Silk Fibroin-Based bioanodes integrating Bacillus subtilis and Prussian blue for Rapidly activated and High-Performance microbial fuel cells

• Silk fibroin provides biocompatible immobilization of electrogenic bacteria maintaining high viability. • Bioanodes integrating Bacillus subtilis and Prussian blue as redox mediator nearly double current output. • Functionalized anodes reduce soil MFCs start-up time from 8 to 4 days. • Immobilized Bacillus subtilis promotes selective microbial colonization and sustained performance. • A green silk fibroin crystallization protocol enables scalable bioanodes up to 95 cm 2 . Soil microbial fuel cells (sMFC) are promising bioelectrochemical systems that convert organic matter in soil into electricity via microbial metabolism. However, their practical deployment is hindered by low efficiency, limited scalability, and the long start-ups time required for biofilm formation. This study introduces a synergistic immobilization strategy that integrates both electrogenic bacteria and redox mediators directly onto the anode surface. Bacillus subtilis , a soil-dwelling electrogenic bacterium, was encapsulated within silk fibroin (SF) membranes using a one-step green chemical process that ensures high bacterial viability and strong adhesion to large-area carbon anodes (95 cm 2 ). Co-immobilization of Prussian blue (PB) within the SF matrix enhanced electron transfer between microorganisms and electrodes. The dual-functionalized bioanodes nearly doubled the maximum current output (2.5 mA vs. 1.5 mA), and reduced start-up time from 8 to 4 days. After one month, SF membranes remained structurally stable and supported active microbial colonization, confirming their durability and biocompatibility. These findings demonstrated that simultaneous immobilization of electrogenic bacteria and redox mediators accelerated biofilm activation and boosted energy generation, positioning SF as a scalable platform for sustainable, field-ready microbial bio-batteries.