Study’s Novelty/Excerpt This study presents an approach to enhancing microbial fuel cell (MFC) performance by employing phototrophic bacteria (PTB) and sustainable electrode materials, specifically a 3D anode electrode fabricated from reduced graphene oxide (rGO) and nickel (Ni) foam. By integrating morphological, biochemical, and molecular techniques to identify the electrochemically active PTB, the research achieved a significant eight-fold increase in power density using rGO-Ni electrodes compared to conventional Ni electrodes. This work underscores the potential of utilizing sustainable materials and PTB to improve MFC efficiency and economic viability, offering a promising direction for sustainable bioelectricity generation. Full Abstract Over the past years, despite intensified research on microbial fuel cells (MFC), low power densities were recorded, reducing the productivity and economic viability of the process. This necessitated testing various MFC configurations, fabricating various electrodes, and evaluating various substrate types and species of electrogenic microorganisms to improve MFC performance. Despite the dual advantage of phototrophic bacteria (PTB), metabolizing organic waste substances and generating electricity, less research was conducted on the bacterium. Although a significant amount of energy is generated using unsustainable (fossil-based) materials in electrode fabrication, this study focuses on using sustainable materials like carbon cloth and graphite to fabricate a 3D anode electrode to exploit the maximum energy generated by PTB. The PTB used in this study was identified through morphological characteristics and biochemical tests (catalase and oxidase) and confirmed using a molecular technique: 16S rRNA sequencing. Preliminary results indicated that the PTB was gram-negative, spherical in shape, non−motile, and facultatively anaerobic bacterium. Analysis of the 16S rRNA partial sequence was conducted in GenBank databases. 100 significant sequences with the lowest and highest similarities of 84.10% and 98.76% were recorded, respectively. Of these, 13 strains had the highest similarities of >90%, all belonging to the genus Dysgonomonas, with D. oryzarvi Dy73 (98.76%) as the closest. Reduced graphene oxide (rGO) used as the anode was prepared using Hummer’s method by depositing the rGO on nickel (Ni) foam which changed the colour of Ni from grey to black after depositing and annealing. In addition to the SEM images, which showed a continuous multi−layered 3D scaffold on the Ni, the cyclic voltammetry (CV) analyses indicated an increase in the electrochemical activities of the rGO−Ni electrode compared to Ni. The CV also confirmed the bacterium to be electrochemically active. The 100 mL glucose−fed two−chamber MFC were separately run with the Ni and rGO–Ni as anode electrodes in a batch mode for 11 days, while carbon cloth was used as the cathode for both runs. An approximate 0.58 W/m2 power density was recorded for Ni, but eight−fold of Ni’s, 4.9 W/m2was generated by rGO−Ni. The study demonstrated that using fabricated 3D rGO–Ni as anode electrode can increase the microbial adhesion and power density of bacterium in MFC, thereby providing a more applicable and sustainable alternative to bioelectricity generation.