Indigofera tinctoria L. leaf powder promotes initiation of indigo reduction by inducing of rapid transition of the microbial community

Abstract

Water-insoluble indigo is solubilized by the reducing action of microorganisms which occurs during fermentation. In natural indigo fermentation, composted leaves of Polygonum tinctorium L. (sukumo) are the raw material that has been used as both the indigo source and the bacterial inoculum. Ideally, indigo reduction occurs shortly after preparation of the fermentation vat. The time-to-reduction depends on the quality of the sukumo and the methods for preparation and management of the fermentation batch. We estimated the effect of adding Indigofera tinctoria L. leaf powder (LP) to indigo fermentation in two fermentations originally exhibiting either rapid or slow time-to-reduction (T-sukumo and D-sukumo, respectively). Alkalihalobacillus spp. (97.7%–98.4% similarities with Alkalihalobacillus macyae) were observed only in the LP-added T-sukumo fermentation liquor. They appeared from day 1 (0.7%) and increased to 24.4% on day 6, and their presence was related to indigo reduction. Differences in functional ratio between LP-added and its control batches revealed enhancement of pathways related to reconstitution of cellular functions and substrate metabolisms, to all of which Alkalihalobacillus spp. contributed intensively. In D-sukumo batch, appearance of bacteria necessary to initiate indigo reduction (principally Anaerobacillus/Polygonibacillus) was comparatively slower. LP promotes earlier indigo reduction in both T- and D-sukumo-based batches, owing to its promotion of microbiota transition. The effect of the LP was intensified from day 1 to day 2 in both sukumo using batches according to the assumed function of the microbiota. The initial effect of LP on the T-sukumo batches was more intense than that in the D-sukumo batches and was continued until day 3, while the duration in the T-sukumo batches was continued until day 5. Based on these observations, we propose that the LP functions through its phytochemicals that eliminate oxygen, stimulate the microbiota, and accelerate its transitional changes toward a suitable function that opens the pathway for the extracellular electron transfer using carbohydrates as a substrate.

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

“…The period from the fermentation preparation to indigo reduction initiation differs depending on the procedure and quality of raw materials. Transitional changes in the redox state of indigo may be related to changes in the microbiota, which is in turn, associated with the indigo reduction initiation, as previously have suggested using clone library analysis (Aino et al, 2010) and nextgeneration sequencing (Tu et al, 2019a(Tu et al, ,b, 2021Lopes et al, 2021Lopes et al, , 2022Nakagawa et al, 2022). On days 0-1, oxygen metabolizable Bacillaceae (e.g., Sutcliffiella cohnii) and Actinobacteria (e.g., Nocardiopsis ganjiahuensis) appear, following which their numbers significantly decrease during days 2-10 as the redox potential (ORP) decreases due to their consumption of oxygen.…”

“… Lopes et al (2021) also reported the importance of “PTS” in fermentation, which accelerated indigo reduction by the addition of Indian indigo leaf powder during early fermentation. The importance of “starch and sucrose metabolism” in indigo reduction has been reported in comparative studies between of rapid and slow time-to-indigo reduction sukumo ( Lopes et al, 2022 ). Using various preparation samples in this study, we clarified the significance of the two subpathways, “PTS” and “starch and sucrose metabolism,” by assessing the relationship between dyeing intensity and functional abundance during early fermentation.…”