Synthesis of g-C₃N₄ was performed by conventional methods (Yan et al., 2009). Melamine was purchased from Wako Pure Chemical Industries. To ensure reproducibility, the heating program was carefully controlled to prevent premature decomposition and promote polymerization of melamine into g-C₃N₄. The sample was placed in a crucible and sealed with a lid to prevent sublimation. First, the sample was heated to 500°C at a heating rate of 20°C/min and held for 2 hours. Then, the heating rate was changed to 5°C/min, and the sample was heated to 520°C and held for 2 hours. After the heat treatment, the sample was cooled naturally to room temperature, and the resulting solid was powdered using a mortar. Figure 1 shows the scanning electron microscopy (SEM) image of the synthesized g-C₃N₄ powder. The synthesized particles ranged in size from approximately 10 to 40µm.

Rice-husk-derived carbon (RHC) was selected for its porous structure, high surface area, and low production cost, making it suitable for microbial colonization. The RHC was subjected to alkaline etching by stirring in a 6 mM sodium hydroxide solution at 90°C for 24 hours. After treatment, the mixture was carefully rinsed with deionized water and then air-dried at 60 °C for 12 hours. The resulting material was then ground into a fine powder using a mortar and pestle.

Electrode fabrication was carried out according to the method described in (Hirose et al., 2023). For the cathode, a mixture consisting of Co-MnO₂ catalyst (0.75 g), RHC (0.525 g), and carbon ink (3 mL of commercial sumi ink) was prepared and used as the catalyst layer. For the anode, a mixture of RHC (0.9 g) and sumi ink (3.5 mL) was homogenized and cast into a 2 cm × 2 cm mold fabricated by 3D printing. A stainless steel mesh was embedded into the mixture, and the electrode was air-dried at 60°C for 24 hours.

To incorporate graphitic carbon nitride (g-C₃N₄) into the anode material, the powdered RHC was modified by dispersing 5 wt%, 10 wt%, and 12 wt% g-C₃N₄ in methanol and then stirring for 24 hours. After dispersion, the solid was collected via centrifugation and dried at 60°C for 24 hours to obtain g-C₃N₄-modified RHC. The resulting modified carbon material was then mixed with sumi ink and used to fabricate the g-C₃N₄-containing anode electrodes.

Luria-Bertani (LB) medium was utilized as the nutrient source for microbial cultivation. The medium was prepared by dissolving 5.0 g of tryptone, 2.5 g of yeast extract, and 5.0 g of sodium chloride (NaCl) in 380-400 mL of deionized water, ensuring that the total volume would not exceed 400 mL after all components were added. Separately, 0.5 g of sodium hydroxide was dissolved in 40 mL of deionized water, and 2.5 mL of this solution was added to the primary mixture. If the total volume was still below 400 mL at this stage, deionized water was added to bring it to 400 mL. Subsequently, an additional 100 mL of deionized water was added, and the final mixture was sterilized by autoclaving to complete the preparation of LB medium.

The microbial fuel cell (MFC) employed in this study was constructed as a single-chamber system equipped with a floating air cathode, as illustrated in Figure 2. In this system, the anode and cathode coexist in the same chamber, simplifying the structure and improving ease of operation. The chamber was filled with a reaction solution composed of 100 mL of Luria-Bertani (LB) medium as a nutrient medium and 500 mL of water for dilution. Mud collected from the natural environment in Japan was used as the microbial source, of which 2.5 mL was added to the reaction system.

Compared with the double-chamber type, the single-chamber MFC has a simpler structure and does not require an intermediate ion-exchange membrane, thereby reducing manufacturing costs and internal resistance. In addition, since oxygen from the air can be supplied directly to the cathode (Li et al., 2011), (Logan et al., 2006), it does not require an external energy supply and has excellent operational efficiency and scalability, making it a promising configuration for practical use.

A fixed 10 kΩ external resistor was continuously connected between the anode and cathode of the MFC throughout the experiment to ensure a stable discharge condition. Experiments were performed under controlled ambient conditions at 25 ± 1 °C. Daily fluctuations in solution level caused by evaporation were compensated for by replenishing with tap water as needed. Electrode potential and power density were used as indicators of performance. The external resistance varied in steps from 10,000 Ω to 200 Ω, and the voltage at both ends at each resistance value was measured. From these voltage values and the corresponding resistance values, the current and power were calculated and further divided by the effective surface area of the anode to obtain the current density and power density. In addition, power density measurements were performed multiple times during the experimental period to evaluate changes in the power generation characteristics of the MFC over time. Ag/AgCl electrodes were employed as reference electrodes to measure the electrode potentials. The power generation voltage (V) of the MFC was defined as the potential difference between the anode and the cathode, and is represented by the following equation.

V =E cat h ode -E anode (1)

The voltage applied to the external resistors was measured using a data acquisition device (DAQ) controlled by LabVIEW, which automatically recorded voltage data at 15-minute intervals.

To investigate whether g-C₃N₄ was successfully incorporated and supported on the rice husk biochar, the elemental composition of the particles was analyzed using Energy Dispersive X-ray Spectroscopy (EDS).

Figures. 3 and 4 provide a comprehensive visualization of the structural and elemental characteristics of biochar modified with 10 wt% g-C₃N₄. Figure 3(a) presents the SEM image of g-C₃N₄-modified biochar, illustrating its surface morphology. Figure 3(b), (c), (d), and (e) show the elemental mapping images of carbon (C), nitrogen (N), oxygen (O), and silicon (Si), illustrating their respective spatial distributions and providing evidence for the successful incorporation of g-C₃N₄ into the biochar structure.

Figure 4 shows the corresponding EDS spectrum of the modified biochar; the X-axis corresponds to the energy of the X-rays, while the Y-axis indicates the signal intensity. The mass percentages of detected elements are summarized in Table 1. The measured elemental composition consisted of carbon (80.83%), nitrogen (6.41%), oxygen (10.93%), and silicon (1.83%).

In contrast, the EDS results for the unmodified biochar, shown in Table 2, indicate the absence of nitrogen. These data clearly demonstrate that g-C₃N₄ was successfully incorporated into the biochar using the experimental method employed in this study.

Figure 5 illustrates the temporal evolution of anode potentials relative to the Ag/AgCl reference, demonstrating the effect of g-C₃N₄ incorporation into the anode material under the same cathode conditions. By keeping the cathode conditions constant, the changes observed are attributed to the anode configuration, as the potential was maintained. Focusing on the anode potential, the anode with 5 wt% g-C₃N₄ had the highest potential at -540 mV at 14 days, higher than the other conditions. The more negative anode potential observed in the 5 wt% electrode indicates greater electron-accepting capability, suggesting improved microbial electron-transfer kinetics. This trend aligns with reports that nitrogen-rich materials facilitate microbial adhesion and redox activity. However, since g-C₃N₄ is not intrinsically highly conductive, increasing the loading to 10 wt% or 12 wt% diminished the electrochemical performance. This suggests that the addition of g-C₃N₄ improves anodic performance only in the appropriate concentration range, beyond which the balance between conductivity and microbial activity may become unbalanced and counterproductive.

This behavior suggests that g-C₃N₄ incorporation enhances microbial electron transfer at moderate loading, while excessive incorporation disrupts conductive pathways within the carbon matrix. The superior performance at 5 wt%, therefore, reflects a more favorable internal distribution of g-C₃N₄ that supports both microbial activity and electron flow.

Figure 6 and Figure 7 show the electrochemical performance of the MFC with g-C₃N₄ added to the anodes at different percentages (5, 10, and 12 wt%). Figure 6 shows the change over time of the maximum power density measured every 2 days starting on day 8 of installation. Across all metrics, the 5 wt% g-C₃N₄ anode exhibited superior performance. Notably, on day 14, the maximum power density reached 43.245 μW/cm², approximately 1.9 times higher than the 23.256 μW/cm² of the anode without addition. In the power density curve, the 5 wt% addition condition also showed a significant increase in power output relative to current density compared to the other conditions, confirming that it has excellent energy conversion efficiency. These results suggest that the moderate addition of g-C₃N₄ (5 wt%) effectively promotes microbial activity and electron transfer on the anode surface and improves MFC performance. On the other hand, higher additions (10 wt% and 12 wt%) tended to suppress performance, suggesting that excess g-C₃N₄ may adversely affect electron transfer and microbial activity.

As shown in Figure 7, the anode containing 5 wt% g-C₃N₄ exhibits the highest power density over the entire current density range. This result suggests that moderate g-C₃N₄ incorporation provides a favorable balance between enhanced microbial activity and sufficient electrical conductivity, whereas excessive loading negatively affects electron transport

These results indicate that the performance trend is governed by how g-C₃N₄ incorporation modifies the internal electron pathways and microbial attachment sites. At 5 wt%, g-C₃N₄ is sufficiently integrated to enhance biofilm formation and electron transport, whereas higher loadings disrupt conductivity and reduce efficiency. The observed performance differences can therefore be interpreted based solely on how g-C₃N₄ incorporation affects internal conductivity and microbial colonization. This explanation strengthens the validity of the case study without requiring additional experiments or simulations. This study provides a clear framework for understanding how nitrogen-rich additives influence both the biological and conductive properties of carbonaceous anodes. By presenting a comparative evaluation based solely on controlled loading variations, the work offers insights that are broadly applicable to the design of future biomass-derived MFC electrodes.

Not applicable.

The author(s) declare that there is no competing interest