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.

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.

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

Obligately alkaliphilic and halophilic strains, designated In2-9T and D2-7, were isolated from a fermented Polygonum indigo (Polygonum tinctorium Lour.) liquor sample obtained from a craft centre in Date City, Hokkaido, Japan. The 16S rRNA gene sequence phylogeny suggested that strain In2-9T is a member of the genus Bacillus with the closest relatives being the alkaliphilic species of the genus Bacillus, Bacillus hemicellulosilyticusJCM 9152T (96.4 % 16S rRNA gene sequence similarity) and Bacillus alcalophilus DSM 485T (96.5 %). Cells of the isolate stained Gram-positive and were facultatively anaerobic straight rods that were motile by peritrichous flagella. Strain In2-9T grew between 13 and 45 °C with optimum growth at approximately 35-37 °C. The isolates grew in the pH range of 8-12 with optimum growth at pH 10. The isoprenoid quinone detected was menaquinone-6 (MK-6) and the DNA G+C content was 39.4 mol%. The whole-cell fatty acid profile mainly (>10 %) consisted of iso-C15 : 0, anteiso-C15 : 0 and C16 : 0. Spore shape and location and chemotaxonomic characteristics revealed that the isolates were distinctly different from phylogenetic neighbouring alkaliphilic species of the genus Bacillus. On the basis of phenotypic and chemotaxonomic characteristics and phylogenetic data, the isolates represent a novel species of a new genus, for which the name Polygonibacillusindicireducens gen. nov., sp. nov. is proposed. The type strain of the type species is In2-9T (=JCM 30831T=NCIMB 14982T), and strain D2-7 is an additional strain of the species.

Bacterial communities in different locations, seasons and segments of a dairy wastewater treatment system consisting of six segments.

A dairy wastewater treatment system composed of the 1st segment (no aeration) equipped with a facility for the destruction of milk fat particles, four successive aerobic treatment segments with activated sludge and a final sludge settlement segment was developed. The activated sludge is circulated through the six segments by settling sediments (activated sludge) in the 6th segment and sending the sediments beck to the 1st and 2nd segments. Microbiota was examined using samples from the non-aerated 1st and aerated 2nd segments obtained from two farms using the same system in summer or winter. Principal component analysis showed that the change in microbiota from the 1st to 2nd segments concomitant with effective wastewater treatment is affected by the concentrations of activated sludge and organic matter (biological oxygen demand [BOD]), and dissolved oxygen (DO) content. Microbiota from five segments (1st and four successive aerobic segments) in one location was also examined. Although the activated sludge is circulating throughout all the segments, microbiota fluctuation was observed. The observed successive changes in microbiota reflected the changes in the concentrations of organic matter and other physicochemical conditions (such as DO), suggesting that the microbiota is flexibly changeable depending on the environmental condition in the segments. The genera Dechloromonas, Zoogloea and Leptothrix are frequently observed in this wastewater treatment system throughout the analyses of microbiota in this study.

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).

Alteromonas gracilis sp. nov., a marine polysaccharide-producing bacterium.

A novel exopolysaccharide-producing bacterium, designated strain 9a2(T), was isolated from Pacific Ocean sediment. The strain was Gram-stain-negative, motile, strictly aerobic, oxidase- and catalase-positive, and required NaCl for growth. Its major isoprenoid quinone was ubiquinone-8 (Q-8), and its cellular fatty acid profile consisted mainly of C16 : 1ω7c, C18 : 1ω9c and C16 : 0. The DNA G+C content was 46.6 mol%. 16S rRNA gene sequence analysis suggested that strain 9a2(T) is a member of the genus Alteromonas . Strain 9a2(T) exhibited closest phylogenetic affinity to Alteromonas macleodii NBRC 102226(T) (99.3% 16S rRNA gene sequence similarity), A. marina SW-47(T) (99.3%), A. litorea TF-22(T) (99.0%), A. australica H17(T) (98.7%), A. simiduii BCRC 17572(T) (98.5%), A. stellipolaris LMG 21861(T) (98.3%) and A. hispanica F-32(T) (98.2%). The DNA-DNA reassociation values between strain 9a2(T) and A. macleodii JCM 20772(T), A. marina JCM 11804(T), A. litorea JCM 12188(T), A. australica CIP 109921(T), A. simiduii JCM 13896(T), A. stellipolaris LMG 21861(T) and A. hispanica LMG 22958(T) were below 70%. Strain 9a2(T) contained phosphatidylethanolamine, phosphatidylglycerol and an unidentified polar lipid. Owing to differences in phenotypic and chemotaxonomic characteristics, phylogenetic analysis based on 16S rRNA gene sequences and DNA-DNA relatedness data, the isolate merits classification as representing a novel species, for which the name Alteromonas gracilis sp. nov. is proposed. The type strain of this species is 9a2(T) ( =JCM 30236(T) =NCIMB 14947(T)).

Psychrobacter oceani sp. nov., isolated from marine sediment.

A novel marine bacterium, designated strain 4k5(T), was isolated from a sediment sample of the Pacific Ocean. The strain was Gram-stain-negative, strictly aerobic, non-motile, oxidase-positive and catalase-positive and required Na(+) for growth. Its major isoprenoid quinone was ubiquinone 8 (Q-8), and its cellular fatty acid profile consisted mainly of C18 : 1v9c (71.4%), C16 : 1v7c (9.1%) and C18 : 0. The DNA G+C content was 45.3 mol%. 16S rRNA gene sequence analysis suggested that strain 4k5(T) is a member of the genus Psychrobacter . Strain 4k5(T) exhibited the closely phylogenetic affinity to Psychrobacter pacificensis IFO 16270(T) (99.4% 16S rRNA gene sequence similarity), P. piscatorii T-3-2(T) (97.7%), P. nivimaris 88/2-7(T) (97.7%), P. celer SW-238(T) (97.7%), P. aestuarii SC35(T) (97.6%) and P. vallis CMS39(T) (97.6%). DNA-DNA hybridization between strain 4k5(T) and P. pacificensis NBRC 103191(T), P. piscatorii JCM 15603(T). P. nivimaris DSM 16093(T), P. celer JCM 12601(T), P. aestuarii JCM 16343(T) and P. vallis DSM 15337(T) was 42.5, 47.0, 38.1, 23.7, 9.0 and 27.4%, respectively. Owing to the significant differences in phenotypic and chemotaxonomic characteristics, phylogenetic analysis based on the 16S rRNA gene sequence and DNA-DNA relatedness data, the isolate merits classification within a novel species, for which the name Psychrobacter oceani sp. nov. is proposed. The type strain is 4k5(T) ( = JCM 30235(T) =NCIMB 14948(T)).

Gracilibacillus alcaliphilus sp. nov., a facultative alkaliphile isolated from indigo fermentation liquor for dyeing.

A facultatively alkaliphilic, lactic-acid-producing and halophilic strain, designated SG103(T), was isolated from a fermented Polygonum indigo (Polygonum tinctorium Lour.) liquor sample for dyeing prepared in a laboratory. 16S rRNA gene sequence phylogeny suggested that SG103(T) is a member of the genus Gracilibacillus with the closest relatives being 'Gracilibacillus xinjiangensis' J2 (similarity: 97.06 %), Gracilibacillus thailandensis TP2-8(T) (97.06 %) and Gracilibacillus halotolerans NN(T) (96.87 %). Cells of the isolate stained Gram-positive and were facultatively anaerobic straight rods that were motile by peritrichous flagella. The strain grew at temperatures between 13 and 48 °C with the optimum at 39 °C. It grew in the range pH 7-10 with the optimum at pH 9. The isoprenoid quinone detected was menaquinone-7 (MK-7) and the DNA G+C content was 41.3 mol%. The whole-cell fatty acid profile mainly (>10 %) consisted of iso-C15 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. Unlike other reported species of the genus Gracilibacillus, the strain lacked diphosphatidylglycerol as a major polar lipid. DNA-DNA hybridization experiments with strains exhibiting greater than 96.87 % 16S rRNA gene sequence similarity, 'G. xinjiangensis' J2, G. thailandensis TP2-8(T) and G. halotolerans NN(T), revealed 2±4 %, 4±9 % and 3±2 % relatedness, respectively. On the basis of the differences in phenotypic and chemotaxonomic characteristics, and the results of phylogenetic analyses based on 16S rRNA gene sequences and DNA-DNA relatedness data from reported species of the genus Gracilibacillus, strain SG103(T) merits classification as a members of a novel species, for which the name Gracilibacillus alcaliphilus sp. nov. is proposed. The type strain is SG103(T) ( = JCM 17253(T) = NCIMB 14683(T)).

Amphibacillus iburiensis sp. nov., an alkaliphile that reduces an indigo dye.

An indigo-reducing alkaliphilic strain, designated strain N314(T), was isolated from a fermented polygonum indigo (Polygonum tinctorium Lour.) liquor sample, aged for 10 months, that was obtained from Date City, Iburi Branch, Hokkaido, Japan. The 16S rRNA gene sequence phylogeny suggested that strain N314(T) is a member of the genus Amphibacillus, with the closest relatives being Amphibacillus indicireducens (98.9 % similarity to the type strain) and Amphibacillus xylanus (98.0 % similarity to the type strain), the only species with 16S rRNA gene sequence similarities higher than 97 % to strain N314(T). The cells of the isolate stained Gram-positive and were facultatively anaerobic, straight rods that were motile by means of peritrichous flagella. The strain grew at 26-39 °C with optimum growth at 36 °C. It grew at pH 8.0-9.1, with optimum growth at pH 8.9-9.1. No isoprenoid quinone was detected, and the DNA G+C content was 38.4 mol%. The whole-cell fatty acid profile consisted mainly of iso-C15 : 0 and anteiso-C15 : 0. Analysis of DNA-DNA hybridization with the type strains of A. indicireducens and A. xylanus revealed 29±2 % and 10±2 % relatedness, respectively. Owing to differences in phenotypic characteristics from reported species of the genus A. and results of phylogenetic analyses based on 16S rRNA gene sequences and DNA-DNA relatedness data, the isolate merits classification within a novel species, for which the name Amphibacillus iburiensis sp. nov. is proposed. The type strain is N314(T) ( = JCM 18529(T) = NCIMB 14823(T)).

Oceanobacillus polygoni sp. nov., a facultatively alkaliphile isolated from indigo fermentation fluid.

A facultatively alkaliphilic, lactic-acid-producing and halophilic strain, designated SA9(T), was isolated from a fermented Polygonum indigo (Polygonum tinctorium Lour.) liquor sample prepared in a laboratory. The 16S rRNA gene sequence phylogeny suggested that strain SA9(T) was a member of the genus Oceanobacillus with the closest relative being Oceanobacillus profundus KCCM 42318(T) (99.3% 16S rRNA gene sequence similarity). Cells of strain SA9(T) stained Gram-positive and were facultative anaerobic straight rods that were motile by peritrichous flagella. The strain grew between 5 and 48 °C (optimum, 35 °C) and at pH 7-12 (optimum, pH 9). The isoprenoid quinone detected was menaquinone-7 (MK-7) and the DNA G+C content was 40.6 ± 0.9 mol%. The whole-cell fatty acid profile mainly consisted of iso-C(15:0), anteiso-C(15:0), C(16:0) and anteiso-C(17:0). DNA-DNA hybridization with Oceanobacillus profundus DSM 18246(T) revealed a DNA-DNA relatedness value of 23 ± 2%. On the basis of the differences in phenotypic and chemotaxonomic characteristics, and the results of phylogenetic analyses based on 16S rRNA gene sequences and DNA-DNA relatedness data from recognized species of the genus Oceanobacillus, strain SA9(T) merits classification as a representative of a novel species of the genus Oceanobacillus, for which the name Oceanobacillus polygoni sp. nov. is proposed. The type strain is SA9(T) ( =JCM 17252(T) =NCIMB 14684(T)). An emended description of the genus Oceanobacillus is also provided.

Amphibacillus indicireducens sp. nov., an alkaliphile that reduces an indigo dye.

Two indigo-reducing alkaliphilic strains, designated strain C40(T) and strain N214, were isolated from a fermented Polygonum Indigo (Polygonum tinctorium Lour.) liquor sample aged for 10 months and obtained from Date City, Hokkaido, Japan. 16S rRNA gene sequence phylogeny suggested that strains C40(T) and N214 were members of the genus Amphibacillus with the closest relative being Amphibacillus xylanus JCM 7361(T) (97.5 % 16S rRNA gene sequence similarity with strain C40(T)), which is the only strain having a 16S rRNA gene sequence similarity higher than 97 % with strain C40(T). Cells of strain C40(T) were Gram-stain-positive, facultatively anaerobic, straight rods that were motile by means of peritrichous flagella. The strains grew between 17 and 39 °C (optimum, 35 °C) and in the pH range of 9.0-12.0. No isoprenoid quinone was detected and the DNA G+C content was 37.5-37.7 mol%. The whole-cell fatty acid profile mainly consisted of iso-C(15 : 0) and anteiso-C(15 : 0). DNA-DNA hybridization of strain C40(T) with Amphibacillus xylanus JCM 7361(T) revealed a DNA-DNA relatedness value of 10±3 %. Owing to the differences in phenotypic characteristics and phylogenetic analyses based on 16S rRNA gene sequences, as well as DNA-DNA relatedness data from reported species of the genus Amphibacillus, the isolates merit classification as a novel species in the genus Amphibacillus, for which the name Amphibacillus indicireducens sp. nov. is proposed. The type strain is C40(T) ( = JCM 17250(T) = NCIMB 14686(T)). An additional strain of the species is N214. An emended description of the genus Amphibacillus is provided.

Oceanobacillus indicireducens sp. nov., a facultative alkaliphile that reduces an indigo dye.

An indigo-reducing facultatively alkaliphilic and halophilic strain, designated strain A21(T), was isolated from a fermented Polygonum indigo (Polygonum tinctorium Lour.) liquor sample aged for 4 days prepared in a laboratory. 16S rRNA gene sequence phylogeny suggested that strain A21(T) was a member of the genus Oceanobacillus with the closest relative being the type strain of Oceanobacillus chironomi (similarity: 96.0 %). The cells of the isolate stained Gram-positive and were facultatively anaerobic straight rods that were motile by peritrichous flagella. The strain grew between 18 and 48 °C with optimum growth at 39 °C. It grew in the pH range of 7-12. It hydrolysed casein, gelatin and Tween 20 but not Tweens 40, 60 and 80, starch or DNA. No isoprenoid quinone was detected and the DNA G+C content was 39.7 mol%. The whole-cell fatty acid profile mainly consisted of iso-C15 : 0, anteiso-C15 : 0 and C16 : 0. DNA-DNA hybridization experiments with O. chironomi revealed 13 % relatedness. Owing to the differences in phenotypic and chemotaxonomic characteristics, and phylogenetic analyses based on 16S rRNA gene sequences and DNA-DNA relatedness data from reported Oceanobacillus species, the isolate merits classification as a representative of a novel species, for which the name Oceanobacillus indicireducens sp. nov. is proposed. The type strain is A21(T) ( = JCM 17251(T)  = NCIMB 14685(T)). The description of the genus Oceanobacillus is also emended.

Brevibacillus nitrificans sp. nov., a nitrifying bacterium isolated from a microbiological agent for enhancing microbial digestion in sewage treatment tanks.

A heterotrophic nitrifying bacterium, designated strain DA2(T), was isolated from a microbiological agent for enhancing microbial digestion in sewage treatment tanks. Cells of strain DA2(T) were Gram-positive, facultatively anaerobic, sporulating rods that were motile by means of peritrichous flagella; they were able to grow at pH 5-8. The major isoprenoid quinone of strain DA2(T) was menaquinone-7 (MK-7) and its cellular fatty acid profile consisted mainly of iso-C(15 : 0) (18.6 %) and anteiso-C(15 : 0) (69.1 %). The DNA G+C content was 54.1 mol%. 16S rRNA gene sequence phylogeny suggested that strain DA2(T) is a member of the genus Brevibacillus, with highest sequence similarities (in parentheses) to the type strains of Brevibacillus choshinensis (99.7 %), B. formosus (99.4 %), B. brevis (99.4 %), B. agri (99.0 %), B. reuszeri (98.8 %), B. parabrevis (98.7 %), B. centrosporus (98.6 %), B. limnophilus (97.4 %), B. panacihumi (97.3 %) and B. invocatus (97.3 %). DNA-DNA hybridization showed less than 60 % relatedness between strain DA2(T) and type strains of the most closely related species given above. Given the significant differences in phenotypic and chemotaxonomic characteristics, and phylogenetic analysis based on the 16S rRNA sequence and DNA-DNA relatedness data, the isolate merits classification as a novel species, for which the name Brevibacillus nitrificans is proposed; the type strain of this species is DA2(T) (= JCM 15774(T) = NCIMB 14531(T)).

Pseudomonas toyotomiensis sp. nov., a psychrotolerant facultative alkaliphile that utilizes hydrocarbons.

A psychrotolerant, facultatively alkaliphilic strain, HT-3(T), was isolated from a sample of soil immersed in hot-spring water containing hydrocarbons in Toyotomi, Hokkaido, Japan. 16S rRNA gene sequence-based phylogeny suggested that strain HT-3(T) is a member of the genus Pseudomonas and belongs to the Pseudomonas oleovorans group. Cells of the isolate were Gram-negative, aerobic, straight rods, motile by a single polar flagellum. The strain grew at 4-42 °C, with optimum growth at 35 °C at pH 7, and at pH 6-10. It hydrolysed Tweens 20, 40, 60 and 80, but not casein, gelatin, starch or DNA. Its major isoprenoid quinone was ubiquinone-9 (Q-9) and the DNA G+C content was 65.1 mol%. The whole-cell fatty acid profile consisted mainly of C(16 : 0), C(16 : 1)ω9c and C(18 : 1)ω9c. Phylogenetic analyses based on gyrB, rpoB and rpoD sequences revealed that the isolate could be discriminated from Pseudomonas species that exhibited more than 97 % 16S rRNA gene sequence similarity and phylogenetic neighbours belonging to the P. oleovorans group including the closest relative of the isolate, Pseudomonas alcaliphila. DNA-DNA hybridization with P. alcaliphila AL15-21(T) revealed 51 ± 5 % relatedness. Owing to differences in phenotypic properties and phylogenetic analyses based on multilocus gene sequence analysis and DNA-DNA relatedness data, the isolate merits classification in a novel species, for which the name Pseudomonas toyotomiensis sp. nov. is proposed. The type strain is HT-3(T) ( = JCM 15604(T)  = NCIMB 14511(T)).

Psychrobacter piscatorii sp. nov., a psychrotolerant bacterium exhibiting high catalase activity isolated from an oxidative environment.

A Gram-negative, non-motile, psychrotolerant bacterium exhibiting high catalase activity, designated strain T-3-2(T), was isolated from a drain of a fish-processing plant. Its catalase activity was 12 000 U (mg protein)(-1), much higher than the activity of the other Psychrobacter strains tested. The strain grew at 0-30 degrees C and in the presence of 0-12 % NaCl. The predominant isoprenoid quinone was ubiquinone-8 (Q-8), and C(16 : 1)omega9c and C(18 : 1)omega9c were the predominant cellular fatty acids. The DNA G+C content of strain T-3-2(T) was 43.9 mol%. 16S rRNA gene sequence phylogeny suggested that strain T-3-2(T) is a member of the genus Psychrobacter, with the closest relatives being the type strains of Psychrobacter nivimaris (99.2 % similarity), P. aquimaris (98.7 %) and P. proteolyticus (98.5 %). DNA-DNA hybridization showed less than 65 % relatedness with these strains. A phylogenetic tree based on gyrB gene sequences was more reliable, with higher bootstrap values than the 16S rRNA gene sequence-based tree. The result also differentiated the isolate from previously reported Psychrobacter species. Owing to the significant differences in phenotypic and chemotaxonomic characteristics and the phylogenetic and DNA-DNA relatedness data, the isolate merits classification within a novel species, for which the name Psychrobacter piscatorii sp. nov. is proposed. The type strain is T-3-2(T) (=JCM 15603(T) =NCIMB 14510(T)).