Optimization of Poly-γ-Glutamic Acid Production by Moderate Electric Field-Assisted Fermentation in a Haematococcus pluvialis-Based Medium

Moderate electric field (MEF) fermentation was investigated as a nonthermal stress strategy to enhance the formation of extracellular polymeric substances (EPS; mucilage) by Bacillus subtilis HA in a Haematococcus-based medium. Response surface methodology (RSM), combined with a Box–Behnken design, was employed to optimize the fermentation medium composition and the MEF operating parameters. Under conventional fermentation (CF), the optimized medium (2.91% Haematococcus pluvialis, 3.10% glucose, and 13.42% L-glutamate) yielded 8.60 ± 0.25% mucilage, closely matching the predicted value (8.54%) and confirming model adequacy. The optimized MEF conditions (85.2 Hz, 0.29 A, and 40.90% duty cycle) increased mucilage formation to 13.59 ± 0.18%, corresponding to approximately 1.6-fold enhancement compared with CF. Statistical analysis indicated that frequency–current interactions and a nonlinear duty cycle response governed EPS accumulation. Substrate–metabolite analysis further showed that MEF significantly reduced residual L-glutamate (1.25 ± 0.03%) while increasing poly-γ-glutamic acid (γ-PGA) content (11.07 ± 0.16%), indicating enhanced conversion of L-glutamate into γ-PGA-rich mucilage. In comparative physicochemical stress experiments, 3% KCl supplementation produced the highest mucilage formation among non-MEF conditions. The MEF-treated system achieved the highest mucilage production, indicating that MEF is a tunable and effective strategy for promoting substrate-to-polymer conversion and regulating EPS production in Bacillus-based fermentation systems.