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

Abstract

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.

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

“…As an initial step in the fermentation, sukumo was treated with hot wood ash extract (60-80 • C, pH > 10.5). It has been reported that high pH has a larger impact on the microbiota in sukumo than in a high-temperature treatment (60 • C) [10]. High temperature is effective if combined with high pH.…”

“…Small-scale batch experiments have shown that indigo-reducing bacterial communities are constructed within five days by replacing the initially appearing aerobic microbes. In addition, the changing velocity of the microbiota is faster until approximately day 30, regardless of the timing of wheat bran introduction [10,13]. This is probably because the influence of the substrates derived from sukumo was effective for approximately 30 days.…”

“…This initial change in fermentation activates microbiota adaptable to alkaline and low-ORP environments (≤−600 mV) in the fermentation fluid. In our previous study, high pH, low redox potential (ORP), and the addition of a substrate (wheat bran) induced microbiota to direct the original microbiota in sukumo to reduce indigo [10]. However, these parameters change depending on the fermentation procedures, such as the fermentation scale and timing of wheat bran addition.…”

“…However, reducing indigo during fermentation using composted indigocontaining plants is sometimes difficult. This suggests that the original microbiota in sukumo changes depending on the preparation and maintenance procedures [10]. Although 0.048% operational taxonomic units (OUTs) belonging to the indigo-reducing genus Alkalibacterium were previously detected in sukumo [9], Alkalibacterium OTUs were not detected in >30,000 reads of 16S rRNA gene sequences in next-generation sequencing (NGS).…”

“…Although understanding the dynamics and ecosystem of the microbiota is directly related to the convergence and sustainability of this indigo-reducing microbiota, understanding the characteristics of the constituent microorganisms is also important for understanding the functions of the microbiota in the ecosystem. Transitional changes in microbiota in indigo fermentation employing sukumo have been analyzed using PCR-DGGE and clone libraries analysis [2] and NGS in small-scale fermentation in the laboratory [9][10][11][12][13][14]. In addition to these uncultured procedures, indigo-reducing bacteria have been isolated [1,15].…”