2025 Volume 40 Issue 3 Article ID: ME24104
In traditional indigo dyeing, water-insoluble indigo is anaerobically converted into soluble leuco-indigo via microbial reduction in alkaline dye suspensions, allowing its use as a fabric dye. Although various indigo-reducing bacteria have been isolated to date, culture-independent microbial community analyses have suggested that bacteria belonging to uncultured clades also contribute to indigo reduction. Therefore, we aimed to isolate previously overlooked indigo-reducing bacteria using an unconventional culture method. We conducted enrichment cultures and single-colony isolation using a medium supplemented with sukumo, an indigo dye source derived from the composted leaves of indigo-containing plants, as the sole energy, carbon, and nitrogen sources. We isolated a previously uncultured bacterium belonging to the family Tissierellaceae, which had been predicted as a major indigo reducer in various indigo dyeing processes solely based on microbial community analyses. The insoluble indigo-reducing activity of the Tissierellaceae isolate, strain TU-1 was significantly higher than that of known indigo-reducing bacteria. The addition of the culture supernatant of strain TU-1 enhanced the reduction of indigo powder by other indigo-reducing bacteria, with similar stimulatory effects to those of the insoluble electron mediator, anthraquinone. These results indicate that strain TU-1 possesses a high capacity for secreting electron mediators, conferring a significant reduction capacity for insoluble indigo. Further investigations, including the discovery of additional unknown indigo-reducing bacteria and the identification of the mediators they produce, will provide a more detailed understanding of the mechanisms underlying indigo reduction in practical dyeing processes.
