The Effect of Electricity Generation on the Performance of Microbial Fuel Cells for Anammox

According to the varying concentrations of COD in the influent, this experiment was divided into three distinct stages. The relevant nitrogen data were meticulously recorded and subsequently analyzed to determine the optimal COD concentration for Anammox AFB-MFC (Figure 3), thereby assessing its nitrogen removal performance.

The system exhibited a high capacity for the removal of both NH₄⁺-N and NO₂⁻-N under three varying influent COD concentration gradients. During phase I (1–21 d), with an influent NH₄⁺-N concentration of 209.98 mg/L, the effluent concentration at the anode for NH₄⁺-N decreased to 58.48 mg/L, while at the cathode, it was measured as 25.10 mg/L. Simultaneously, with an influent NO₂⁻-N concentration of 200.13 mg/L, the effluent concentration at the anode for NO₂⁻-N reduced to 54.40 mg/L, and at the cathode, it reached a level of 8.51 mg/L. Anammox AFB-MFC also achieved lower nitrogen effluent concentrations in the other two stages, although there were slight differences in the NRE of the three stages (Figure 4a). At an influent COD concentration of 100 mg/L, the average NRE of the anode was 68.13%, while that of the cathode stood at 65.56%. Moreover, the overall average NRE reached 89.04%. During phase II (22–41 d), at an influent COD concentration of 200 mg/L, the average NRE of the anode was 69.90%, the average NRE of the cathode was 69.58%, and the average NRE of the reactor was 90.85%. Increasing the influent COD concentration to 300 mg/L resulted in an average NRE of 66.18% for the anode; the average NRE of the cathode was 62.98%; and the average NRE of the reactor was 87.48%. Therefore, for optimal performance of the Anammox AFB-MFC system, it is recommended to maintain a consistent influent COD concentration at around 200 mg/L. The same conclusion can be inferred from the observation of the cathode NRE and the coupling system NRE: when the influent COD concentration was 200 mg/L, the reactor exhibited the highest NRE, indicating its strongest removal capacity for NH₄⁺-N and NO₂⁻-N. This finding aligned with previous research by Chamchoi et al. [19], who investigated Anammox-coupled nitrogen removal in an UASB reactor using aquaculture wastewater. They observed a decrease in the NH₄⁺-N removal rate by AnAOB when COD concentration exceeded 200 mg/L.

In addition, a small amount of NO₃⁻-N was inevitably generated during the reaction process. According to the Anammox reaction mechanism, the production of NO₃⁻-N should account for 11% of the influent TN. However, in this experiment, when the influent TN concentration was approximately 400 mg/L, the observed production of NO₃⁻-N never exceeded 13 mg/L, significantly deviating from its theoretical value. This phenomenon can be attributed to the endogenous DNRA electron transfer mechanism exhibited by AnAOB bacteria. Specifically, AnAOB possesses the capability to sequentially convert NO₃⁻-N into NO₂⁻-N and NH₄⁺-N through the enzymes Nar and NrfA [22]. Denitrifying bacteria can utilize intracellular carbon sources, such as polyhydroxyfatty acid esters (PHA) and glycogen, as electron donors to substitute external carbon sources in order to achieve NO₃⁻-N removal.

During this stage, the output voltage of the Anammox AFB-MFC exhibited fluctuations, characterized by significant decreases and increases (Figure 4b). It was hypothesized that variations in organic matter concentration may impact the power generation of the reactor, leading to a decline in the output voltage and power due to disruptions in the stable state of electricity-producing bacteria caused by changes in influent organic matter concentration. Gradually enriched methanogenic bacteria started competing with electricity-producing bacteria for organic substrates, resulting in reduced system power generation. Throughout this process, the output voltage ranged from a minimum of 356.6 mV to a maximum of 439.1 mV. In the final stage, MFC power generation tended to stabilize within a range of 380–410 mV. Hassan et al. [23] constructed an MFC reactor inoculated with AnAOB and employed the MFC anode for landfill leachate treatment. They observed that under high influent COD concentrations (800 mg/L), the power density of the MFC was enhanced, which is consistent with our experimental findings. The average output voltage and power density of the MFC reached their peak values at the highest influent COD concentration. Furthermore, the formation process of bioparticles in the reactor is closely linked to electron and proton transfers at both the anode and cathode, potentially influencing voltage output performance, as influent substrate concentration and environmental conditions vary.

With the continuous variations in experimental conditions, corresponding fluctuations in MLSS and MLVSS concentrations were observed within the anode and biological cathode regions of the system (Figure 6). By monitoring both concentration changes, it can be inferred that during the influent COD concentration alteration stage alone, both MLSS and MLVSS exhibited an increasing trend. The initial MLSS value at the anode increased from 17.73 g/L to 23.65 g/L on 56 d, while the initial MLVSS value rose from 13.82 g/L to 19.16 g/L on day 56 as well. Similar upward trends were observed for the cathode region, with a gradual increase in the MLVS/MLSS ratio indicating enhanced microbial activity during this phase. This enhancement may be attributed to a progressive rise in influent organic matter concentration, which promoted microbial growth and metabolism within the cathodic chamber.

Due to the presence of heme in AnAOB cells, AnAOB appears red [24], and its bioparticles are also red [25]. Research has shown that AnAOB bioparticles exhibit excellent reaction performance [26], but their growth is greatly limited by substrate transfer. Anammox bioparticles cease to grow when they reach a certain particle size. As the influent substrate is consumed, cells inside the anaerobic bioparticles immediately become inactive and die. Some dead sludge is discharged from the reactor outlet, increasing the pollutant concentration in the effluent, while others become a new component of the bioparticles as nutrients enter the next life cycle of anaerobic bioparticles. Zhu et al. [27] found that Anammox bioparticles exhibited the highest activity when the particle size was between 0.5 and 0.9 mm. To improve the stability of Anammox and reduce the dissolution rate of bioparticle cells, fillers were added to the reactor for the attachment of anaerobic ammonia oxidation bacteria to fillers and the cultivation of anaerobic ammonia oxidation bioparticles.

In the initial stage, the liquid in both anode and cathode chambers appeared yellow-brown, while the mixture of mud and water in the cathode chamber had a lighter color. Upon the addition of Anammox sludge, initially both polar chambers retained their yellow-brown hue. However, with the continuous operation of the MFC coupling system, it was observed that the sludge gradually transitioned to orange-red and eventually turned reddish-brown, with particle sizes ranging from 1 to 5 mm. Simultaneously, there was a noticeable increase in the volume of bioparticles within the anode chamber. Due to the limited AFB volume, the bioparticles could not grow indefinitely; consequently, some bioparticles experienced cracking and loss of activity. Eventually, in the anode chamber, anaerobic bioparticles coexisted with flocculent sludge upon the cathode outlet discharge. Furthermore, slight discoloration was observed on the surface of a small amount of spherical packing in the cathode chamber, transitioning from white and light yellow to red. It is hypothesized that red AnAOBs on the packing’s surface initiated growth. The MFC operation resulted in the transformation of some red particles into a black-brown color, accompanied by the peeling off of a layer of black film from the packing material. This phenomenon is speculated to be attributed to the inactivation and cracking of AnAOB, with the detached film eventually being discharged through the cathode outlet. Figure 7a illustrates an image depicting the bioparticles within the reactor, while Figure 7b presents a visual representation of the bioparticles present in the reactor. It can be observed that these bioparticles formed within the MFC exhibit a spherical shape with predominantly orange-red coloration and particle sizes ranging from 1 to 3 mm.

The overall morphology of the anaerobic bioparticles is depicted in Figure 7c. From the figure, it can be observed that the particle size ranges between 1 and 3 mm, exhibiting a spherical shape with some surface irregularities such as grooves, which facilitate microbial attachment (Figure 7d). The red circle in Figure 7e represents a spherical bacterium measuring approximately 1 μm in diameter, featuring a central depression that resembles a crater structure. This observation aligns with the previously reported morphology of AnAOB and thus suggests its potential identification as such. Furthermore, numerous spherical bacteria can be observed on the surface of bioparticles (Figure 7f), highlighted within the red circle, exhibiting diameters ranging from 0.9 to 1.4 μm, which is consistent with the defined size range for AnAOB. Therefore, these spherical bacteria are likely to be classified as AnAOB.

In order to comprehensively investigate the evolutionary dynamics of microbial communities and account for the columnar architecture of AFB, a metagenomic analysis was conducted on bioparticle samples collected from the upper, middle, and lower regions of the anode AFB reactor, as well as sludge particles in the cathode chamber. Figure 8 illustrates the microbial composition and relative abundance at both the phylum and genus levels.

At the phylum level (Figure 8a), regarding the anode (bottom), the main bacterial communities in MFC sludge particles are Planctomycota (23.30%), Pseudomonadota (18.33%), Chloroflexota (14.48%), Bacteroidota (12.49%), and Ignavibacteriota (10.43%). The main bacterial communities in MFC cathode anaerobic sludge are Chloroflexota (28.60%), Pseudomonadota (16.99%), Planctomycota (14.66%), Ignavibacteriota (6.91%), and Actinomycetota (5.67%). The research findings indicate that Anammox bioreactors harbor not only Planctomycotes but also Proteobacteria, Bacteroidetes, and Chloroflexota, aligning with the outcomes of this study [28,29]. In these advantageous communities, the current consensus recognizes AnAOB as a member of Planctomycotes, including Candidatus Kunenia and Candidatus Brocadia. The presence of a substantial population of AnAOB in MFC can be deduced in both anode and cathode. Chloroflexota is recognized as a pivotal microbial community in the degradation of carbohydrates [30]. It is also accountable for facilitating the maintenance of granular biofilms to a certain extent. Ignavibacterota is an anaerobic bacterium that is widely present in humid and hot environments. Bacteroidota, a microbial phylum closely associated with the formation of AnGS granulation, plays a crucial role in maintaining granulation through the secretion of EPS [31]. Furthermore, Thermodesulfobacteriota (0.69% in anode; 0.23% in cathode) exerts a predominant influence on the oxidation community of acetic acid substrates in bioelectrochemical systems and actively participates in bacteria’s extracellular electron transfer process [32].

At the genus level (Figure 8b), the polar chambers harbor abundant and uniformly distributed microbial communities. The AnAOB bacteria dominated both electrode chambers of the MFC, constituting a significant proportion. Candidatus Brocadia exhibited an abundance of 12.62% in the lower layer of the anode, 8.84% in the middle layer, and 9.75% in the upper layer. Candidatus Kunenia demonstrated an abundance of 1.37%, 3.49%, and 5.29% in the respective layers of the anode. Regarding the cathode, Candidatus Brocadia exhibited an abundance of 4.23%, and Candidatus Kunenia accounted for 0.34%. The prevalence of these two types of AnAOB bacteria can be attributed to favorable and stable environmental conditions provided by the reactor. It can be observed that, irrespective of the anode or cathode chamber, Candidatus Brocadia exhibited significantly higher abundance in this reactor compared to Candidatus Kunenia, indicating a subtle complementary relationship. When the abundance of Candidatus Brocadia was high, the relative abundance of Candidatus Kunenia was low. Furthermore, the uneven distribution of these two types of AnAOB bacteria in AFB suggests that despite incorporating a reflux device, there still exists non-uniform mixing at the anode, resulting in selective enrichment of these bacteria at distinct positions. Research findings imply that at a low substrate concentration, Candidatus Kuenenia exhibits superior growth compared to Candidatus Brocadia. Conversely, at a high substrate concentration, Candidatus Brocadia demonstrates enhanced proliferation over Candidatus Kuenenia [33]. The nitrogen content in the wastewater prepared for this experiment is significantly elevated, resulting in a higher abundance of Candidatus Brocadia. Additionally, the cathode chamber exhibits a lower abundance of AnAOB bacteria compared to the anode chamber, potentially attributed to the continuous closure of the anode with minimal dissolved oxygen (DO) presence. Consequently, the anaerobic environment at the anode demonstrates enhanced stability. This finding further substantiates previous experimental outcomes and underscores that nitrogen removal capability is superior at the anode when contrasted with the cathode. In addition, Thermomonas, Aquimonas, Thauera, and Simplicispira also possess the capability to reduce nitrate [34]. Hydrogenophaga is recognized as a versatile denitrifying bacterium responsible for nitrate removal [35], and its enrichment confirms the occurrence of nitrogen removal in the reactor. Nitrosomonas and Nitrospira, representing AOB and NOB in bioparticles, respectively, play pivotal roles in driving nitrification reactions.

The activity of electricity-generating microorganisms directly determines the electricity production capacity of MFC. Previous experiments have demonstrated a positive correlation between MFC’s electricity production capacity and system nitrogen removal, highlighting the significance of analyzing microorganisms associated with electricity generation in Anammox AFB-MFC for efficient nitrogen removal. Paracoccus (0.019%) is classified as a Gram-negative bacterium and exhibits diverse nitrogen metabolism pathways. It demonstrates the ability to utilize NO₃⁻-N, NO₂⁻-N, NO, and N₂O as substrates for metabolic processes. Furthermore, apart from nitrogen removal, Paracoccus has been acknowledged for its electrochemical activity. Hydrogenophaga (0.023%) is a Gram-negative bacterium capable of utilizing the byproducts of acetic acid metabolism in electrogenic bacteria. Consequently, Hydrogenophaga can establish a symbiotic relationship with electricity-producing bacteria, commonly coexisting with Geobacter (0.033%), which are known for their ability to generate electrical current in MFC systems. Ideonella (0.014%) is classified as a Gram-negative bacterium and exhibits the ability to utilize various organic acids, amino acids, and carbohydrates as carbon sources. Moreover, this bacterium demonstrates anaerobic growth capabilities by employing chlorate as an electron acceptor. Notably, its strain, Ideonelladechlorirans sp., also possesses the capacity to utilize nitrate as a terminal electron acceptor [36]. Klebsiella (0.0039%) is a newly discovered electrochemically active bacterium [37], which has been identified as the dominant microbial community on the electrode surface in non-inoculated MFC electrochemical cathodes [38]. Flavobacterium (0.023%) is classified as a Gram-negative bacterium and has been detected in the majority of MFC. Previous literature has demonstrated its capacity for extracellular electron transfer [39]. Acinetobacter (0.032%) is classified as a Gram-negative bacterium and represents a prevalent electrogenic microorganism found in the cathode biofilm of MFC [40,41]. Thauera (0.027%) is a predominant bacterial genus in solid-phase nitrogen removal processes and also exhibits the ability to generate electricity through organic degradation in both aerobic and anaerobic MFC anodes [42]. This further enhances microbial aggregation, aerobic granulation, and partial nitrification–denitrification. Extensive research has demonstrated that Pseudomonas (0.092%) possesses the capability to generate a significant amount of electron shuttle mass, thereby significantly enhancing the power generation capacity of MFC [43]. Shewanella, commonly detected in MFC (0.0023%), were also observed in the reactor; however, their abundance was significantly diminished. The observed phenomenon could potentially be attributed to the prevailing anaerobic conditions within the reactor and the presence of Anammox bacteria specifically cultivated for nitrogen removal. Further investigation into this matter would yield valuable insights.

The distribution of species present in the four microbial samples is illustrated in Figure 8c, while Figure 8d displays the functional distribution within these samples. On the left side of both figures, the samples and their respective groups are depicted, whereas on the right side, the top ten dominant species are highlighted. The abundance distribution of different species across the samples is visualized through interconnected inner-colored bands.

In order to facilitate a more comprehensive comparison and analysis of the relationship between microbial communities in the anode and cathode chambers, we established a correlation network (Figure 9). The top 30 species were selected with the highest abundance for analysis. AnAOB was highlighted within a black box, with Candidatus Brocadia represented by a larger circle indicating higher abundance and Candidatus Kunenia represented by a smaller circle. In Figure 9, AnAOB exhibited a significant positive correlation with Planctomycetota and Ignavibacteriota while showing negative correlations with Pseudomonadota, Actinomycetota, Chloroflexota, and Nitrospirota. The coexistence of electricity-producing bacteria in Ignavibacteriota and AnAOB could potentially explain the synergistic effect between nitrogen removal and electricity production in the system. The presence of abundant AnAOB on the anode surface might inhibit the growth of other nitrogen-related bacteria, such as Actinomycetota with nitrogen-fixing ability. Additionally, Nitrospirota, as a typical NOB, displayed a negative correlation with AnAOB. Microbial correlation networks for both anode and cathode revealed that AnAOB was present in both polar chambers, particularly Candidatus Brocadia showed potential mutual promotion with electricity-producing bacteria but had a negative correlation with NOB.