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.

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.

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.

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.

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.

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.

Overexpressed Superoxide Dismutase and Catalase Act Synergistically to Protect the Repair of PSII during Photoinhibition in Synechococcus elongatus PCC 7942.

The repair of PSII under strong light is particularly sensitive to reactive oxygen species (ROS), such as the superoxide radical and hydrogen peroxide, and these ROS are efficiently scavenged by superoxide dismutase (SOD) and catalase. In the present study, we generated transformants of the cyanobacterium Synechococcus elongatus PCC 7942 that overexpressed an iron superoxide dismutase (Fe-SOD) from Synechocystis sp. PCC 6803; a highly active catalase (VktA) from Vibrio rumoiensis; and both enzymes together. Then we examined the sensitivity of PSII to photoinhibition in the three strains. In cells that overexpressed either Fe-SOD or VktA, PSII was more tolerant to strong light than it was in wild-type cells. Moreover, in cells that overexpressed both Fe-SOD and VktA, PSII was even more tolerant to strong light. However, the rate of photodamage to PSII, as monitored in the presence of chloramphenicol, was similar in all three transformant strains and in wild-type cells, suggesting that the overexpression of these ROS-scavenging enzymes might not protect PSII from photodamage but might protect the repair of PSII. Under strong light, intracellular levels of ROS fell significantly, and the synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein, was enhanced. Our observations suggest that overexpressed Fe-SOD and VktA might act synergistically to alleviate the photoinhibition of PSII by reducing intracellular levels of ROS, with resultant protection of the repair of PSII from oxidative inhibition.

Contribution of intracellular negative ion capacity to Donnan effect across the membrane in alkaliphilic Bacillus spp.

To elucidate the energy production mechanism of alkaliphiles, the relationship between the H(+) extrusion rate by the respiratory chain and the corresponding ATP synthesis rate was determined in the facultative alkaliphile Bacillus cohnii YN-2000 and compared with those in the obligate alkaliphile Bacillus clarkii DSM 8720(T) and the neutralophile Bacillus subtilis IAM 1026. Under high aeration condition, much higher ATP synthesis rates and larger Δψ in the alkaliphilic Bacillus spp. grown at pH 10 than those in the neutralophilic B. subtilis grown at pH 7 were observed. This high ATP productivity could be attributed to the larger Δψ in alkaliphiles than in B. subtilis because the H(+) extrusion rate in alkaliphiles cannot account for the high ATP productivity. However, the large Δψ in the alkaliphiles could not be explained only by the H(+) translocation rate in the respiratory chain in alkaliphiles. There is a possibility that the Donnan effect across the membrane has the potential to contribute to the large Δψ. To estimate the contribution of the Donnan effect to the large Δψ in alkaliphilic Bacillus spp. grown at pH 10, intracellular negative ion capacity was examined. The intracellular negative ion capacities in alkaliphiles grown at pH 10 under high aeration condition corresponding to their intracellular pH (pH 8.1) were much higher than those in alkaliphiles grown under low aeration condition. A proportional relationship is revealed between the negative ion capacity and Δψ in alkaliphiles grown under different aeration conditions. This relationship strongly suggests that the intracellular negative ion capacity contributes to the formation of Δψ through the Donnan effect in alkaliphilic Bacillus spp. grown at pH 10.

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).

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.

Isolation of acetogenic bacteria that induce biocorrosion by utilizing metallic iron as the sole electron donor.

Corrosion of iron occurring under anoxic conditions, which is termed microbiologically influenced corrosion (MIC) or biocorrosion, is mostly caused by microbial activities. Microbial activity that enhances corrosion via uptake of electrons from metallic iron [Fe(0)] has been regarded as one of the major causative factors. In addition to sulfate-reducing bacteria and methanogenic archaea in marine environments, acetogenic bacteria in freshwater environments have recently been suggested to cause MIC under anoxic conditions. However, no microorganisms that perform acetogenesis-dependent MIC have been isolated or had their MIC-inducing mechanisms characterized. Here, we enriched and isolated acetogenic bacteria that induce iron corrosion by utilizing Fe(0) as the sole electron donor under freshwater, sulfate-free, and anoxic conditions. The enriched communities produced significantly larger amounts of Fe(II) than the abiotic controls and produced acetate coupled with Fe(0) oxidation prior to CH4 production. Microbial community analysis revealed that Sporomusa sp. and Desulfovibrio sp. dominated in the enrichments. Strain GT1, which is closely related to the acetogen Sporomusa sphaeroides, was eventually isolated from the enrichment. Strain GT1 grew acetogenetically with Fe(0) as the sole electron donor and enhanced iron corrosion, which is the first demonstration of MIC mediated by a pure culture of an acetogen. Other well-known acetogenic bacteria, including Sporomusa ovata and Acetobacterium spp., did not grow well on Fe(0). These results indicate that very few species of acetogens have specific mechanisms to efficiently utilize cathodic electrons derived from Fe(0) oxidation and induce iron corrosion.

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)).