Volume 25

DOI:http://doi.org/10.65613/741896 Li Chen HongKong Baptist University ,Hongkong Article submission 5-01 –2026Article acception 29-04-2026Publication 31—05- 2026 Abstract: Microbial fuel cells (MFCs) represent a promising bioelectrochemical technology for sustainable energy recovery coupled with wastewater treatment. In this study, aerobic halophilic bacteria isolated from china were evaluated for their electrochemical performance in an MFC system. Aluminum electrodes were employed, generating a stable electrical potential of approximately 550 mV. Six isolates were individually screened, revealing generally low extracellular electron transfer (EET) efficiency; however, one strain exhibited markedly enhanced electroactivity, with a maximum voltammetric signal of 612 mV. Phylogenetic characterization based on 16S rRNA gene sequencing identified this strain as belonging to the genus Salinivibrio. Biofilm formation by Salinivibrio sp. on both anodic and cathodic surfaces was found to be a critical factor governing electron transfer and current generation. Electrical performance was normalized to reactor volume, which may partially account for variability in power output interpretation. Furthermore, the application of Salinivibrio sp. in sterile wastewater under controlled in vitro conditions significantly improved electricity production. These findings highlight the potential of halophilic electroactive bacteria in MFC applications and emphasize the importance of operational parameters, including aeration and substrate homogeneity, in optimizing bioelectrochemical system performance.. Keywords: Microbial fuel cell; Bioelectrochemistry; Extracellular electron transfer; Saltivibrio sp.; Halophilic bacteria; Biofilm-mediated electron transfer 1.Introduction Fossil fuels, comprising coal, natural gas, and oil, were formed from the remains of deceased flora and fauna that underwent compression and thermal alteration over millions of years due to overlying sedimentary and rock layers  . ]1[These fossil fuels can generate high-efficiency energy to operate vehicles, electronic devices, and support daily human activities ] 2[]3[. Nonetheless, their limited availability renders them unsustainable energy sources. Therefore, there is a necessity for renewable and sustainable fuels to supplant fossil fuels and advance a contemporary industrial civilization ]4[]5[. The microbial fuel cell exemplifies an eco-friendly method for power generation while concurrently treating wastewater, facilitating the reduction of chemical oxygen demand and electrical energy densities ]6[]7[. Electroactive bacteria significantly influence many surroundings by establishing electrical connections with other cells and minerals at their exterior surfaces or by reducing soluble extracellular redox-active compounds, including flavins and humic chemicals ]8[. A burgeoning corpus of research underscores their significant phylogenetic variety and demonstrates that these germs are integral to several biogeochemical cycles, as well as to the gut microbiome, anaerobic waste digesters, and metal corrosion ]9[]10[.. The objective of this study is to identify indigenous electroactive bacteria and to construct a bacterial biopile. 2. Methodology 2.1. Sampling Water samples were initially collected from the saline lake (Sebkha) located in Aïn Beïda. Sampling was performed randomly using a sterile spatula at a depth of approximately 20 cm. The collected sample was then transferred into a sterile glass flask to avoid contamination. 2. 2. Physico-chemical analyses Conductivity quantifies a solution’s capacity to facilitate the flow of electric charges, which is contingent upon the mobility of ions. The pH measurement relies on the potential difference between a reference electrode and an indicator electrode responsive to H⁺ ions, with the resultant potential translated to pH using a pH meter. 3. Microbiological Analysis 3.1. Isolation of aerobic bacteria and preparation of biofuel cell reactors 3.2. Isolation of electroactive bacteria The isolation of electroactive strains is conducted using biopiles seeded with aerobic bacteria sourced from Sebkhat Aïn Beïda. Bacteria adhered to the surfaces of the aluminum electrodes are swabbed, followed by isolation on Chapman agar and salt agar. The plates are incubated for 96 hours at 30 degrees Celsius. The colonies that emerge are subjected to multiple rounds of purification under identical conditions. 3.3. Strain selections Precultures were established by inoculating two colonies from each purified strain into 50 mL of saline BN medium to identify strains with the highest electroactive potential. The strains included S1, S2, S3, and S4. Cultures were maintained at 30 °C with agitation at 150 rpm for 48 hours. The bio-batteries were subsequently constructed using 150 mL of saline BN medium, incorporating aluminum electrodes with a surface area of 35 cm² (5 cm × 7 cm). Cultures were maintained at 30 °C with agitation at 100 rpm for a duration of 10 days. 3.4.Identification of selected strain 3.4.1. Morphological characterization The macroscopic analysis of isolated colonies is a crucial initial step in their characterization, aiding in the identification process. A variety of phenotypic factors are taken into account, including: surface characteristics, elevation, and pigmentation. The microscopic assessment of the colony morphology is conducted using Gram staining 1]1[. 3.4. Identification of selected strain Bacterial isolates were identified using direct nucleotide sequencing of the 16S rRNA gene, followed by a comparison of nucleotide identity with sequences available in the international Gen Bank database. The PCR process utilized universal primers (27F: 5′-AGA GTT TGA TCC TGG CTC AG-3’ and 1492R: 5′-CCG TCA ATT CCT TTG AGT TT-3’). The resultant DNA pellet was air-dried, re-suspended in 15 μl of formamide, and subsequently analyzed with a 3730 XL Genetic Analyzer Capillary Array (Applied Biosystems). The sequences were examined using CHROMAS PRO software. Isolates were identified by comparing sequences to those in the GeneBank database with NCBI’s BLAST tool (accessible at https://blast.ncbi.nlm.nih.gov/Blast.cgi), according to percent homology with reference strains. 3.5.  Biofuel cell optimization The electroactive capacity of isolate S1 and the effect of electrode surface area were investigated using microbial fuel cells equipped with aluminum electrodes of different sizes (16, 35, and 70 cm²). Pre-cultures were grown in saline BN medium at 30°C under agitation, then transferred into glass reactors and incubated for 10 days. Biomass growth and electrochemical activity were monitored daily by measuring optical density (OD600) and electrical current production. 3.6. Wastewater Treatment Precultures were established using a combination of the two strains, S7 and Sab, in 400 cc of BN saline medium. The cultures were incubated at 30°C with shaking at 100 rpm for a duration of 48 hours. Subsequent to incubation, the cultures underwent centrifugation at 3000 rpm at 4°C. The bacterial pellet was collected and subsequently washed multiple times with sterile saline to eliminate any remnants of the growing media. The cleaned bacterial cells were subsequently resuspended