Importance rapid initial decline in oxidation-reduction potential, followed by an increase in extracellular electron transport activities, for the rapid onset of indigo reduction.

In most complex microbial systems, the ideal process underlying transitional microbial changes that lead to the formation of functional states is not fully elucidated. To understand the basis for the occurrence of indigo reduction, we analyzed the prerequisites causing transitional shifts in microflora that lead to the indigo-reducing state. To this end, timing of wheat bran (WB) addition, during indigo fermentation process using sukumo (composted leaves of Polygonum tinctorium L.) as the inoculum, substrate, and indigo source, were varied. Early initiation of indigo reduction was achieved through the early proliferation of obligate anaerobic Alkalicella caledoniensis followed by Alkalibacterium spp. or Evansella vedderi. Although it can be predicted that Alkalicella caledoniensis exhibits extracellular electron transport (EET) activity, to promote even effective reduction of indigo, Alkalibacterium spp. or E. vedderi, which have the EET gene sequence series and exert strong metabolic abilities, should emerge using WB. The emergence of Alkalicella caledoniensis was associated with drastic a decrease in bacterial diversity and a concurrent rapid decline in oxidation-reduction potential (ORP). The rate and extent of Alkalicella caledoniensis appearance depended on the rate of ORP reduction. Multivariate analysis (i.e., RDA) revealed that Alkalicella caledoniensis directed the initial drastic changes of microbiota, aligning with the decline in ORP. Prior to these major microbial shifts oxygen consumption by aerobic bacteria utilizing sukumo initiated the ORP decrease. These findings contribute to understanding the approach to steer the initially highly diverse bacterial community during early fermentation toward rapid induction of indigo reduction.