Enhanced extracellular respiration of engineered Bacillus subtilis via anodic electro-fermentation with pH optimisation

Leveraging alternative electron acceptors to support anaerobic metabolism in industrially relevant microorganisms holds substantial biotechnological potential, especially when coupled to anodic electro-fermentation, which provides a non-depleting electron sink to the microorganisms. Bacillus subtilis is a widely used industrial workhorse for biochemical production, valued for its genetic tractability and environmental stress tolerance. However, the anaerobic, anodic metabolism of B. subtilis has been constrained by limited redox flexibility. Here, deletion of ldh (lactate dehydrogenase) to restrict fermentative NAD⁺ regeneration enabled engineered B. subtilis to survive anaerobically via anodic respiration, partially oxidising glucose while steering the metabolism toward 2,3-butanediol. The anodic metabolism showed enhanced extracellular electron transfer mediated by ferricyanide, with the highest current density of 0.77 mA/cm² reached within 2 h. Carbon flux was directed predominantly to 2,3-butanediol (0.49 ± 0.07 mol_product/mol_glucose) under incomplete glucose oxidation and without pH control. In addition, pH control further improved anodic electro-fermentation performance. At pH 6.5, 66% of the added glucose was consumed, and 2,3-butanediol carbon selectivity rose to 77.1 ± 0.6%, whereas at pH 7.5, cells consumed 89% of the glucose with 73.4 ± 0.7% 2,3-butanediol carbon selectivity. To our knowledge, this is the first investigation of anodic electro-fermentation in B. subtilis that integrates metabolic engineering with pH optimisation strategies, demonstrating a new route to produce high-purity biochemicals from renewable resources.