cathode. The subsequent increase in cathode current was caused by a decrease in nitrate ion concentration due to the limited amount of nitrate ions available in the fed-batch mode. The cathode Coulomb efficiency for nitrate ion reduction exceeded 100% under all applied potential conditions (data not shown). These high values ​​indicate that heterotrophic denitrification using organic substrates in BES as electron donors can also contribute to nitrate ion removal rates. Nitrite ions, as an intermediate product of denitrification, were detected on day 1 of the experiment, increased until day 2, and then decreased as the accumulation period progressed (Figure 1C). The reduction of nitrate ions to nitrite ions indicates that it occurred in the cathode on days 1 and 2, when the denitrification process had just begun. Subsequently, the nitrite ions were bioelectrochemically reduced to the next denitrification step, namely NO, N2O to N2, by cathode microorganisms, consistent with previous reports (Puig et al., 2011). Furthermore, ammonia inflow from the anode to the cathode through the membrane was detected on days 1 and 2 (Figure 1D). Ammonia may be oxidized to nitrite ions during this period (see Section 3.4.1). Under electrochemically stimulated conditions, control BES (2-12 mg L) -1 NO2 - Compared to -N), higher concentrations of nitrite ions were detected (25-46.7 mg L). -1 NO2 --N) This was consistent with faster nitrate ion removal under applied potential. GC / MS analysis revealed that N2O, a potent greenhouse gas, was detected only at low concentrations (data not shown). This suggests that the denitrification cycle likely completed with nitrogen release. The denitrification reaction in BES likely contributes to the increase in alkalinity to pH 8.0. This pH remains within the optimal range for conventional denitrification (Sun et al., 2020). Generally, these results demonstrate the potential advantages of biocathode denitrification systems using aerated wastewater, combined with simultaneous treatment of raw wastewater by controlled electron delivery. Furthermore, this study highlights the importance of using sludge as inoculant. In a previous study, Khilyas et al. (Khilyas et al., 2017) compared different types of sludge as inoculants for treating pig farm wastewater and found that sludge collected from the same aeration tank performed better as an anode inoculant for microbial fuel cells than sludge from a brewery. In addition, high electron utilization efficiency, low sludge production, and easy handling are all promising features for nitrate ion removal using livestock wastewater for large-scale reactors.

[0049] 1.2. Treatment of raw wastewater from pig farming in the anode tank During these experiments, the removal of COD and VFA in the anode chamber was continuously monitored (Table 3). Under the applied cathode potential and OCP mode conditions, the COD consumption rate was similar (approximately 0.87 ± 0.07 g COD L). -1 d -1 ), on the first day, 1.62g L -1 d -1 ±0.03g L -1 d -1This was the highest removal rate. The overall average efficiency under these conditions was 58.5±2.6%, with the highest being achieved at -0.4V (61.4±0.5%), but there was no statistical difference within the BES under applied potential and OCP mode (data not shown). This may indicate that the potential applied to the cathode does not significantly affect the COD removal rate in the anode chamber in this system. 1.6±0.7g COD L -1 d -1 and 2.1±0.5g COD L -1 d -1 Similar COD removal rates have been previously reported (Vilajeliu-Pons et al., 2017). In this report, pig manure was treated in a 6-layer microbial fuel cell (MFC) in continuous mode. These results indicate that denitrifying BES was able to achieve similar processing rates to MFCs specifically designed for treating organic matter. On the other hand, in a reactor without inoculant (sludge) under an applied potential of -0.6V, the COD removal rate was 0.53 g L -1 d -1 ±0.21g L -1 d -1 The efficiency was 38.5 ± 2.1%. This result demonstrates the importance of sludge from the aeration tank as initial bacterial inoculum, contributing to the rapid accumulation of microbial communities. The best VFA removal was detected at applied potentials of -0.2V and -0.4V, with efficiencies of 41.5±8.3% and 39.7±6.2%, respectively. (486±18.5 mg VFA L) -1 d -1 The highest removal rate was observed at -0.2V. This indicates that the activity of the electrogenic bacteria reached its maximum when -0.2V was applied to the cathode. In this case, the BES cell voltage stabilized at 0.13±0.05V (data not shown). This means that the anode potential was 0.33±0.05V. It has been previously shown that when the anode potential is higher than 0.2V versus the Ag / AgCl reference electrode, the electrogenic bacteria operate at their highest electron transfer rate, resulting in a higher percentage of VFA removal (Prokhorova et al., 2017). Under other tested conditions, the potential at the anode did not exceed the value of 0.2V. To further investigate the water quality of wastewater after BES treatment in the anode tank, a coliform bacterial test, commonly used as an indicator of water pathogenicity or fecal contamination, was performed. After 7 days of experimental operation, a more than 1000-fold reduction in coliform density was observed (data not shown). This suggests that treatment with BES was able to suppress pathogenic bacteria in the wastewater. This is consistent with previous findings (Vasieva et al., 2019) that BES was able to disinfect wastewater concentrated with Enterobacteriaceae (Shigella, Yersinia, Vibrio). Further experiments are needed to understand whether different potentials at the anode affect the reduction in coliform density. In addition, suspended solids (SS) were also removed from the wastewater with an efficiency of 89% (Table 1).

[0050] 2. Optimization of nitrate ion removal in BES (Stage II) As a result of ion transfer through the cation exchange membrane (CEM), ammonium is transported from the anode to the cathode, reaching a maximum concentration of 162.5 ± 7.5 mg NH4. + -NL -1 It was detected (Figure 1D). The accumulation of ammonium increased the total nitrogen concentration in the cathode tank. To overcome this limiting factor, the CEM was replaced with an anion exchange membrane (AEM) in Stage II of the experimental operation. 300 mg L -1 NO3 - -The initial total amount of N was completely removed in the AEM reactor within 5 days (maximum 99±2 mg NO3) - -NL -1 d -1 ). On the other hand, in the CEM reactor, when -0.6V was applied, it took approximately 8-9 days (maximum 34mg L). -1 d -1 ±7mg L -1 d -1 (Figure 2A). Overall, the BES with AEM also achieved better organic matter removal in the anode tank (0.8g COD L). -1 d -1)。In this case, only a negligible amount of nitrate ions (about 1.1 mg NO3 - -N L -1 ) was detected. This indicates that either only a small amount of diffusion occurred or the nitrate ions that migrated through the AEM were reduced to nitrogen gas by denitrifying bacteria in the anode tank. This is consistent with previous studies based on pig farm wastewater (Vilajeliu-Pons et al. 2015). In stage II of the experimental operation, only a slight increase in ammonium was observed in the cathode tank (about 3.8 mg L -1 ±2.2 mg NH4 + -N L -1 d -1 ).

[0051] 3. Adaptability for long-term operation (stage III) Currently, little is known about the long-term adaptability of electroautotrophic denitrifying bacteria using actual livestock wastewater and the denitrification efficiency over time. In stage III of this study, long-term denitrification focusing on the stability and accumulation of denitrifying bacteria was investigated. The BES with AEM was operated for 45 cycles. Each cycle had a 3-day hydraulic retention time (HRT). Every odd cycle, the wastewater was changed in both tanks (the anode was filled with untreated wastewater and the cathode was filled with aerated wastewater). Every even cycle, only the cathode tank was filled with a new batch of aerated wastewater. The BES was operated at an applied potential of -0.6 V and in the OCP mode used as a control. Up to the first 10 cycles, a high nitrate ion removal rate: 9 mg L -1 d -1 (under the applied potential conditions) was demonstrated. Since the absorption of nitrate ions by the anion exchange membrane was confirmed (data not shown), the initial high removal rate might be due to membrane absorption along with the denitrification process. After 10 cycles, the nitrate ion removal rate stabilized at an average value of 60 mg NO3 - -N L -1 d -1 (the highest value was 78 mg NO3 - -N L -1 d -1These results indicate an increase in nitrate ion removal efficiency during long-term operation compared to previous studies (Gregoire et al., 2014; Tang et al., 2017). Once stabilized, the removal efficiency of each odd cycle (changing both anode and cathode wastewater) and even cycle (changing only the cathode wastewater and operating with less organic matter in the anode tank) was compared (Figure 3). The average reduction in nitrate ions over 3 days was 177.2 ± 58 mg L in the even cycle (at a potential of -0.6 V). -1 and 133.8±56 mg L under OCP -1 ) compared to odd cycles (198±51 mg L under a potential of -0.6V) -1 and 160±42 mg L under OCP -1 The levels were significantly higher in this case. This indicates the importance of the anodic organic material as an electron source for faster nitrate ion removal. Furthermore, a significant decrease in nitrate ion concentration coincided with a clear current consumption (Figure 4). The current in the initial cycle tended to have a higher peak value on day 1 after the wastewater was changed, and then gradually decreased as the nitrate ion concentration decreased (Figures 4A and 4B). After longer operating times, the current stabilized regardless of changes in the wastewater (Figure 4C). This effect may be attributed to the rapid consumption of electrons by denitrifying bacteria supplied by the cathode. In summary, these results suggest that cathode denitrification in a BES with an AEM using actual wastewater has a very promising removal rate. Long-term experimental operation promoted the growth of desired denitrifying bacteria exhibiting good electrochemical activity, and a higher nitrate ion reduction rate was observed. To facilitate the scaling up of pig farms, we are developing reactors with lower-cost components, lower maintenance requirements, and appropriate microbial community stability over long-term operation.

[0052] 4. Structure of bacterial groups The taxonomic composition of the microbial communities occupying the cathode from Stage I experiments using CEM (under applied potentials of -0.6V and -0.2V, under OCP mode, and without electrodes) and Stage III experiments using AEM (under applied potential of -0.6V and OCP mode after 6 months of operation) was evaluated using a shotgun metagenomic sequencing approach, compared to the original microbial communities from activated sludge (Figure 5). In Stage I, the effects of different electrochemical conditions on the cathode microbial communities were investigated. In the latter half of Stage III, the main objective was to investigate the adaptation of established electrotrophic and denitrifying microbial communities on the cathode after 6 months. Approximately 800,000 sequences were obtained under each condition. Genera with relative abundances greater than 1% were considered to constitute the core communities. The taxonomic distribution of the samples was analyzed at the genus-species level.

[0053] 4.1. Comparative analysis of microbial communities under different electrochemical conditions The activated sludge used as inoculum represented the initial bacterial population, with Acidovorax spp. (10.3%) of the Comamonadaceae family and Mycobacterium spp. (5.9%) of the Mycobacteriaceae family, along with Thauera (31.7%) and Azoarcus (4.3%) of the Zoogloeaceae family being predominant. Previously, two specific families, Zoogloeaceae and Comamonadaceae, were identified as being primarily involved in the denitrification process in activated sludge (Khan et al., 2002). Furthermore, Thauera and Azoarcus were shown to account for approximately 16% of the total viable bacteria in activated sludge (Juretschko et al., 2002). During BES operation, the bacterial community shifted from a heterotrophic anaerobic predominant group to an anaerobic group with diverse metabolic pathways, including both heterotrophic and autotrophic organisms. In samples from Stage I experiments, microbial diversity and their functions varied depending on the applied electrochemical conditions. Under conditions of an applied potential of -0.6V, a significant change in taxonomic distribution was observed, and this condition recorded the fastest denitrification. The most abundant bacteria belonged to the genus Pseudomonas (21.7%), indicating the presence of various denitrifying and electron-producing bacteria (Deng et al., 2020; Vo et al., 2020). Deng et al. (2020) also showed that in the MFC-granular sludge coupling system, denitrification was mainly carried out by the very dominant Pseudomonas (14.79%) and Thauera spp. (26.21%). Thauera exhibited the highest relative abundance in activated sludge samples in reactors under OCP mode (22.6%) and a potential of -0.2V (26.6%), but its abundance decreased to 9.8% under a potential of -0.6V. It is a heterotrophic facultative anaerobic and obligate respiratory bacterium that can use nitrate and nitrite ions as electron acceptors (Deng et al., 2020; Yang et al., 2019). Instead of the heterotrophic Thauera being dominant, the microbial community was further concentrated with autotrophic denitrifying bacterial genera Sideroxydans (9.9%) and Galionella (8.6%), which possess the potential to accept electrons from electrodes. Both are adapted to chemoautotrophy, including pathways for CO2 fixation and electron transport pathways for growth on Fe(II) at low O2 levels (Emerson et al., 2013). The ability to oxidize extracellular Fe(II) is based on specific fimbriae and cytochrome sets, which enable these bacteria to receive electrons from the cathode and transport them to nitrate ions (Emerson et al., 2013). The main difference between these bacteria is the ability of Sideroxydans to grow on reduced S-compounds and fix nitrogen. On the other hand, Galionella is tolerant of heavy metals in livestock wastewater treatment environments (Irshad et al., 2013), where heavy metals are generally present (Fabisch et al., 2013). Interestingly, the nitrite / nitric oxide reductase operon of Sideroxydans is almost identical to that of Acidovorax. Previous studies had confirmed that some Acidovorax spp. can be propagated by denitrification using inorganic electron donors, such as Fe(II) (Chakraborty et al., 2011; Park et al., 2017), but our taxonomic compositional analysis revealed that the abundance of Acidovorax decreased to 8.4% in a reactor with an applied potential of -0.6V. This may be due to their suppression by the dominant Pseudomonas spp. Under an applied potential of -0.2V, the core microbial community consisted of Thauera (26.6%), Nitrosomonas (12.4%), Thiobacillus (12%), Acidovorax (9.8%), and Pseudomonas (8.9%). All of these are involved in the nitrogen cycle. Nitrosomonas is the most well-known ammonia-oxidizing bacterium, being electrochemically active and capable of accepting electrons from the cathode electrode (Wang et al., 2013) (Holmes et al., 2004). In this study using CEM, ammonium inflow from the anode to the cathode was observed (Figure 1D), potentially creating ideal conditions for the vigorous growth of Nitrosomonas, supporting the conversion of ammonium to nitrogen gas. In OCP mode, the core microbial community remained closely related to the microbial community of the original inoculum. In this case, the most dominant genera were Thauera (22.6%), Acidovorax (11%), and Azoarcus (5.6%), but there was also a very abundant Geobacter (19.9%). The effective coexistence of electron-generating Geobacter (6.5%) and denitrifying Thauera (59.9%) has been previously reported in long-term tests of a single-chamber air cathode system with an external resistance of 1000Ω (Huang et al., 2019). However, this nutrient symbiotic relationship is still under investigation and requires further research. In this study, Geobacter was observed to be more abundant under OCP mode than under any other conditions. These results suggest that OCP conditions may be related to the ferric reduction process. From all of the above, the biofilms that developed on the surface of the biocathode and were investigated consisted of a very diverse group of microorganisms, including microorganisms with opposing functions (e.g., Fe3 + Reducing microorganisms / Fe2 +It is suggested that oxidative microorganisms may coexist and interact on complementary processes. The relationship between species diversity and ecosystem function has been debated for decades, but there is a growing consensus that greater diversity leads to increased functional productivity and stability of microbial communities (Tilman et al., 2014). Increased overall diversity of electrotrophic denitrifying bacteria in reactors at -0.6V is associated with improved ecosystem function and stability in bacterial denitrifying communities with comparable accumulations, thereby improving BES performance for nitrate ion removal.

[0054] 4.2. Cathode microbial communities after long-term adaptation It was interesting to investigate how the microbial community changed during long-term operation in fed-batch mode. In Stage III, we examined the adaptation of the microbial community on the cathode under an applied potential of -0.6V and OCP mode for 6 months. To the best of our knowledge, this study is the first to investigate pre-growing denitrifying biofilms in a fed-batch system using actual wastewater during long-term operation. After 6 months under an applied potential of -0.6V on the cathode, the microbial community was mainly composed of Thiobacillus (60.7%) (Figure 5). These bacteria utilize sulfur (S) from iron sulfide (FeS) as an electron donor and oxygen as an electron acceptor. In this case, S is sulfur (SO4). 2- It is oxidized to ). Some species of this genus are Fe 2+It can oxidize and use nitrate ions as electron acceptors (Straub et al., 1996). Thiobacillus denitrificans has been reported as an electroactive denitrifying bacterium that can directly utilize solid electrodes as the sole electron donor and accumulate on the cathode to promote denitrification (Pous et al., 2014; Yu et al., 2015). Furthermore, it has been previously reported that Thiobacillus (75-80%) was dominant in cathode biofilms in BES using pre-accumulated inoculum (Pous et al., 2014). These results demonstrate the importance of Thiobacillus and its ability to create robust and stable biofilms on electrodes for efficient and continuous denitrification processes using wastewater. Under OCP mode, the main contributing factors were uniformly distributed among the following bacteria: Thauera spp. (14.3%), Nitrospira (13.7%), Mycobacterium (10.6%), and Acidovorax (8.2%). Interestingly, Acidovorax (3.9%), Mycobacterium (2.9%), and Nitrospira (2.7%) were also found to be the second most abundant after Thiobacillus in the reactor under -0.6V. Mycobacterium includes pathogens known to cause serious diseases in mammals and humans. This genus has been previously found during autotrophic microbial denitrification (Broman et al., 2017). The decrease in their abundance is likely related to the bactericidal capacity of BES, as previously reported by Vasieva et al. (Vasieva et al., 2019), but further investigation is still needed. Nitrospira is known to play a crucial role in nitrification as an aerobic, chemoautotrophic nitrite-oxidizing bacterium (Mehrani et al., 2020). These results have shown that such bacteria can develop physically stable and biologically active biofilms during prolonged treatment, but under electrically stimulated environments, they are overwhelmed by Thiobacillus, which acts as the primary consumer of electrons on the electrode surface.

[0055] 4.3 Cathode microorganisms at the species level Further analysis of the taxonomic distribution at the species level was conducted, and the top 30 species were selected to create a logarithmic heatmap (Figure 5B). In short-term experiments (Stage I), two Pseudomonas species, P. putida and P. aeruginosa, were found most abundantly under a potential of -0.6V. Both are pathogenic bacteria and are very closely related. However, under anaerobic conditions, P. aeruginosa can undergo complete denitrification accompanied by excessive production of nitrite ions (Arat et al., 2015), while the heavy metal-resistant P. putida can achieve both nitrification and aerobic denitrification simultaneously (Zhang et al., 2019). Nitrosospira multiformis, a chemoautotrophic bacterium that oxidizes ammonia to nitrite ions and assimilates CO2 as its main carbon source, was mainly found in BES tanks with CEM and where an ammonium flux to the cathode tank was detected. The abundant presence of P. aeruginosa and N. multiformis at -0.6V compared to other conditions indicated a higher NO2 level. - This can explain the high concentration of [substance]. Consistent with the genus-level analysis, the two most abundant autotrophic denitrifying bacteria (Gallionella and Syderoxydans) were identified as Gallionella capsiferriformans and Syderoxydans lithotrophicus. Another known denitrifying bacterium, Dechloromonas aromatica, is capable of promoting N2O production under salt or stress conditions (Han et al., 2019). In the long-term experiment (stage III), Thiobacillus denitrificans was the most abundant strain, consistent with the genus-level study. It is interesting to note that the growth of bacteria such as Sorangium cellulosum, known for its ability to inhibit the growth of its partners (Li et al., 2013), was reduced under long-term operation. The microbial composition structure of the accumulated biofilm on the cathode at -0.6V was visualized using SEM analysis (Supplementary Material).

[0056] 5. Analysis of nitrogen metabolism in cathode biofilms Further nitrogen cycle-related processes in each bioreactor—denitrification, nitrification, ammoniaization, and nitrogen fixation—were analyzed. Overall, denitrification had the highest hit rate among the four processes. To demonstrate the expression of denitrification genes during Stage I, the metatranscriptome was analyzed from samples under an applied potential of -0.6V and under OCP mode as a control. The expression of six representative genes—nitrate reductases napAB and narGHI, nitrite reductases nirS and nirK, nitric oxide reductase norBC, and nitrous oxide reductase nosZ—was investigated to analyze the bacterial species involved in each stage of the denitrification process (Figure 6). Considering the high microbial diversity, only gene abundances exceeding 3% of the total copy were counted. Depending on the different electrochemical conditions, the bacteria involved in each stage of the denitrification process changed. Of the two types of nitrate reductases, resparatriate (NarGHI) and periplasmic (NapAB), periplasmic nitrate reductase accounted for the total number of readings per species. The napAB gene, which was highly abundant in the strain most closely related to Thauera sp. MZ1T, was expressed only under the applied potential conditions (15.5%). On the other hand, Azoarcus sp. BH72 (19.4% vs. 6.7% in OCP mode) and Bordetella petrii (12.4% vs. 6.7% in OCP mode) were found to be present under both conditions, but were clearly more abundant at -0.6V. These results indicate the ability to electrotrophically advance the first step of denitrification. Regarding resparatrinitrate reductase (NarGHI), Aromatoleum aromaticum was the most dominant species under the applied potential (39%). Both A. aromaticum and T. denitrificans possess enzymes that reduce all intermediates of the denitrification process, but under some conditions in this study, the abundance of these lines was below 3%. Thauera sp., Acidovorax sp., Alicycliphilus denitrificans, and Dechloromonas aromatica also possess all the genes necessary for complete denitrification and were captured in high abundance under the applied potential conditions in this study. Two structurally distinct nitrite reductases are found in denitrifying bacteria, but neither is ever expressed in the same cell (Zumft, 1997): one contains copper (Cu-Nir) encoded by the nirK gene, and the other contains heme c and heme d1 (cd1-Nir) encoded by the nirS gene. T. denitrificans and Burkholderia pseudomallei are the two main sources of the nirSK gene in samples at -0.6V, while A. aromaticum and Thauera sp. were dominant under OCP mode. On the other hand, the abundance of the nirSK gene in P. putida (4%) and S. lithotrophicus (6.1%) was determined only under applied potential conditions. This is consistent with previous findings in Section 3.4.1, where the microbial community in BES at -0.6V was dominated by these autotrophic bacteria. Furthermore, the dominance of Thauera under OCP conditions was demonstrated at the transcriptome level: the expression of nitrite reductase (nirSK), nitric oxide reductase (norBC), and nitrous oxide reductase (nosZ) was clearly dominated by species closely related to Thauera sp. MZ1T. Nitric oxide reductase has two subunits, NorC and NorB. Here, NorC, as c-type cytochrome, receives electrons from the periplasmic donor and transfers them to NorB, which contains two b-type heme and non-heme iron (Vaccaro et al., 2015). Potential electrotrophic denitrifying bacteria, closely related to the following species: Maribacter sp. HTCC2170 (10.3%), Methylococcus capsulatus (8.1%), Roseobacter denitrificans (3.7%), and Dechloromonas aromatica (3.7%), were identified at this denitrification stage in BES at -0.6V. However, further investigation of these accumulated potential electrotrophic bacteria is needed. On the other hand, under OCP mode, norBC was mainly represented by Thauera sp. (20.9% vs. 5.9% under applied potential) and T. denitrificans (14% vs. 11% under applied potential). The final stage of denitrification is completed by the catalytic action of soluble periplasmic Cu-containing N2O reductase nosZ. Notably, the bacterial composition of norBC and nosZ genes is highly diverse in samples under applied potential and OCP. This indicates that these conditions contribute to the final two stages of the denitrification process.

[0057] In one embodiment of this application, the improvement of nitrate ion removal in the cathode tank of a bioelectrochemical system (BES) using aerated swine wastewater under high nitrate ion levels and low organic carbon conditions was investigated, focusing on the relationship between nitrogen and the bacterial communities involved in the denitrification pathway. As a result, a BES with an anion exchange membrane (AEM) under a cathode applied potential of -0.6V relative to an AgCl / AgCl reference electrode achieved 99±2 mg L -1 d -1 The removal rate was shown. Furthermore, when measured over 7 days, organic compounds from untreated, undiluted wastewater were removed at an efficiency of 61.4 ± 0.5%, with an achieved efficiency of approximately 5 g COD L. -1 From the initial concentration, 0.46g of COD L -1 d -1The substances were simultaneously removed in the anode tank at the specified removal rate. The greatest microbial diversity was detected in BES under a potential of -0.6V. This diversity included autotrophic denitrifying bacteria, such as Syderoxidans, Gallionela, and Thiobacillus.

[0058] Example 2 56L reactor operation Materials and methods 1. BES design, inoculation, and system operation A 56L reactor system (38L cathode tank and 18L anode tank) was constructed and installed on-site next to the pig farm and aeration tank at the Okinawa Prefectural Livestock Research Center (Figures 8-11). In this system, two types of wastewater flows are treated simultaneously: high-level organic matter raw wastewater that serves as an electron source in the anode tank, and aerated wastewater containing low levels of organic nitrate ions (low C / N ratio (approximately 0.1)) in the cathode tank. The reactor consists of two anode tanks and four cathode tanks separated by an anion exchange membrane (AMI-7001 Membrane Internationals) (Figures 8-9). The same carbon brush electrodes as in Example 1 were used, except that a 1-meter-long electrode was installed in each tank (Figures 9-11). This reactor enabled meandering wastewater flow. Activated sludge collected from the aeration tank was inoculated into each tank at a concentration of 25% of the tank volume. A potentiostat (Hokuto HA-151B) was used, and the cathode was matched to -0.4V versus Ag / AgCl.

[0059] The solid-separated raw pig wastewater collected from the farm was pumped into sedimentation tank #1, where it was stored anaerobically for 3-5 hours. It was then transferred to sedimentation tank #2 by another pump. From there, it was sent to anode tank #1 in the BES via a 1-day HRT. After treatment in the anode tank, the treated wastewater was placed in an aeration tank for oxidation and nitrification. From the aeration tank, the aerated wastewater was pumped into sedimentation tank #3, where NO3 treatment was performed. - -N 300mg L -1The value was adjusted to [value]. The aerated wastewater stored in sedimentation tank #3 was pumped to the cathode tank in the BES for final treatment with a 2-day HRT (Figure 9). However, the residence time was changed to 1 day in the 4th and 5th months after the start of operation, and then returned to a 2-day HRT. Samples were taken from both tanks before treatment (inflow) and after treatment (outflow).

[0060] 2.Chemical analysis COD, ammonium (NH4) + -N), nitrate ion (NO3 - -N), nitrite ion (NO2 - The concentration of -N) was analyzed using the HACH test kit (USA). BOD, suspended solids, total nitrogen, soluble phosphorus, total phosphorus, and calcium were analyzed according to the environmental standards of the Water Pollution Control Act (Japan). Odor was measured using an odor sensor (XP-329IIIR, New Cosmos Electric Company, Japan).

[0061] 3.Results The results from operating a 56L reactor are shown in Figures 12 to 14. Figure 14 shows the BES measurements for BOD, COD, SS, and odor in the anode tank, and NO3 in the cathode tank. - This indicates successful removal of total nitrogen, soluble phosphorus, total phosphorus, and calcium. On average, approximately 1 g / L of BOD was removed from the raw wastewater using this system. Removing 1 g / L of BOD is calculated to reduce operating costs by 50%, and this cost saving is mainly due to reduced excess sludge removal and power consumption, made possible by shorter aeration times. This also leads to a reduction in CO2 emissions from the aeration tank.

[0062] Example 3 Operation of a BES with external resistance The experiments were conducted using the same 2L reactor as described in Example 1, in a three-electrode setup with a cathode potential applied at -0.4V, as shown in Figure 7i, and in a two-electrode setup for an external resistance experiment (R=500Ω), as shown in Figure 7ii. In the three-electrode setup, the cathode was used as the working electrode controlled by chronoamperometry, and the Ag / AgCl electrode was used as the reference electrode (Radiometer XR300 Reference Electrode, Hach, 0.197V vs. a standard hydrogen electrode). The cell voltage during the external resistance experiment was monitored with a data logger (GRAPHTEC Midi Logger GL240). All experiments were conducted using an AEM as a separator, and raw pig wastewater in the anode tank and aerated wastewater without nitrate ion adjustment in the cathode tank, which resulted in a change in nitrate ion concentration at the start of the experiment. After inoculation, BES was pre-incubated under open-circuit potential (OCP) to allow the bacterial biofilm to adapt to the environment. Figure 7E compares the removal of nitrate ions in a 3-day experiment using an applied cathode potential of -0.4V and an external resistor R=500Ω for Ag / AgCl. Figure 7F shows the cathode potential, anode potential, and current at the external resistor R=500Ω. Figures 7E and 7F show NO3 removal without power supply using a BES. - This indicates that the removal was successful. This means that the BES can function as a battery. [Industrial applicability]

[0063] The biocathode denitrification BES described in this application is a promising technology for treating two types of pig farm wastewater: raw wastewater for the removal of organic matter, odor, and pathogens, and aerated wastewater for the removal of nitrate ions. Under conditions where the cathode was set to -0.6V, the growth of autotrophic denitrifying bacteria was promoted, and at the same time, the removal rate of nitrate ions was improved. Electrotrophic Syderoxydans lithotrophicus and Gallionella capsiferriformans were dominant in short-term operation, while Thiobacillus denitrificans was dominant in long-term operation. This system may include a pretreatment system (a continuous sedimentation tank for removing large particles from wastewater) and a subsequent wastewater flow to a BES bioreactor. If the cathode wastewater contains phosphorus and calcium, phosphorus is also removed in the cathode tank via electrocrystallization. In this case, phosphates, such as calcium phosphate, precipitate on the cathode. In some embodiments, more than 30%, preferably more than 50%, of the phosphate phosphorus present in the aerated wastewater is removed by weight after treatment in the cathode tank. This system has also been shown to reduce fecal bacteria, using E. coli as an indicator. This system can operate using pig farm wastewater, one of the harshest types of wastewater. Because it is an anaerobic treatment, there is little need to remove excess sludge, which leads to reduced operating costs.

[0064] This device utilizes chemical energy from raw wastewater with a high COD in the anode tank to reduce nitrate ions in aerated wastewater in the cathode tank. Electrons generated by oxidizing COD under anaerobic conditions are supplied to the anode by electrogenic bacteria, and then flow to the cathode via an external circuit, where they are used by denitrifying bacteria to reduce nitrate ions to N2. This setup allows for the reduction of nitrate ions even when the COD of the wastewater in the cathode is low. The pH in the cathode tank increases as nitrate ions are reduced to N2, causing phosphate precipitation. The advanced wastewater treatment described herein allows for the removal of COD from raw wastewater in the anode tank under anaerobic conditions, resulting in significantly less excess sludge generation than conventional activated sludge processes. This apparatus can be installed in addition to existing aeration wastewater facilities, typically resulting in increased treatment capacity and reduced operating costs (Figure 10). In other words, the apparatus of the present invention can denitrify aerated wastewater, which may have low COD levels, using the energy collected by treating the raw wastewater, without the need to add further chemicals for denitrification or adjust COD and oxygen levels.

Claims

1. An apparatus for simultaneously denitrifying aerated wastewater and treating raw wastewater, At least one anode tank having at least one anode therein for treating raw wastewater; at least one cathode tank having at least one cathode therein for denitrifying the aerated wastewater; wherein the anode chamber is attached to the cathode chamber via a separator for transporting anions and / or cations between the anode chamber and the cathode chamber; Device.

2. The device according to claim 1 , wherein the cathode chamber and / or the anode chamber further comprises a reference electrode therein.

3. The device of claim 1 , wherein the anode and / or the cathode are conductive electrodes.

4. An apparatus as described in claim 1, wherein the cathode tank and / or the anode tank are inoculated with activated sludge inside.

5. 10. The apparatus of claim 1, wherein at least one of the anode and cathode chambers comprises means for continuous or periodic agitation within the chamber.

6. 10. The apparatus of claim 1, wherein the apparatus is connected to an aeration tank.

7. 1. A system for simultaneously performing anaerobic removal of organic compounds, such as organic matter, suspended solids, and volatile fatty acids, and pathogens, from raw wastewater containing organic compounds, and removal of nitrate and / or nitrite ions from aerated wastewater containing nitrate and / or nitrite ions, comprising: 1) A device according to claim 1 or 2, In the anode tank, the power-generating bacteria decompose organic compounds in the raw wastewater and supply electrons through the anode; and In the cathode tank, denitrifying bacteria receive electrons via the cathode and convert nitrate ions and / or nitrite ions in the aerated wastewater into N ２ reduced to gas, system.

8. 2) further comprising means for monitoring the potential between the cathode and the anode or between the cathode or the anode and a reference electrode; wherein the means is connected to the anode and cathode, or the anode, cathode and reference electrode. The system of claim 7.

9. 8. The system of claim 7, wherein the power-generating bacteria comprises at least one bacterium selected from the group consisting of Geobacter, Desulfovibrio, Syntrophobacter, Clostridium, Alicycliphilus, Thauera, Acidovorax, Xanthomonas, Bacteroides, Rhodopseudomonas, Thiomonas, Acinetobacter, Stenotrophomonas, Dechloromonas, Pseudomonas, Azoarcus, and Ralstonia species.

10. 8. The system of claim 7, wherein the denitrifying bacteria comprise at least one bacterium selected from the group consisting of Syderoxidans, Gallionela, Thiobacillus, Thauera, Mycobacterium, Alicycliphilus, Azoarcus, Acidovorax, Psudomonas, Dechloromonas, Methylibium, Burkholderia, Leptothrix, Ralstonia, Aromatoleum, Cupriavidus, Delfia, Nitrosomonas, Methylococcus, and Maribacter species.

11. at least one anode cell having at least one anode therein; at least one cathode cell having at least one cathode therein; wherein the anode chamber is attached to the cathode chamber via a separator to transport anions and / or cations between the anode chamber and the cathode chamber; 1. A method for simultaneously performing anaerobic removal of organic compounds, such as organic matter, suspended solids, and volatile fatty acids, and pathogens, from raw wastewater containing organic compounds, and removal of nitrate and / or nitrite ions from aerated wastewater containing nitrate and / or nitrite ions, comprising: adding raw wastewater to the anode chamber and aerated wastewater to the cathode chamber; Here, in the anode compartment, the power-generating bacteria decompose organic compounds, thereby supplying electrons through the anode; and in the cathode compartment, the denitrifying bacteria receive electrons through the cathode and convert nitrate ions and / or nitrite ions into N ２ reduced to gas, method.

12. at least one anode cell having at least one anode therein; at least one cathode cell having at least one cathode therein; wherein the anode chamber is attached to the cathode chamber via a separator to transport anions and / or cations between the anode chamber and the cathode chamber; 1. A method for simultaneously performing anaerobic removal of organic compounds, such as organic matter, suspended solids, and volatile fatty acids, and pathogens, from raw wastewater containing organic compounds, and removal of nitrate and / or nitrite ions from aerated wastewater containing nitrate and / or nitrite ions, comprising: adding raw wastewater to the anode chamber and aerated wastewater to the cathode chamber; Here, in the anode compartment, the power-generating bacteria decompose organic compounds, thereby supplying electrons through the anode; and in the cathode compartment, the denitrifying bacteria receive electrons through the cathode and convert nitrate ions and / or nitrite ions into N ２ reduced to gas, method.

13. 13. The method of claim 11 or 12, wherein the power-generating bacteria comprise at least one bacterium selected from the group consisting of Geobacter, Desulfovibrio, Syntrophobacter, Clostridium, Alicycliphilus, Thauera, Acidovorax, Xanthomonas, Bacteroides, Rhodopseudomonas, Thiomonas, Acinetobacter, Stenotrophomonas, Dechloromonas, Pseudomonas, Azoarcus, and Ralstonia species.

14. 13. The method of claim 11 or 12, wherein the denitrifying bacteria comprise at least one bacterium selected from the group consisting of Syderoxidans, Gallionela, Thiobacillus, Thauera, Mycobacterium, Alicycliphilus, Azoarcus, Acidovorax, Psudomonas, Dechloromonas, Methylibium, Burkholderia, Leptothrix, Ralstonia, Aromatoleum, Cupriavidus, Delfia, Nitrosomonas, Methylococcus, and Maribacter species.

15. 13. The method according to claim 11 or 12, wherein the raw wastewater is livestock wastewater or its supernatant.

16. The method according to claim 11 or 12, wherein the raw wastewater is swine farm wastewater or its supernatant.

17. 13. The method of claim 11 or 12, wherein the aerated wastewater is an aerated livestock wastewater or its supernatant containing low levels of organic compounds.

18. 13. The method of claim 11 or 12, wherein the aerated wastewater is aerated swine wastewater or its supernatant containing low levels of organic compounds.

19. The apparatus further comprising a reference electrode in the cathodic or anodic chamber; 13. The method of claim 12, wherein the method further comprises monitoring the potential of either the cathode or the anode relative to a reference electrode, and the potential of the cathode relative to the reference electrode is adjusted to between −0.2 and −0.8 V.

20. The apparatus further comprising a reference electrode in the cathodic or anodic chamber; 13. The method of claim 12, wherein the method further comprises monitoring the potential of either the cathode or the anode relative to a reference electrode, and the potential of the cathode relative to the reference electrode is adjusted to between −0.4 and −0.6 V.

21. The method of claim 11 or 12, further comprising inoculating the anode tank and / or the cathode tank with activated sludge at a volume of 0% to 60% of the anode tank and / or the cathode tank.

22. The method described in claim 21, wherein inoculating the activated sludge comprises inoculating the activated sludge into the anode tank and / or the cathode tank at a volume of 20% to 25% of the anode tank and / or the cathode tank.

23. The aerated wastewater after being added to the cathode tank contains a total of 100 mg / L or less of NO as nitrogen equivalent. ３ － and NO ２ － 13. The method of claim 11 or 12, comprising:

24. at least one anode cell having at least one anode therein; at least one cathode cell having at least one cathode therein; wherein the anode chamber is attached to the cathode chamber via a separator to transport anions and / or cations between the anode chamber and the cathode chamber; A method for simultaneously performing anaerobic removal of organic compounds, such as organic matter, suspended solids, and volatile fatty acids, and pathogens, from raw wastewater containing power-generating bacteria and organic compounds, and removal of nitrate ions and / or nitrite ions and phosphorus from aerated wastewater containing denitrifying bacteria, nitrate ions and / or nitrite ions, and phosphorus, comprising: adding raw wastewater to the anode chamber and aerated wastewater to the cathode chamber; Here, in the anode compartment, the power-generating bacteria decompose organic compounds, thereby supplying electrons through the anode; and in the cathode compartment, the denitrifying bacteria receive electrons through the cathode and convert nitrate ions and / or nitrite ions into water-insoluble N. ２ The phosphate is reduced to gas and precipitated in the cathode chamber. method.

25. at least one anode cell having at least one anode therein; at least one cathode cell having at least one cathode therein; wherein the anode chamber is attached to the cathode chamber via a separator to transport anions and / or cations between the anode chamber and the cathode chamber; A method for simultaneously performing anaerobic removal of organic compounds, such as organic matter, suspended solids, and volatile fatty acids, and pathogens from raw wastewater containing power-generating bacteria and organic compounds, and removal of nitrate ions and / or nitrite ions from aerated wastewater containing denitrifying bacteria and nitrate ions and / or nitrite ions, comprising: adding raw wastewater to the anode chamber and aerated wastewater to the cathode chamber; Here, in the anode compartment, the power-generating bacteria decompose organic compounds, thereby supplying electrons through the anode; and in the cathode compartment, the denitrifying bacteria receive electrons through the cathode and convert nitrate ions and / or nitrite ions into water-insoluble N. ２ The phosphate is reduced to gas and precipitated in the cathode chamber. method.

26. 26. The method of claim 24 or 25, wherein more than 30% of the phosphate phosphorus present in the aerated wastewater is removed by weight of phosphorus.

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