Isolation and characterization of indigo-reducing bacteria and analysis of microbiota from indigo fermentation suspensions

ABSTRACT In natural indigo dyeing, the water-insoluble indigo included in the composted indigo leaves called sukumo is converted to water-soluble leuco-indigo through the reduction activities of microorganisms under alkaline conditions. To understand the relationship between indigo reduction and microorganisms in indigo-fermentation suspensions, we isolated and identified the microorganisms that reduce indigo and analyzed the microbiota in indigo-fermentation suspensions. Indigo-reducing microorganisms, which were not isolated by means of a conventional indigo carmine-reduction assay method, were isolated by using indigo as a direct substrate and further identified and characterized. We succeeded in isolating bacteria closely related to Corynebacterium glutamicum, Chryseomicrobium aureum, Enterococcus sp. for the first time. Anthraquinone was found to be an effective mediator that facilitated the indigo-reduction activity of the isolated strains. On analysis of the microbiota in indigo-fermentation suspensions, the ratio of indigo-reducing bacteria and others was found to be important for maintaining the indigo-reduction activity.

The Mechanism Underlying of Long-Term Stable Indigo Reduction State in Indigo Fermentation Using Sukumo (Composted Polygonum tinctorium Leaves)

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.

Indigofera Tinctoria Leaf Powder As A Promising Additive To Improve Indigo Fermentation Prepared With Sukumo (Composted Polygonum Tinctorium Leaves)

Being insoluble in the oxidize form, indigo dye must be solubilized by reduction for it to penetrate textile. One of the procedures is the reduction by natural bacterial fermentation. Sukumo, composted leaves of Polygonum tinctorium, is a natural source of indigo in Japan. Although sukumo has an intrinsic bacterial seed, the onset of indigo reduction with this material may vary greatly. Certain additives improve indigo fermentation. Here, we studied the effects of Indigofera tinctoria leaf powder (LP) on the initiation of indigo reduction, bacterial community, redox potential (ORP), and dyeing intensity in the initial stages and in aged fermentation fluids prepared with sukumo. I. tinctoria LP markedly decreased ORP at day 1 and stabilised it during early fermentation. These effects could be explained by the phytochemicals present in I. tinctoria LP that act as oxygen scavengers and electron mediators. Using next generation sequencing results, we observed differences in the bacterial community in sukumo fermentation treated with I. tinctoria LP, which was not influenced by the bacterial community in I. tinctoria LP per se. The concomitant decrease in Bacillaceae and increase in Proteinivoraceae at the onset of fermentation and the ratio of facultative to obligate anaerobes are vital to the for initiation and maintenance of indigo reduction. Hence, I. tinctoria LP improved early indigo reduction by decreasing the ORP and hasten the appropriate transitions in the bacterial community in sukumo fermentation.

The synergetic effect of α-hydroxycarbonyls mixtures used as green reducing agent on the indigo dyeing process

Textile Industries use different chemicals in dyeing processes, consuming large quantities of water and producing large volumes of wastewater. For the particular case of indigo dyeing processes, its reduction is performed chemically by the addition of sodium dithionite. However, this is considered environmentally unfavorable because of the resulting contaminated wastewaters. Therefore, it is important to replace sodium dithionite with other alternatives in order to achieve cleaner processes. ?-hydroxycarbonyls have been suggested as possible environmentally friendly alternatives to reduce indigo. However, each one applied alone is enable to attain the dyeing performances offered by the conventional reductant. Thus , the study of the synergy of some selected ?-hydroxycarbonyls was proposed. In this paper, a mixture design of experimental (DOE) methods was used to determine the optimum combination of ?-hydrxycarbonyls to be applied in the indigo reduction process. Based on the design expert software, quadratic models were established as functions of ?-hydroxycarbonyls proportions. The diagnostics of models were investigated by using mixture contour plots. Finally, a model was proposed to predict the optimum conditions leading to dyeing performances exceeding those obtained from the reduction of indigo by the conventional sodium dithionite.