Complete genome sequence of Opitutales bacterium strain ASA1, isolated from soil.

We present the complete genome of Opitutales bacterium ASA1, isolated from soil. The genome is 5,821,695 bp with 4,638 protein-coding sequences. The genome data suggest that this strain belongs to the class Opitutae of the phylum Verrucomicrobiota, and its genome has six unique biosynthetic gene clusters associated with secondary metabolites.

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.

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.

Corrigendum: Differences in Bioenergetic Metabolism of Obligately Alkaliphilic Bacillaceae Under High pH Depend on the Aeration Conditions.

[This corrects the article DOI: 10.3389/fmicb.2022.842785.].

Differences in Bioenergetic Metabolism of Obligately Alkaliphilic Bacillaceae Under High pH Depend on the Aeration Conditions.

Alkaliphilic Bacillaceae appear to produce ATP based on the H+-based chemiosmotic theory. However, the bulk-based chemiosmotic theory cannot explain the ATP production in alkaliphilic bacteria because the H+ concentration required for driving ATP synthesis through the ATPase does not occur under the alkaline conditions. Alkaliphilic bacteria produce ATP in an H+-diluted environment by retaining scarce H+ extruded by the respiratory chain on the outer surface of the membrane and increasing the potential of the H+ for ATP production on the outer surface of the membrane using specific mechanisms of ATP production. Under high-aeration conditions, the high ΔΨ (ca. -170 mV) of the obligate alkaliphilic Evansella clarkii retains H+ at the outer surface of the membrane and increases the intensity of the protonmotive force (Δp) per H+ across the membrane. One of the reasons for the production of high ΔΨ is the Donnan potential, which arises owing to the induction of impermeable negative charges in the cytoplasm. The intensity of the potential is further enhanced in the alkaliphiles compared with neutralophiles because of the higher intracellular pH (ca. pH 8.1). However, the high ΔΨ observed under high-aeration conditions decreased (∼ -140 mV) under low-aeration conditions. E. clarkii produced 2.5-6.3-fold higher membrane bound cytochrome c in the content of the cell extract under low-aeration conditions than under high-aeration conditions. The predominant membrane-bound cytochrome c in the outer surface of the membrane possesses an extra Asn-rich segment between the membrane anchor and the main body of protein. This structure may influence the formation of an H+-bond network that accumulates H+ on the outer surface of the membrane. Following accumulation of the H+-bond network producing cytochrome c, E. clarkii constructs an H+ capacitor to overcome the energy limitation of low aeration at high pH conditions. E. clarkii produces more ATP than other neutralophilic bacteria by enhancing the efficacy per H+ in ATP synthesis. In low H+ environments, E. clarkii utilizes H+ efficiently by taking advantage of its high ΔΨ under high-aeration conditions, whereas under low-aeration conditions E. clarkii uses cytochrome c bound on its outer surface of the membrane as an H+ capacitor.

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).

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.

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.

Relationship Between Main Channel Structure of Catalases and the Evolutionary Direction in Cold-Adapted Hydrogen Peroxide-Tolerant Exiguobacteium and Psychrobacter.

Catalase has crucial role in adaptive response to H2O2. Main channel structure responsible for substrate selectivity was estimated to understand the relationship between the evolutionary direction of catalases from Exiguobacterium oxidotolerans and Psychrobacter piscatorii which survive in cold and high concentration of hydrogen peroxide, and their catalytic property. E. oxidotolerans catalase (EKTA) exhibited a higher ratio of compound I formation rate using peracetic acid (a substrate lager than H2O2)/catalase activity using H2O2 as the substrate than P. piscatori catalase (PKTA). It was considered that the ratio was attributed to the size of the amino acid residues locating at the bottle neck structure in the main channel. The differences in the ratio of the compound I formation rate with peracetic acid to catalase activity with H2O2 between the deeper branches in the phylogenetic tree in both EKTA and PKTA were large. This indicates that catalases from the hydrogen peroxide-tolerant bacteria have evolved in different directions, exhibiting effective catalytic activity and allowing broader substrates size or H2O2-specific substrate acceptability in EKTA and PKTA, respectively. It is considered that the main channel structure reflected the difference in the evolutionary direction of clade 1 and clade 3 catalases.

Genomic characterization of closely related species in the Rumoiensis clade infers ecogenomic signatures to non-marine environments.

Members of the family Vibrionaceae are generally found in marine and brackish environments, playing important roles in nutrient cycling. The Rumoiensis clade is an unconventional group in the genus Vibrio, currently comprising six species from different origins including two species isolated from non-marine environments. In this study, we performed comparative genome analysis of all six species in the clade using their complete genome sequences. We found that two non-marine species, Vibrio casei and Vibrio gangliei, lacked the genes responsible for algal polysaccharide degradation, while a number of glycoside hydrolase genes were enriched in these two species. Expansion of insertion sequences was observed in V. casei and Vibrio rumoiensis, which suggests ongoing genomic changes associated with niche adaptations. The genes responsible for the metabolism of glucosylglycerate, a compound known to play a role as compatible solutes under nitrogen limitation, were conserved across the clade. These characteristics, along with genes encoding species-specific functions, may reflect the habit expansion which has led to the current distribution of Rumoiensis clade species. Genome analysis of all species in a single clade give us valuable insights into the genomic background of the Rumoiensis clade species and emphasize the genomic diversity and versatility of Vibrionaceae.

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

The duration for which the indigo-reducing state maintenance in 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 2 months; Batch 2: nearly 1 year) 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 4.5 months, 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 5 days. 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.

Formation of Proton Motive Force Under Low-Aeration Alkaline Conditions in Alkaliphilic Bacteria.

In Mitchell's chemiosmotic theory, a proton (H+) motive force across the membrane (Δp), generated by the respiratory chain, drives F1Fo-ATPase for ATP production in various organisms. The bulk-base chemiosmotic theory cannot account for ATP production in alkaliphilic bacteria. However, alkaliphiles thrive in environments with a H+ concentrations that are one-thousandth (ca. pH 10) the concentration required by neutralophiles. This situation is similar to the production of electricity by hydroelectric turbines under conditions of very limited water. Alkaliphiles manage their metabolism via various strategies involving the cell wall structure, solute transport systems and molecular mechanisms on the outer surface membrane. Our experimental results indicate that efficient ATP production in alkaliphilic Bacillus spp. is attributable to a high membrane electrical potential (ΔΨ) generated for an attractive force for H+ on the outer surface membrane. In addition, the enhanced F1Fo-ATPase driving force per H+ is derived from the high ΔΨ. However, it is difficult to explain the reasons for high ΔΨ formation based on the respiratory rate. The Donnan effect (which is observed when charged particles that are unable to pass through a semipermeable membrane create an uneven electrical charge) likely contributes to the formation of the high ΔΨ because the intracellular negative ion capacities of alkaliphiles are much higher than those of neutralophiles. There are several variations in the adaptation to alkaline environments by bacteria. However, it could be difficult to utilize high ΔΨ in the low aeration condition due to the low activity of respiration. To explain the efficient ATP production occurring in H+-less and air-limited environments in alkaliphilic bacteria, we propose a cytochrome c-associated "H+ capacitor mechanism" as an alkaline adaptation strategy. As an outer surface protein, cytochrome c-550 from Bacillus clarkii possesses an extra Asn-rich segment between the region anchored to the membrane and the main body of the cytochrome c. This structure may contribute to the formation of the proton-binding network to transfer H+ at the outer surface membrane in obligate alkaliphiles. The H+ capacitor mechanism is further enhanced under low-aeration conditions in both alkaliphilic Bacillus spp. and the Gram-negative alkaliphile Pseudomonas alcaliphila.

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).

Correction: Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed.

[This corrects the article DOI: 10.1371/journal.pone.0180053.].

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.