Tomodane Promotes Microbial Community Convergence and Indigo Reduction During Laboratory-Scale Indigo Fermentation

Indigo dyeing is a traditional microbial fermentation process practiced worldwide using plants that produce indican, a precursor of indigo. In Okinawa Prefecture, Japan, indigo dyeing is traditionally performed using a muddy suspension containing indigo, prepared from Strobilanthes cusia. In this region, the addition of a fully fermented dye liquid, Tomodane, is believed to promote fermentation, although its microbial basis remains unclear. In this study, we developed a laboratory-scale indigo fermentation system with biological replication to investigate the effects of Tomodane supplementation on the dyeing intensity, bacterial community dynamics, and indigo particle size. Fermentation supplemented with Tomodane showed an earlier onset of fabric dyeing than fermentation without Tomodane. Microbial community analyses revealed that the bacterial communities in Tomodane-supplemented fermentation converged more rapidly toward a stable community structure. Additionally, bacterial taxa putatively associated with extracellular electron transfer (EET), a process relevant to indigo reduction, were more abundant in the bacterial community at earlier fermentation stages in the presence of Tomodane. Indigo particle size decreased more rapidly during Tomodane-supplemented fermentation, coinciding with an earlier dyeing onset. These results suggest that Tomodane facilitates indigo fermentation by facilitating the early establishment of a functionally competent microbial community capable of efficient indigo reduction.