Indigo Fermentation Research Articles

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

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.

Fermentation Blues: Analyzing the Microbiota of Traditional Indigo Vat Dyeing in Hunan, China.

ABSTRACTTraditional indigo dyeing through anaerobic fermentation has recently gained worldwide attention in efforts to address concerns regarding the sustainability of industrial indigo dyeing and the impact of toxic reducing agents such as sodium dithionite (Na2S2O4) on human health and the ecological environment. Intriguingly, changes in the microbiota during indigo fermentation are known to potently affect the onset of indigo reduction, and thus elucidation of the microbial community transitions could help develop methods to control the initiation of indigo reduction. Here, we investigated the microbiota associated with the traditional indigo dyeing practiced in Hunan, China. Specifically, we identified the bacterial and fungal components of the microbiota at distinct stages in the indigo fermentation process by analyzing 16S rRNA gene and internal transcribed spacer sequences. Our analyses revealed two substantial changes in the microbiota during the traditional indigo fermentation process. The first change, which was probably caused by the introduction of Chinese liquor (featuring a high alcohol concentration), resulted in decreased bacterial diversity and increased proportions of Pseudomonas, Stenotrophomonas, and Bacillaceae family members. The second change, which could be attributed to the addition of specific plant species, led to an increase in the abundance of Alkalibacterium, Amphibacillus, the obligate anaerobe Turicibacter, the facultative anaerobe Enterococcus, and ZOR0006, as well as to a decrease in the pH and redox potential values. Our results indicate that the specific plant mixture included in the procedure here could be used as an effective additive to accelerate the initiation of indigo reduction during the fermentation process. To the best of our knowledge, this is the first report revealing the fungal diversity during the indigo fermentation process and, furthermore, showing that the fungal diversity has remained in transition despite the relatively stable bacterial diversity in the proper indigo fermentation process. Although traditional indigo fermentation in China is challenging to manage, we can benefit from local knowledge of the fermentation process, and understanding the scientific bases of traditional indigo fermentation will facilitate the development of environmentally friendly procedures.IMPORTANCE Chemical reducing agents included in modern indigo dyeing to initiate indigo reduction can be harmful to both human health and the environment. Given that traditional indigo dyeing involves natural fermentation in a dye vat using natural organic additives without the use of toxic chemicals and that changes in the microbiota during traditional indigo fermentation potently affect the onset of indigo reduction, elucidation of these microbial community transitions could help develop methods to control the initiation of indigo reduction. This study on the microbiota associated with the traditional indigo dyeing practiced in Hunan, China, has identified the bacterial and fungal communities at distinct stages of the indigo fermentation process. Notably, the addition of specific plant species might yield the desired microbial communities and appropriate fermentation conditions, which could be used as an effective additive to accelerate the initiation of indigo reduction. This study has also revealed the fungal diversity during the indigo fermentation process for the first time and shown that the fungal diversity has remained in transition despite the relatively stable bacterial diversity. Thus, this work provides new insights into the traditional indigo fermentation process used in China and substantially enhances current efforts devoted to designing environmentally friendly methods for industrial indigo dyeing.

Fundicoccus fermenti sp. nov., an indigo-reducing facultative anaerobic alkaliphile isolated from indigo fermentation liquor used for dyeing.

Two facultative anaerobic and facultative alkaliphilic indigo-reducing strains, designated F-1T and F-2, were isolated from indigo fermentation liquor produced from couched woad fermentation-based Indian indigo fermentation fluid. The 16S rRNA gene phylogeny showed that Fundicoccus ignavus WS4937T (99.5%) was the closest neighbour of F-1T. The isolated bacterial cells were Gram-stain-positive and facultative anaerobic coccoids. Strain F-1T grew at between 5 and 37 °C with optimum growth between 28‒32 °C. The isolate grew in a pH range of 7.0‒10.5, with optimum growth between pH 9.0‒10.5. The DNA G+C content was 37.6 mol% (HPLC). The whole-cell fatty acid profile mainly consisted (>10 %) of C16 : 0, C16 : 1 ω9c, C18 : 0 and C18 : 1 ω9c. The digital DNA-DNA hybridization value between strain F-1T and F. ignavus WS4937T was 52.9 %. Based on their physiological and biochemical characteristics, and phylogenetic and genomic data, the isolates can be discriminated from F. ignavus WS4937T. The name Fundicoccus fermenti sp. nov. is proposed. The type strain of this species is F-1T (JCM 34140T=NCIMB 15255T).

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

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, and 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.

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, increase in the ratio of facultative to obligate anaerobes (F/O ratio), or the total abundance of facultative anaerobes (F) or obligate anaerobes (O) (designated F + O) are vital for the 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.

Relationship of Respirable Dust Exposure to Pulmonary Function among Informal-sector Weavers using Indigo-dyed Cotton

Abstract Informal-sector weavers using indigo-dyed cotton are occupationally exposed to respirable dust which may contain contaminants from chemicals used for pH adjustment in the natural indigo fermentation process. The major health problems associated with respirable dust induce pulmonary function impairment and respiratory disease. However, there have been few studies into the respiratory problems of informal-sector weavers in Thailand. This study investigated the link between occupational respirable dust exposure and pulmonary function among weavers using indigo-dyed cotton in Thailand. A cross-sectional study was conducted of 147 weavers located in Sakon Nakhon province. Respiratory dust (RD) samples were collected from the immediate breathing zone of all weavers. Lung function was measured using a portable spirometer operated by a trained physician and the spirometric results were examined by a pulmonologist before reporting. Most participants were female (98.6%) and the median reported interquartile age (IQR) was 58 (50–62.75) years. Average predicted values for FVC, FEV1 and FEV1/FVC were 92.9% (±20.7), 96.7% (±17.9) and 88.3% (±10.8). Of the respiratory function patterns of the 147 weavers, 20 (13.6%) were restrictive, 11 (7.5%) were obstructive and 1 (0.7%) was combined. The average RD concentration (mean ± SD) was 47.9 ± 28.3 μg/m3. Multivariate linear regression models showed an increase of 1 μg/m3 in RD exposure was associated with a 0.179% lower level of FEV1 (95% confidence interval (CI) -0.278 to -0.080) and a 0.068 % lower level of FEV1/FVC (95% CI -0.128 to -0.008). Our findings suggested that exposure to respirable dust might impair pulmonary function in informal-sector cotton weavers. Keywords: Indigo-dyed Cotton, Informal-sector Weavers, Occupational Exposure, Pulmonary function, Respirable Dust

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.

Analysis of bacterial flora of indigo fermentation fluids utilizing composted indigo leaves (sukumo) and indigo extracted from plants (Ryukyu-ai and Indian indigo)

Indigo is a fabric dye that requires reduction by microbial activity or chemical reagents to render it soluble in water. Sources of indigo for fermentation are primarily divided into composted indigo-containing plants and indigo extracted from plants. To elucidate the factors responsible for bacterial diversity, and for sustaining reduced state of indigo in different preparations, this study assessed fermentation-derived fluids using composted plant leaves, sukumo, and extracted indigo (Ryukyu-ai paste, and Indian indigo cake) prepared using different procedures. Regardless of the indigo source, obligate anaerobic bacteria, including the families Proteinivoraceae and Tissierellaceae, predominate (16.9-46.1%), suggesting their high affinity for this fermentation ecosystem (hyperalkaline and low redox potential). Moreover, bacterial communities in sukumo fermentations are more diverse than those from indigo extracts with the diversity tending to increase based on the fermentation period. Our results further suggest that the microbiota composition in sukumo fermentation is associated with the various bacterial nutrients derived from sukumo, including seed microorganisms. In addition, the debris derived from sukumo can reduce the pH stress experienced by the microorganisms. Further, regardless of 5.4 years difference in the fermentation age, the bacterial flora in two Ryukyu-ai batches exhibit similar features with low microbial diversities. The uniformity of the nutrient, along with the simple, yet strong, bacterial network in Ryukyu-ai fluids may be responsible for the stable bacterial flora composition. Taken together, these results indicate that the microbiota in indigo fermentation is highly influenced by the seed culture, the nutrient derived from raw materials, and the fermentation conditions.

Respiratory symptoms and their association with exposure to respiratory dust among indigo-dyed cotton workers

Indigo-dyed cotton weavers are exposed to various dust particles that may contain chemical contaminants from indigo fermentation process and can be related to respiratory symptoms. This cross-sectional study thus investigates the prevalence of respiratory symptoms and their association with respiratory dust (RD) exposure among workers in northeastern Thailand. It recruited 146 indigo-dyed cotton weavers. The prevalence of respiratory symptoms was collected by a self-report questionnaire included cough, phlegm, chest tightness, wheezing, and dyspnea. The average RD was 47.9 ± 28.3 μg/m3. Logistic regression models revealed a strong association between RD exposure and respiratory symptoms after adjustment. An exposure to RD ≥50 µg/m3 was associated with 5.05-, 3.58-, 2.88-, and 3.30-fold increased odds ratio of cough, phlegm, chest tightness, and dyspnea, respectively. The study results reveal that RD exposure may contribute to respiratory symptoms among indigo-dyed cotton weavers.

Mechanistic Insights into Indigo Reduction in Indigo Fermentation: A Voltammetric Study

Indigo is one of the oldest natural blue dyes. Microorganisms and their enzymatic activities are deeply involved in the traditional indigo staining procedure. To elucidate the mechanism of the microbial indigo reduction, we directly performed cyclic voltammetry on alkaline fermenting dye suspensions. A pair of characteristic redox peaks of leuco-indigo was observed in a supernatant fluid of the fermenting dye suspension. On the other hand, it was found that the indigo/leuco-indigo redox couple mediated two-way microbially catalyzed oxidation and reduction in a sediment-rich suspension of the fermenting suspension. Acetaldehyde was supposed to be the electron donor and acceptor of the catalytic reactions. In order to verify the bioelectrocatalytic reaction, we isolated indigo-reducing bacterium K2-3′ from the fermenting suspension, and the two-way bioelectrocatalysis was successfully restaged in a model system containing K2-3′ and methyl viologen (as a soluble mediator instead of indigo) as well as acetaldehyde at pH 10.

Characterization of the microbiota in long- and short-term natural indigo fermentation.

The duration for which the indigo-reducing state maintenancein indigo natural fermentation in batch dependent. The microbiota was analyzed in two batches of sukumo fermentation fluids that lasted for different durations (Batch 1: less than 2months; Batch 2: nearly 1year) to understand the mechanisms underlying the sustainability and deterioration of this natural fermentation process. The transformation of the microbiota suggested that the deterioration of the fermentation fluid is associated with the relative abundance of Alcaligenaceae. Principal coordinates analysis (PCoA) showed that the microbial community maintained a very stable state in only the long-term Batch 2. Therefore, entry of the microbiota into a stable state under alkaline anaerobic condition is an important factor for maintenance of indigo fermentation for long duration. This is the first report on the total transformation of the microbiota for investigation of long-term maintenance mechanisms and to address the problem of deterioration in indigo fermentation.

Analysis of the microbiota involved in the early changes associated with indigo reduction in the natural fermentation of indigo.

Constituents of the seed microbiota and initial changes in the microbiota in fermentations are important in fermentation progression. To identify the origin of indigo-reducing bacteria and understand the initial changes in the microbiota that occur concomitantly with the initiation of indigo reduction during indigo fermentation, we analysed the initial changes in the microbiota. The proportions of the reported indigo-reducing taxa Alkalibacterium, Amphibacillus and Polygonibacillus increased to 24.0% on the 5th day, to 15.2% on the 7th day and to 42.8% at 4.5months, and the relative abundances of these taxa were 0.048%, 0.14% and 0.02%, respectively, in sukumo (composted Japanese indigo plant material used for fermentation). In the early phase of the microbiota transition, two substantial changes were observed. The first change may be attributed to the substantial environmental changes caused by the introduction of heated wood ash extract (pH ≥ 10.5, temperature ≥ 60°C). This change increased the proportions of Alkalibacterium and the family Bacillaceae. The second change in microbiota might be initiated by the consumption of oxygen by aerobic microorganisms until the 5th day followed by an increase in the abundance of the obligate anaerobe Anaerobranca and the aerotolerant Amphibacillus and a decrease in the abundance of Bacillaceae. This experiment demonstrated that the 0.048% Alkalibacterium in the original material was augmented to 23.6% of the microbiological community within 5days. This means that using the appropriate material and performing appropriate pretreatment and adjustment of fermentation conditions are important to increase the abundance of the taxa that reduce indigo.

Microbial Communities Associated With Indigo Fermentation That Thrive in Anaerobic Alkaline Environments.

Indigo fermentation, which depends on the indigo-reducing action of microorganisms, has traditionally been performed to dye textiles blue in Asia as well as in Europe. This fermentation process is carried out by naturally occurring microbial communities and occurs under alkaline, anaerobic conditions. Therefore, there is uncertainty regarding the fermentation process, and many unknown microorganisms thrive in this unique fermentation environment. Until recently, there was limited information available on bacteria associated with this fermentation process. Indigo reduction normally occurs from 4 days to 2 weeks after initiation of fermentation. However, the changes in the microbiota that occur during the transition to an indigo-reducing state have not been elucidated. Here, the structural changes in the bacterial community were estimated by PCR-based methods. On the second day of fermentation, a large change in the redox potential occurred. On the fourth day, distinct substitution of the genus Halomonas with the aerotolerant genus Amphibacillus was observed, corresponding to marked changes in indigo reduction. Under open-air conditions, indigo reduction during the fermentation process continued for 6 months on average. The microbiota, including indigo-reducing bacteria, was continuously replaced with other microbial communities that consisted of other types of indigo-reducing bacteria. A stable state consisting mainly of the genus Anaerobacillus was also observed in a long-term fermentation sample. The stability of the microbiota, proportion of indigo-reducing microorganisms, and appropriate diversity and microbiota within the fluid may play key factors in the maintenance of a reducing state during long-term indigo fermentation. Although more than 10 species of indigo-reducing bacteria were identified, the reduction mechanism of indigo particle is riddle. It can be predicted that the mechanism involves electrons, as byproducts of metabolism, being discarded by analogs mechanisms reported in bacterial extracellular solid Fe3+ reduction under alkaline anaerobic condition.

Sodium requirement in indigo fermentation

Sodium requirement in indigo fermentation

Bacillus fermenti sp. nov., an indigo-reducing obligate alkaliphile isolated from indigo fermentation liquor for dyeing

The indigo-reducing, facultatively anaerobic and obligately alkaliphilic strains Bf-1T, Bf-2 and Bf-4 were isolated from an indigo fermentation liquor used for dyeing, which uses sukumo [composted Polygonum indigo (Polygonum tinctorium Lour.) leaves] as a basic ingredient and was obtained from a craft centre in Date City, Hokkaido, Japan. The 16S rRNA gene sequence analyses indicated that the closest neighbours of strain Bf-1T are Bacillus maritimus DSM 100413T (98.3 % 16S rRNA gene sequence similarity), Bacillus persicus DSM 25386T (98.2 %) and Bacillus rigiliprofundi LMG 28275T (97.7 %). The 16S rRNA gene sequence of strain Bf-1T was almost identical to the sequences of strains Bf-2 and Bf-4 (99.9 %). Cells of strain Bf-1T stained Gram-positive and formed straight rods that achieved motility through a pair of subpolar flagella. Strain Bf-1T grew at temperatures of between 15 and 45 °C with optimum growth at 33‒40 °C. The strain grew in the pH range of pH 8‒12, with optimum growth at pH 10. The isoprenoid quinone detected was menaquinone-7 (MK-7), and the DNA G+C content was 41.7 %. The whole-cell fatty acid profile mainly (>10 %) consisted of iso-C15 : 0 and iso-C16 : 0. Phylogenetically related neighbours, although demonstrating high 16S rRNA gene sequence similarity (>97.6 %) with strain Bf-1T, exhibited less than 9 % relatedness in DNA-DNA hybridization experiments. Based on evidence from this polyphasic study, the isolates represent a novel species, for which the name Bacillus fermenti sp. nov. is proposed. The type strain of this species is Bf-1T (=JCM 31807T=NCIMB 15079T).

Paralkalibacillus indicireducens gen., nov., sp. nov., an indigo-reducing obligate alkaliphile isolated from indigo fermentation liquor used for dyeing.

Obligately alkaliphilic, indigo-reducing strains, designated Bps-1T, Bps-2 and Bps-3, were isolated from an indigo fermentation liquor used for dyeing, which was produced from sukumo (composted Polygonum indigo leaves) obtained from a craft centre in Data City, Hokkaido, Japan, by using medium containing cellulase-treated sukumo. The 16S rRNA gene sequence phylogeny suggested that Bps-1T has a distinctive position among the alkaliphilic species of the genus Bacillus, with its closest neighbours being Bacillus pseudofirmus DSM 8715T, Bacillus lindianensis DSM 26864T and Bacillus alcalophilus DSM 485T (96.1, 95.8 and 95.5 % 16S rRNA gene sequence similarities, respectively). The 16S rRNA sequence of strain Bps-1T was identical to those of strains Bps-2 and Bps-3. Cells of the novel isolate were Gram-stain-positive and were facultatively anaerobic straight rods that were motile by means of a pair of flagella (subpolar and centre sides). Spherical endospores were formed in the terminal position. Strain Bps-1T grew between 18 and 40 °C with optimum growth at 33 °C. The isolate grew in the pH range 8‒11, with optimum growth at pH 9‒10. The isoprenoid quinone detected was menaquinone-7 (MK-7), and the DNA G+C content was 40.3 %. The whole-cell fatty acid profile (>10 %) mainly consisted of anteiso-C15 : 0, iso-C15 : 0 and C16 : 0. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, the isolates represent a novel species of a novel genus, for which the name Paralkalibacillus indicireducens gen. nov., sp. nov. is proposed. The type strain of this species is Bps-1T (JCM 31808T=NCIMB 15080T), with strains Bps-2 and Bps-3 representing additional strains of the species.

Development of media to accelerate the isolation of indigo-reducing bacteria, which are difficult to isolate using conventional media.

Indigo-reducing bacteria perform natural fermentation in indigo fermentation fluid. Owing to the stochastic nature of the process, the constituent in indigo fermentation fluid differ depending on the prepared batch and fermentation period. To identify new indigo-reducing bacteria, isolation of the bacteria is indispensable. However, isolation of indigo-reducing bacteria is difficult because conventional media are often unsuitable to isolate these slow-growing bacteria that also exist in low numbers. Hydrolysates of polysaccharides and mixtures of plant base constituents are candidates to accelerate the isolation of indigo-reducing bacteria that cannot be isolated using conventional media. In this current study, wheat bran hydrolysate and composted indigo leaves (sukumo) were used as ingredients in the fermentation fluid in the selective medium for indigo-reducing bacteria in anaerobic culture. The results suggested that obligate and oxygen-non-metabolizing facultative anaerobes are difficult to isolate using conventional media, whereas oxygen-metabolizing facultative anaerobes, relatively rapid-growing and major bacterial strains are relatively easy to isolate. Media containing sukumo hydrolysate facilitated the isolation of novel species of Bacillus pseudofirmus-related strains, whereas media containing wheat bran hydrolysate facilitated the isolation of Amphibacillus spp. (including new species). Seven species (including two new species) of indigo-reducing bacteria were isolated using wheat bran hydrolysate-containing media, whereas six species (including three new species) of indigo-reducing bacteria were isolated using media containing both wheat bran and sukumo hydrolysates. These newly developed culture media will facilitate the isolation of unknown bacteria in indigo fermentation and in environments similar to indigo fermentation fluid.

Analysis of microbiota involved in the aged natural fermentation of indigo.

Although the indigo reduction process is performed via natural fermentation and maintained under open-air condition, the indigo-reducing reactions continue for 6 months (on average) or longer. Identifying the mechanism underlying the maintenance of this process could lead to the development of a novel, long-lasting, unsterilized bioprocesses. To determine the mechanisms underlying the maintenance of the indigo fermentation system microbiota for more than 6 months in a reduced state in an anaerobic alkaline environment, we examined changes in the microbiota in one early-phase batch and two aged batches of indigo fermentation fluid. The microbiota in the aged fermentation fluid consisted mainly of the genera Alkalibacterium, Amphibacillus, Anaerobacillus and Polygonibacillus and the family Proteinivoraceae. The genera Alkalibacterium, Amphibacillus and Polygonibacillus are known to include indigo-reducing bacteria. Although the transition speed was slower in the aged fermentation fluid than in the early-stage fluid, the microbiota in the aged fermentation fluid maintained for more than 6 months was drastically changed within a period of 3 months. The results of this study indicate that the bacterial consortia consisted of various indigo-reducing species that replace the previous group of indigo-reducing bacteria. The notable transitional changes may be concomitant with changes in the environmental conditions, such as the nutritional conditions, observed over 3 months. This flexibility may lead to important changes in the microbiota that allow for the maintenance of a fermentation-reducing state over a long period.

Fermentibacillus polygoni gen. nov., sp. nov., an alkaliphile that reduces indigo dye.

Facultatively alkaliphilic strains, designated as strains IEB3T and IEB14, were isolated as indigo-reducing strains from a fermented Polygonum indigo (Polygonum tinctorium Lour) liquor sample prepared in our laboratory using a medium containing an indigo fermentation liquor as a sole substrate. The 16S rRNA gene sequence phylogeny and similarity suggested that strains IEB3T and IEB14 exhibit distinctive positions among the members of the genus Bacillus, and their closest neighbour was Bacillus nanhaiisediminis NH3T (similarity: 97.4 %) among the species with validly published names. The 16S rRNA sequence of strain IEB3Twas identical to that of strain IEB14. The cells of the isolates stained Gram-positive and were facultatively anaerobic, straight rods that were motile by a pair of subpolar flagella. Strains IEB3T and IEB14 grew at temperatures between 12 and 40 °C with optimum growth at 30‒33 °C and in the range of pH 7.5-12. Menaquinone-7 (MK-7) was detected as the major isoprenoid quinone. The DNA G+C contents of strains IEB3T and IEB14 were 39.0 and 39.1 mol%, respectively. The whole-cell fatty acid profile mainly (>10 %) consisted of iso-C14:0, iso-C15:0 and anteiso-C15:0. DNA-DNA hybridization revealed a low relatedness value between strain IEB3T and the phylogenetically most closely related species, Bacillus nanhaiisediminis JCM 16507T (<7 % ). On the basis of phenotypic and chemotaxonomic characteristics and phylogenetic data, the isolates represent a novel species within a novel genus, for which the name Fermentibacillus polygoni gen. nov., sp. nov. is proposed. The type strain is IEB3T (=JCM 30817T=NCIMB 14984T).

자연발효 과정에서 인디고에 환원력을 지닌 미생물 커뮤니티 분석과 농화배양

Indigo is utilized in various industries including textile dyeing, cosmetics, printing and medicinal products and its reduced form, leuco-indigo, is mainly used in these process. Chemical reducing agent (sodium dithionite, sodium sulfide, etc.) is preferred to use for the formation of leucoindigo in industry. In traditional indigo fermentation process, microorganisms can participate in the reduction of indigo and thus it has been known to reduce environmental pollution and noxious byproducts. However, in fermentation method using microorganisms it is difficult to standardize large scale production process due to low yield and reproducibility. In this study, we attempted to develop the indigo reduction process using microbial flora which was isolated from naturally fermented indigo vat or deduced by metagenomic approach. From the results of library analyses of PCR-amplified 16S rRNA genes from the traditional indigo fermentation vat sample (metagenome), it was confirmed that Alkalibacteriums (71%) was distinctly dominant in population. Some strains were identified after confirming that they become pure culture in nutrient media modified slightly. Four strains were separated in this process and each strain showed obvious reducing ability toward indigo in dyeing test. It is expected that the analyzed results will provide important data for standardizing the natural fermentation of indigo and investigating the mechanism of indigo reduction.