Indigo fermentation, which depends on the indigo-reducing action of microorganisms, has traditionally been performed to dye textiles blue in Asia as well as in Europe. This fermentation process is carried out by naturally occurring microbial communities and occurs under alkaline, anaerobic conditions. Therefore, there is uncertainty regarding the fermentation process, and many unknown microorganisms thrive in this unique fermentation environment. Until recently, there was limited information available on bacteria associated with this fermentation process. Indigo reduction normally occurs from 4 days to 2 weeks after initiation of fermentation. However, the changes in the microbiota that occur during the transition to an indigo-reducing state have not been elucidated. Here, the structural changes in the bacterial community were estimated by PCR-based methods. On the second day of fermentation, a large change in the redox potential occurred. On the fourth day, distinct substitution of the genus Halomonas with the aerotolerant genus Amphibacillus was observed, corresponding to marked changes in indigo reduction. Under open-air conditions, indigo reduction during the fermentation process continued for 6 months on average. The microbiota, including indigo-reducing bacteria, was continuously replaced with other microbial communities that consisted of other types of indigo-reducing bacteria. A stable state consisting mainly of the genus Anaerobacillus was also observed in a long-term fermentation sample. The stability of the microbiota, proportion of indigo-reducing microorganisms, and appropriate diversity and microbiota within the fluid may play key factors in the maintenance of a reducing state during long-term indigo fermentation. Although more than 10 species of indigo-reducing bacteria were identified, the reduction mechanism of indigo particle is riddle. It can be predicted that the mechanism involves electrons, as byproducts of metabolism, being discarded by analogs mechanisms reported in bacterial extracellular solid Fe3+ reduction under alkaline anaerobic condition.

The duration for which the indigo-reducing state is maintained in indigo natural fermentation batch dependent. The microbiota was analyzed in two batches of sukumo fermentation fluids that lasted for different durations (Batch 1: less than 2 months; Batch 2: nearly 1 year) to understand the mechanisms underlying the sustainability and deterioration of this natural fermentation process. The transformation of the microbiota suggested that the deterioration of the fermentation fluid is associated with the relative abundance of Alcaligenaceae. Principal coordinates analysis (PCoA) showed that the microbial community maintained a very stable state in only the long-term Batch 2. Therefore, entry of the microbiota into a stable state under alkaline anaerobic condition is an important factor for maintenance of indigo fermentation for long duration. This is the first report on the total transformation of the microbiota for investigation of long-term maintenance mechanisms and to address the problem of deterioration in indigo fermentation.

Indigo fermentation fluid maintains its indigo-reducing state for more than 6 months under open-air. To elucidate the mechanism underlying the sustainability of this indigo reduction state, three indigo fermentation batches with different durations for the indigo reduction state were compared. The three examined batches exhibited different microbiota and consisted of two phases. In the initial phase, oxygen-metabolizing-bacteria derived from sukumo established an initial network. With decreasing redox potential (ORP), the initial bacterial community was replaced by obligate anaerobes (mainly Proteinivoraceae; phase 1). Approximately 1 month after the beginning of fermentation, the predominating obligate anaerobes were decreased, and Amphibacillus and Polygonibacillus, which can decompose macromolecules derived from wheat bran, were predominantly observed, and the transition of microbiota became slow (phase 2). Considering the substrate utilization ability of the dominated bacterial taxa, the transitional change from phase 1 to phase 2 suggests that this changed from the bacterial flora that utilizes substrates derived from sukumo, including intrinsic substrates in sukumo and weakened or dead bacterial cells derived from early events (heat and alkaline treatment and reduction of ORP) to that of wheat bran-utilizers. This succession was directly related to the change in the major substrate sustaining the corresponding community and the turning point was approximately 1 month after the start of fermentation. As a result, we understand that the role of sukumo includes changes in the microbial flora immediately after the start of fermentation, which has an important function in the start-up phase of fermentation, whereas the ecosystem comprised of the microbiota utilizing wheat bran underpins the subsequent long-term indigo reduction.