The MFC device was constructed with cathode and anode chambers. The anode and cathode chambers were both made of organic glass tube 8 cm high and 9 cm in diameter and had an effective volume of 0.452 L (Figure 1). Each chamber used a carbon brush as the electrode. The two chambers were separated by a proton exchange membrane (Nafion 117) and placed in a water bath. The temperature was initially set to 31 ± 1°C and then changed to a dynamic temperature (36–48°C) later in the operation. The cathode and anode chambers were connected with a manual variable resistor (0–9999 Ω) to close the circuit. The cathode chamber was exposed to air, and blast aeration was used. The influent was injected into the anode chamber using a peristaltic pump (YZ1515X, Lange), and the effluent from the anode was fed into the cathode chamber.

The influent for the MFC reactor was an artificially simulated high strength ammonia sludge digestion solution with components of 0.38 g/L CH₃COONa, 2.708 g/L NH₄HCO₃, 0.33 g/L KH₂PO₄, 1 g/L K₂HPO₄·3H₂O, 1 g/L KCl, 1.5 g/L NaHCO₃, 0.016 g/L CaCl₂, and 1 ml/L trace nutrient solution. CH₃COONa and NH₄HCO₃ were added to maintain the chemical oxygen demand (COD) and the ammonia nitrogen concentrations in the influent at 300 and 480 mg/L, respectively.

The anode and cathode chambers of the MFC were inoculated with aerobic sludge from the aeration tank of the Fourth Wastewater Treatment Plant in Xi'an, China. Before operating the MFC, the anode and cathode were soaked in the inoculation sludge for 24 h. Once the reactor was filled with synthetic wastewater, the MFC entered the stage of static culture without the influent while keeping the cathode aerated and the inner circuit open. The concentrations of DO, NO3−-N, NO2−-N, and NH4+-N and the pH of the cathode electrolyte were measured daily. After more than half of the original NH4+-N was converted to NO2−-N in the cathode electrolyte, the aeration mode was changed from continuous to intermittent (2 h aeration and 2 h static), and the wastewater was pumped continuously into the anode chamber with hydraulic retention times (HRTs) of 10.4 h for both the cathode and the anode chambers. After a period of continuous operation, stable partial nitrification was obtained in the cathode chamber of the MFC. An external resistance of 100 Ω was then connected, following which the operation of MFC with SND started.

The anode potential was monitored with a saturated calomel electrode (SCE, +0.242 V standard hydrogen electrode; Type 232, Leici Instrument Factory, Shanghai, China). NO3−-N, NO2−-N and NH4+-N were measured according to Standard Methods for the Examination of Water and Wastewater (Clesceri et al., 1998). DO was determined using a Hach-HQ30d DO analyzer (HACH, America). The voltage and anode potential were monitored and recorded using a PCI1717 voltage collector (Yanhua Company, Shenzhen, China). An N₂O microsensor (Unisense, Denmark) was used for the N₂O analysis.

Samples from the biofilm of the cathode were collected on day 27 and day 83 to investigate the microbial community with DGGE, and DNA was extracted using a fast DNA spin kit (SK8233) for soil according to the manufacturer's instructions. The bacterial 16S rRNA genes were amplified by PCR with the universal primers F357-GC (5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCGCCCCCCTACGGGAGGCAGCAG-3′) and R518 (5′-ATTACCGCGGCTGCTGG-3′). A polyacrylamide gel (8%) with a 30–60% denaturing gradient was used to separate the PCR products (7 mol L-1 urea and 40% formamide comprising 100% denaturant), which were analyzed using DGGE technology and washed with ultrapure water to flush the gel and dye. Eight representative DGGE strips were selected by a clean scalpel and transferred to a 1.5 mL centrifuge tube. The target DNA fragments were then excised and reamplified using the primer sets F357 (5′-CCTACGGGAGGCAGCAG-3′) and R518 (5′-ATTACCGCGGCTGCTGG-3′), and the obtained sequence was matched with the Seqmatch database for sequence alignment. The homology information of each strip was obtained by Sangon Biotech Co., Ltd. (Shanghai, China).

The results of the continuous operation test are shown in Figure 2.

In stage 1 (first 5 days of the test), the MFC with SND was operated at 31 ± 1°C, 100 Ω resistance and intermittent aeration (2 h aeration and 2 h static), and the DO of the catholyte was 0.5–1.0 mg/L. The concentrations of NH4+-N and NO2−-N decreased with a gradual increase in the release voltage of the MFC. However, the removal of total nitrogen (TN) was much greater than that with the electrical current of the MFC. This finding implies that the traditional heterotrophic microbial denitrification with COD was more efficient at removing nitrogen than the electrode denitrification in the cathode chamber. The moderate temperature (31 ± 1°C) and low DO (0.5–1.0 mg/L) in the cathode may be beneficial for SND with COD still present in the anode effluent.

In stage 2 (days 6–34), the temperature of the reactor increased and fluctuated over the range of 36–48°C. The removal of TN began to increase sharply, and the concentration of NO2−-N in the cathode effluent began to decrease correspondingly from the 6th day, while the voltage of the MFC increased slightly and then stabilized. This might have been due to the high temperature in the cathode (36–48°C), which was harmful for the growth of normal ammonia oxidizing bacteria (AOB). Because the suitable range of temperatures for AOB metabolism is 20–30°C, the nitrification rate decreased, and the NO2−-N concentration decreased to approximately 30 mg/L. The increase in the TN removal was mainly caused by the volatilization of ammonium at high temperature. The TN removal from the denitrification in the electrode during stage 2 was lower and similar to that at the end of stage 1 (not greater than 7 mg/L·d based on the MFC voltage). The biofilm was sampled, and the bacteria were identified with PCR-DGGE. The predominant species was found to be Ureibacillus thermosphaericus of the genus Ureibacillus, which grows at temperatures of 37° to 60°C (Fortina et al., 2001).

In stage 3 (days 35–77), the reactor was set to continuous aeration instead of intermittent aeration. As a result, the DO increased to 3 ± 0.6 mg/L. The amount of TN removed per day began to decrease, but the voltage continued to increase. This might have occurred because the heterotrophic denitrification with COD as the electron donor was inhibited by the increase of DO, and the partial oxygen accepted electrons from the electrode. In stage 3, the release voltage initially increased, then dropped and finally steadied at approximately 100 mV, which was much higher than that in stage 2. The following strain sampling clearly demonstrated that the predominant species changed to aerobic denitrifiers (a detailed analysis is provided later). The curves in Figure 2 suggest that the aerobic denitrifiers might have replaced the anaerobic denitrifiers in the latter phase of stage 3. The aerobic denitrifiers appeared to be much more receptive to the electrons from the electrode than the anaerobic denitrifiers, which was determined by comparing the voltage of stage 2 with that of stage 3, in which the effects of electron acceptance by oxygen was taken into account. In contrast, TN removal by anaerobic denitrifiers was much greater than that by aerobic denitrifiers. The heterotrophic denitrification with COD might have mainly caused TN removal, excluding the effect of volatilization in the cathode. Depending on the voltage, the TN removal by electrode denitrification was 8.5–9.0 mg/(L·d), which represented only a small part of the TN removal in stage 3.

The conditions in stage 4 (days 78–88) remained the same as those in stage 3 except for the change in the resistance of the MFC from 100 to 50 Ω. The results showed that the concentrations of NH4+-N and NO2−-N in the cathode effluent decreased, and the amount of TN removed increased in the latter part of this stage (Figure 2). The voltages were greater than those in stage 3, but the TN removal by electrode denitrification was 13.5–14.6 mg/(L·d), which was greater than that in stage 3.

The electricity production performance of the MFC under high DO conditions and different external resistances was investigated as a case study (Figures 3–5, which correspond to days 75, 82, and 97 in Figure 2, respectively). The voltage was positively related to the increase in temperature under external resistances of 50 Ω and 100 Ω, while it was independent of the temperature under open circuit conditions. Regardless of whether the resistances were applied or an open circuit was used, the N₂O emissions were always positively related to the increase in temperature, while the DO was always negatively related to the increase in temperature. The characteristics of denitrification and electricity production of the MFC can be evaluated using these factors.

The fluctuations in the concentrations of DO and N₂O caused by the temperature (Figures 3, 5) show that the DO fluctuation amplitude was approximately 1.2 mg/L, whereas the corresponding N₂O fluctuation was approximately 0.4 mg/L in the open circuit. With a 100 Ω resistance, the DO and N₂O fluctuation amplitudes were 1.5 mg/L and 0.3 mg/L, respectively. This demonstrated that the reduction in oxygen caused by the voltage fluctuation was approximately 0.3 mg O₂/L, and no N₂O was produced by electrode denitrification. The concentrations of N₂O were similar in both scenarios (0.7–1.1 mg/L), whereas the concentrations of DO with a 100 Ω resistance were 0.7–1.0 mg/L, which was lower than that in the open circuit (Figures 3, 5). This indicated that the decrease in DO was dependent on the electrode reaction, while the production of N₂O was independent of the electrode reaction. Using Coulomb's law, with a 100 Ω resistance, the electrode reduction rate of oxygen was calculated to be 1.6–2.3 mg O₂/(L·d), whereas the rate of electrode reduction of nitrite to nitrogen gas was 8.5–9.0 mg N/(L·d) with no production of N₂O.

The fluctuations in the concentrations of DO and N₂O caused by temperature (Figures 4, 5) show that the decrease of DO and the increase of N₂O were both dependent on the electrode reaction. Using Coulomb's law, with a 50 Ω resistance, the electrode reduction rate of oxygen was calculated to be 2.8–4.4 mg O₂/(L·d), whereas the rate of electrode denitrification was 13.5–14.6 mg N/(L·d), in which approximately 10% of the nitrogen removed was converted to N₂O and 90% was converted to nitrogen gas.

The N₂O production and TN removal efficiencies with a 50 Ω external resistance were higher than those with a 100 Ω resistance, which illustrates that electrode denitrification occurred in the cathode of the MFC. These analyses indicate that both oxygen and nitrite can obtain electrons simultaneously from the electrode in the cathode of the MFC.

Biofilm samples were collected from the cathode chamber of the MFC on day 27 in stage 2 (Figure 2) and day 83 in stage 4 (Figure 2). The representative DGGE strips are shown in Figure 6. The closest species and classification of each representative band in the DGGE profile were deposited in the GenBank database (Tables 1, 2). Sequences from DGGE bands are shown in Supplementary Material.

In the first microbial identification (27th day), which corresponded to the low DO and high temperature MFC operating conditions (stage 2 in Figure 2), 12 bands were identified (Table 1). The results showed that the microbial community could be divided into 4 phyla, 4 classes and 8 genera. The phylum Firmicutes (bands 2, 3, 4, 5, 7, 8, 9, 10, and 13) was the predominant bacteria, while the phyla Ignavibacteriae (band 1), Chloroflexi (band 6) and Proteobacteria (band 14) were the subdominant groups.

Within the Firmicutes phylum, bands 2, 4, 5, 7, 8, and 9 belonged to the genus Ureibacillus, related to a species of U. thermosphaericus (similarity 91.2–100%), which was reported to be a thermophilic bacteria (37–60°C, optimum 50–60°C) with heterotrophic growth in aerobic environments and no ability for nitrate reduction (Fortina et al., 2001). This species was inferred to dominate the nitrification in the cathode of the MFC under low DO and high temperature conditions.

Bands 1 and 6 belonged to the genera Ignavibacterium and Unclassified Anaerolineaceae, respectively, which grow under anaerobic conditions, are heterotrophic and cannot utilize nitrate as electron acceptors (Yamada et al., 2006; Iino et al., 2010). These species were inferred to dominate the anaerobic degradation of COD.

Bands 3 (1) and 13 belonged to the genera Geobacillus and Anoxybacillus, respectively, which grow under aerobic conditions, are heterotrophic and can reduce nitrate (Poli et al., 2006; Inan et al., 2013). These species were inferred to dominate the aerobic denitrification.

Bands 3 (2) and 10 belonged to the genera Anoxybacillus and Bacillus, respectively, which grow under anaerobic conditions, are heterotrophic and can reduce nitrate (Kim et al., 1998; Cihan et al., 2014). These two species were inferred to be responsible for the anaerobic denitrification in stage 2 (Figure 2).

Band 14 belonged to the genus Cucumibacter, which grows under aerobic conditions, is heterotrophic and cannot utilize nitrate as electron acceptors (Hwang and Cho, 2008). This species was inferred to be responsible for the aerobic degradation of COD in stage 2 (Figure 2).

In the second microbial identification (83th day), which corresponded to the high DO and high temperature MFC operating conditions (stage 4 in Figure 2), 7 bands were identified (Table 2). The results showed that the microbial community could be divided into 1 phylum, 7 classes and 8 genera. The phylum Proteobacteria (bands 15, 18, 24, 25, 26, 27, and 28) was the predominant and unique phylum. This result agrees with other studies that indicated that Proteobacteria dominates some cathodic denitrifying biofilms (Wrighton et al., 2010).

In the phylum Proteobacteria, bands 15, 24 and 27 belonged to the genera Aquamicrobium, Alishewanella, and Brachymonas or Comamonas, respectively. These three species can grow under aerobic conditions, are heterotrophic, can utilize nitrate as electron acceptors (Hiraishi et al., 1995; Roh et al., 2009; Jin et al., 2013)and were inferred to dominate the aerobic denitrification under the high DO conditions of stage 4 (Figure 2). In particular, Comamonas denitrificans (band 27) has been reported to switch the metabolic pathway for extracellular electron transfer (Xing et al., 2010). A species in the Comamonas genus is known to be an aerobic denitrifier, and C. denitrificans is the only species in the Comamonas genus that can reduce nitrate to nitrogen gas and contains cd1-type nitrite reductase (Gumaelius et al., 2001). Therefore, we propose that C. denitrificans be considered a species of ADB. The other two species were inferred to belong to ADB.

Bands 18, 26 and 28 belonged to the genera Pelomonas, Altererythrobacter and Acinetobacter, respectively. These three species can grow under aerobic conditions, are heterotrophic, cannot utilize nitrate as electron acceptors (Xie and Yokota, 2005; Kwon et al., 2007; Nemec et al., 2009) and might have dominated the aerobic degradation of COD in stage 4 (Figure 2).

Band 25 belonged to the genus Brevundimonas, which grows under aerobic conditions and cannot reduce nitrate, and 90% of the strains are autotrophic (Segers et al., 1994). This species was inferred to be a nitrifier.

Based on this analysis, at high temperatures, the increase in DO resulted in a change in the predominant species from thermophilic autotrophic nitrifiers and facultative heterotrophic denitrifiers at low DO concentrations to thermophilic ADB at high DO concentrations during the operation of the MFC. Three species from the genera Aquamicrobium, Brachymonas or Comamonas, and Alishewanella were inferred to belong to ADB and dominate the aerobic denitrification at high levels of DO. Some aerobic denitrifiers were known to be heterotrophic nitrifiers, which might benefit SND under aerobic conditions. Therefore, autotrophic nitrifiers were replaced, and ADB evolved to be the predominant bacteria at high DO concentrations in stage 4 of the operation. This result is similar to the study by Feng et al., who indicted that aerobic denitrification in the cathode chamber is an important pathway for nitrite and nitrate removal (Feng et al., 2015).

Based on the analysis of the composition of the microbial community and the experimental results, we speculated on the possible reactions in the cathode of the MFC (Figure 7).

The mechanism of aerobic denitrification was determined by studying the aerobic denitrifier T. pantotropha. The cooperative breathing theory, which was proposed by Robertson et al. (1988), is widely recognized as the aerobic denitrification mechanism. Cooperative breathing theory means that both NO3− and O₂ can be used as the final electron receptors. Therefore, the denitrifiers can transfer electrons from the reduced substance to NO3− or O₂, and the denitrification can occur in an aerobic environment. According to the electron transport model proposed by Willson and Bouwer (1997), both NO3− and O₂ can be used as the final electron receptors, while the denitrifiers can transfer electrons from the reduced substance to O₂ or NO3− through nitrate reductase. Moreover, a carbon source is required for ADB. The higher the concentration of the carbon source is, the faster the aerobic denitrification rate will be (Robertson and Kuenen, 1983). Huang and Tseng (2001) indicated that the denitrification rate was highest when C/N was 5 and decreased with increasing C/N when C/N was greater than 5 and acetate was used as the carbon source. However, ADB did not appear to be required at such a high C/N ratio in our study. The influent C/N for the cathode chamber of the MFC was less than 0.3, possibly because the electrons provided by the electrode of the MFC were sufficient for the growth of aerobic bacteria. The aeration cathode chamber of the MFC may be beneficial for the growth of ADB.

Huang et al. indicated that DO was a key factor for aerobic denitrification (Xing et al., 2010). DO concentrations of 2–6 mg/L were beneficial for the growth of aerobic bacteria and the denitrification performance. As a result of the coexistence of aerobic respiration and the denitrifying reductase in one system, both O₂ and NOx− were indispensable for the growth of ADB and were used as electron acceptors. Therefore, at the high DO levels in this study (3.0–4.2 mg/L), the activity of ADB improved, and the denitrification performance of ADB was enhanced. The results that showed that the production of N₂O and the TN removal efficiency were higher with a 50 Ω external resistance than with a 100 Ω resistance at high levels of DO demonstrated that electrode denitrification with ADB occurred in the cathode of the MFC.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.