Adaptive laboratory evolution of Komagataeibacter nataicola for formate-tolerant bacterial cellulose biosynthesis and altered membrane properties.

Formate is a representative weak-acid inhibitor in lignocellulosic hydrolysates and impairs bacterial cellulose (BC) fermentation. Here, adaptive laboratory evolution (ALE) was used to improve formate tolerance in Komagataeibacter nataicola DHU-1 under a defined formate-stress model. Parental DHU-1 was strongly inhibited at ≥30 mM formate, with almost unchanged glucose and formate concentrations and no recoverable BC. After 82 weeks of ALE, the evolved strain DHU-1-F exhibited tolerance to 300 mM formate. Under the tested conditions, no statistically significant changes in the measured fermentation traits were observed after 20 consecutive passages in formate-free medium. Under 300 mM formate, extracellular formate decreased by 164.94 mM within 7 days, while DHU-1-F produced 2.51 g/L BC, corresponding to 71.31% of its formate-free BC-producing capacity. Transcriptomic and reverse transcription quantitative polymerase chain reaction analyses implicated membrane remodeling, respiratory and redox regulation, transport adaptation, and tetrahydrofolate-linked one‑carbon metabolism. Resequencing identified five candidate mutations shared by three isolates from a single ALE population, including a truncating mutation in an EAL domain-containing protein. The resulting BC retained its cellulose I structure but showed increased transparency, a more open nanofibrous morphology, lower apparent bulk density, and reduced mechanical strength. These findings established a strain-level strategy for formate-tolerant BC biosynthesis under defined formate-stress conditions and supported evaluation in authentic or synthetic lignocellulosic hydrolysates containing mixed inhibitors.