Analysis of microbiota involved in the aged natural fermentation of indigo

Takahiro Okamoto, Kenichi Aino, Takashi Narihiro, Hidetoshi Matsuyama, Isao Yumoto

Although the indigo reduction process is performed via natural fermentation and maintained under open-air condition, the indigo-reducing reactions continue for 6 months (on average) or longer. Identifying the mechanism underlying the maintenance of this process could lead to the development of a novel, long-lasting, unsterilized bioprocesses. To determine the mechanisms underlying the maintenance of the indigo fermentation system microbiota for more than 6 months in a reduced state in an anaerobic alkaline environment, we examined changes in the microbiota in one early-phase batch and two aged batches of indigo fermentation fluid. The microbiota in the aged fermentation fluid consisted mainly of the genera Alkalibacterium, Amphibacillus, Anaerobacillus and Polygonibacillus and the family Proteinivoraceae. The genera Alkalibacterium, Amphibacillus and Polygonibacillus are known to include indigo-reducing bacteria. Although the transition speed was slower in the aged fermentation fluid than in the early-stage fluid, the microbiota in the aged fermentation fluid maintained for more than 6 months was drastically changed within a period of 3 months. The results of this study indicate that the bacterial consortia consisted of various indigo-reducing species that replace the previous group of indigo-reducing bacteria. The notable transitional changes may be concomitant with changes in the environmental conditions, such as the nutritional conditions, observed over 3 months. This flexibility may lead to important changes in the microbiota that allow for the maintenance of a fermentation-reducing state over a long period.