Effect of Fermentation Scale on Microbiota Dynamics and Metabolic Functions for Indigo Reduction.

During indigo dyeing fermentation, indigo reduction for the solubilization of indigo particles occurs through the action of microbiota under anaerobic alkaline conditions. The original microbiota in the raw material (sukumo: composted indigo plant) should be appropriately converged toward the extracellular electron transfer (EET)-occurring microbiota by adjusting environmental factors for indigo reduction. The convergence mechanisms of microbiota, microbial physiological basis for indigo reduction, and microbiota led by different velocities in the decrease in redox potential (ORP) at different fermentation scales were analyzed. A rapid ORP decrease was realized in the big batch, excluding Actinomycetota effectively and dominating Alkalibacterium, which largely contributed to the effective indigo reduction. Functional analyses of the microbiota related to strong indigo reduction on approximately day 30 indicated that the carbohydrate metabolism, prokaryotic defense system, and gene regulatory functions are important. Because the major constituent in the big batch was Alkalibacterium pelagium, we attempted to identify genes related to EET in its genome. Each set of genes for flavin adenine dinucleotide (FAD) transportation to modify the flavin mononucleotide (FMN)-associated family, electron transfer from NADH to the FMN-associated family, and demethylmenaquinone (DMK) synthesis were identified in the genome sequence. The correlation between indigo intensity reduction and metabolic functions suggests that V/A-type H+/Na+-transporting ATPase and NAD(P)H-producing enzymes drive membrane transportations and energization in the EET system, respectively.

Environmental factors contributing to the convergence of bacterial community structure during indigo reduction.

Indigo is solubilized through the reducing action of the microbiota that occurs during alkaline fermentation of composted leaves of Polygonum tinctorium L. (sukumo). However, the environmental effects on the microbiota during this treatment, as well as the mechanisms underlying the microbial succession toward stable state remain unknown. In this study, physicochemical analyses and Illumina metagenomic sequencing was used to determine the impact pretreatment conditions on the subsequent initiation of bacterial community transition and their convergence, dyeing capacity and the environmental factors critical for indigo reducing state during aging of sukumo. The initial pretreatment conditions analyzed included 60°C tap water (heat treatment: batch 1), 25°C tap water (control; batch 2), 25°C wood ash extract (high pH; batch 3) and hot wood ash extract (heat and high pH; batch 4), coupled with successive addition of wheat bran from days 5 to 194. High pH had larger impact than heat treatment on the microbiota, producing more rapid transitional changes from days 1 to 2. Although the initial bacterial community composition and dyeing intensity differed during days 2-5, the microbiota appropriately converged to facilitate indigo reduction from day 7 in all the batches, with Alkaliphilus oremalandii, Amphibacillus, Alkalicella caledoniensis, Atopostipes suicloalis and Tissierellaceae core taxa contributing to the improvement of when the dyeing intensity. This convergence is attributed to the continuous maintenance of high pH (day 1 ~) and low redox potential (day 2~), along with the introduction of wheat bran at day 5 (day 5~). PICRUSt2 predictive function profiling revealed the enrichment of phosphotransferease system (PTS) and starch and sucrose metabolism subpathways key toward indigo reduction. Seven NAD(P)-dependent oxidoreductases KEGG orthologs correlating to the dyeing intensity was also identified, with Alkalihalobacillus macyae, Alkalicella caledoniensis, and Atopostipes suicloalis contributing significantly toward the initiation of indigo reduction in batch 3. During the ripening period, the staining intensity was maintained by continuous addition of wheat bran and the successive emergence of indigo-reducing bacteria that also contributed to material circulation in the system. The above results provide insight into the interaction of microbial system and environmental factors in sukumo fermentation.