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The current practice of using stanchion breeding cow houses results in a semi-dry manure that ideally should be used as the substrate for the anaerobic fermentation. The conventional liquid-slurry biogas fermentation is disadvantageous with high running costs due to requirement such as water addition and running efficiency, among others. This study aimed to establish a high-speed semi-dry methane fermentation system for cow manure, by comparing productivity in wet and semi-dry conditions (> 90 and 83-85% water content, respectively), and mesophilic and thermophilic conditions (38 and 55°C, respectively). The highest methane productivity was obtained for 30 days in semi-dry or wet, thermophilic conditions (0.4-16.2 L‧Kg-1VS‧day-1). The highest accumulated production was 60 days (192.1 L‧Kg-1VS) in semi-dry thermophilic conditions. The pronounced difference in methane production at different temperatures in semi-dry digesters was studied by characterizing the microbial community changes in mesophilic and thermophilic digesters. For bacteria, the class Clostridia (including family Clostridiaceae and the genus Clostridium) was dominant in the thermophilic digester. In the thermophilic digester, a succession of taxa from the genus Methanobrevibacter and family Methanobacteriaceae to genus Methanoculleus in archaea was observed. The convergence of archaeal microbiota in thermophilic conditions was more pronounced than in mesophilic conditions. These results indicate that the proposed procedure is capable of improving methane productivity per scale and lowering the running costs concomitant and downsizing the fermentation scale. Key words: Semi-dry fermentation, methane yield, anaerobic digestion, cow manure, biogas.

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.

Catalase has crucial role in adaptive response to H(2)O(2). 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 H(2)O(2))/catalase activity using H(2)O(2) 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 H(2)O(2) 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 H(2)O(2)-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.

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.

The oxidation ditch process is one of the most economical approaches currently used to simultaneously remove organic carbon, nitrogen, and also phosphorus (P) from wastewater. However, limited information is available on biological P removal in this process. In the present study, microorganisms contributing to P removal in a full-scale oxidation ditch reactor were investigated using culture-dependent and -independent approaches. A microbial community analysis based on 16S rRNA gene sequencing revealed that a phylotype closely related to Dechloromonas spp. in the family Rhodocyclaceae dominated in the oxidation ditch reactor. This dominant Dechloromonas sp. was successfully isolated and subjected to fluorescent staining for polyphosphate, followed by microscopic observations and a spectrofluorometric analysis, which clearly demonstrated that the Dechloromonas isolate exhibited a strong ability to accumulate polyphosphate within its cells. These results indicate the potential key role of Dechloromonas spp. in efficient P removal in the oxidation ditch wastewater treatment process.

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 CH 4 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.

A hydrogen peroxide (H2O2)-resistant bacterium, Exiguobacterium oxidotolerans T-2-2T, secretes extracellular catalase. A low shaking frequency (60 rpm) was employed following the observation that a higher amount of extracellular catalase was produced under this condition compared with the previous shaking frequency (120 rpm). The cell concentration reached approximately 4.5 × 106 cells mL−1, at which the initiation of extracellular catalase production was triggered, while the production of the extracellular catalase was largely dependent on that of the intracellular catalase. The best initial aeration condition for extracellular catalase production was 7.5 mg O L−1, which is 95 % of that of the air-saturated state measured under the low shaking frequency. In addition, although the total productivity of catalase was unchanged, introduction of aminolevulinic acid and Tween 60 promoted extracellular production. The extracellular and intracellular catalase productivities were 16,000 and 6,000 U mL−1 of spent culture medium, respectively.

The inhibitory effects of ammonia on two different degradation pathways of methanogenic acetate were evaluated using a pure culture (Methanosaeta thermophila strain PT) and defined co-culture (Methanothermobacter thermautotrophicus strain TM and Thermacetogenium phaeum strain PB), which represented aceticlastic and syntrophic methanogenesis, respectively. Growth experiments with high concentrations of ammonia clearly demonstrated that sensitivity to ammonia stress was markedly higher in M. thermophila PT than in the syntrophic co-culture. M. thermophila PT also exhibited higher sensitivity to high pH stress, which indicated that an inability to maintain pH homeostasis is an underlying cause of ammonia inhibition. Methanogenesis was inhibited in the resting cells of M. thermophila PT with moderate concentrations of ammonia, suggesting that the inhibition of enzymes involved in methanogenesis may be one of the major factors responsible for ammonia toxicity. Transcriptomic analysis revealed a broad range of disturbances in M. thermophila PT cells under ammonia stress conditions, including protein denaturation, oxidative stress, and intracellular cation imbalances. The results of the present study clearly demonstrated that syntrophic acetate degradation dominated over aceticlastic methanogenesis under ammonia stress conditions, which is consistent with the findings of previous studies on complex microbial community systems. Our results also imply that the co-existence of multiple metabolic pathways and their different sensitivities to stress factors confer resiliency on methanogenic processes.

Despite their importance as a biofuel production platform, only a very limited number of butanol-tolerant bacteria have been identified thus far. Here, we extensively explored butanol- and isobutanol-tolerant bacteria from various environmental samples. A total of 16 aerobic and anaerobic bacteria that could tolerate greater than 2.0% (vol/vol) butanol and isobutanol were isolated. A 16S rRNA gene sequencing analysis revealed that the isolates were phylogenetically distributed over at least nine genera: Bacillus , Lysinibacillus , Rummeliibacillus , Brevibacillus , Coprothermobacter , Caloribacterium , Enterococcus , Hydrogenoanaerobacterium , and Cellulosimicrobium , within the phyla Firmicutes and Actinobacteria . Ten of the isolates were phylogenetically distinct from previously identified butanol-tolerant bacteria. Two relatively highly butanol-tolerant strains CM4A (aerobe) and GK12 (obligate anaerobe) were characterized further. Both strains changed their membrane fatty acid composition in response to butanol exposure, i.e., CM4A and GK12 exhibited increased saturated and cyclopropane fatty acids (CFAs) and long-chain fatty acids, respectively, which may serve to maintain membrane fluidity. The gene ( cfa ) encoding CFA synthase was cloned from strain CM4A and expressed in Escherichia coli . The recombinant E. coli showed relatively higher butanol and isobutanol tolerance than E. coli without the cfa gene, suggesting that cfa can confer solvent tolerance. The exposure of strain GK12 to butanol by consecutive passages even enhanced the growth rate, indicating that yet-unknown mechanisms may also contribute to solvent tolerance. Taken together, the results demonstrate that a wide variety of butanol- and isobutanol-tolerant bacteria that can grow in 2.0% butanol exist in the environment and have various strategies to maintain structural integrity against detrimental solvents.

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.

Two strains of alkaliphilic lactic acid bacteria, L-120 and AY103, which can produce L-lactic acid extensively, were isolated during trials of L-lactic acid production under unsterilized conditions at pH 9. Strains L-120 and AY103 are similar to Enterococcus casseliflavus and Enterococcus faecalis, respectively, as determined by 16S rRNA gene sequencing. It was found that wheat bran hydrolysate (1.5 or 3.0% dry w/v) strongly stimulates the production of lactic acid in both strains. Strain L-120 produced 149 g L-1 L-lactic acid at 35°C under pH 9 from 180 g L-1 glucose with the production rate of 3.9 g L-1 h-1. Strain AY103 produced 153 g L-1 L-lactic acid from 180 g L-1 glucose with the production rate of 4.2 g L-1 h-1. Both strains produced high-optical-purity (100%) L-lactic acid. In addition, strain L-120 produced 12% (w/v) L-lactic acid from degraded inedible materials, i.e., rice straw and soy bean curd refuse, within 72 h under unsterilized conditions. The obtained results indicate that these two strains are very useful for Llactic acid production with the advantages of high productivity and rapid production with reduced costs of product purification, raw materials and fermenter sterilization.

A psychrotolerant bacterium, strain T-3 (identified as Psychrobacter piscatorii), that exhibited an extraordinarily high catalase activity was isolated from the drain pool of a plant that uses H(2)O(2) as a bleaching agent. Its cell extract exhibited a catalase activity (19,700 U·mg protein(-1)) that was higher than that of Micrococcus luteus used for industrial catalase production. Catalase was approximately 10% of the total proteins in the cell extract of the strain. The catalase (PktA) was purified homogeneously by only two purification steps, anion exchange and hydrophobic chromatographies. The purified catalase exhibited higher catalytic efficiency and higher sensitivity of activity at high temperatures than M. luteus catalase. The deduced amino acid sequence showed the highest homology with catalase of Psycrobacter cryohalolentis, a psychrotolelant bacterium obtained from Siberian permafrost. These findings suggest that the characteristics of the PktA molecule reflected the taxonomic relationship of the isolate as well as the environmental conditions (low temperatures and high concentrations of H(2)O(2)) under which the bacterium survives. Strain T-3 efficiently produces a catalase (PktA) at a higher rate than Exiguobacterium oxidotolerans, which produces a very strong activity of catalase (EktA) at a moderate rate, in order to adapt to high concentration of H(2)O(2).

Indigo fermentation has been traditionally performed for dyeing textiles in Japan. Limited information is available on the microbiota involved and the succession of the bacterial community structure during indigo reduction. We investigated the bacterial community structure associated with indigo fermentation using denaturing gradient gel electrophoresis and clone library analyses of a PCR-amplified 16S rRNA gene in the early phase of fermentation carried out in our laboratory. A marked substitution of Halomonas spp. by Amphibacillus spp. was observed corresponding to the marked change in the state of indigo reduction. Although the reported indigo-reducing bacteria, Alkalibacterium spp., were not predominant in the early phase of fermentation, they were predominant in fermentation liquor aged for 10 months obtained from Date City, Japan, as determined by culture-dependent and culture-independent analyses. Novel indigo-reducing strains, Amphibacillus spp. strain C40 and Oceanobacillus spp. strain A21, were isolated from fermentation liquor aged for 10 months and from liquor aged for 4 days, respectively. It is considered that, in addition to the strains belonging to the genus Alkalibacterium, strains belonging to genera Amphibacillus and Oceanobacillus play important roles in sustaining the reduced state of indigo during fermentation.

Alkaliphiles grow under alkaline conditions that might be disadvantageous for the transmembrane pH gradient (Delta pH, outside acidic). In this study, the behaviors of extruded protons by the respiration of obligate alkaliphilic Bacillus clarkii K24-1U were investigated by comparison with those of neutralophilic Bacillus subtilis IAM 1026. Although whole-cell suspensions of both Bacillus species consumed oxygen immediately after the addition of air, there were lag times before the suspensions were acidified. Under alkaline conditions, the lag time for B. clarkii significantly increased, whereas that for B. subtilis decreased. In the presence of valinomycin or ETH-157, which disrupts the membrane electrical potential (Delta psi), the cell suspensions of both Bacillus species acidified immediately after the addition of air. Artificial electroneutral antiporters (nigericin and monensin) that eliminate the Delta pH exhibited no significant effect on the lag times of the two Bacillus species except that monensin increased the lag times of B. clarkii. The inhibition of ATPase and the Na(+) channel also exhibited little effects on the lag times. The increased lag time for B. clarkii may represent the Delta psi-dependent proton retention on the outer surface of the cytoplasmic membrane to generate a sufficient Delta pH under alkaline conditions.

Glutaminase from Micrococcus luteus K-3 [Micrococcus glutaminase (Mglu); 456 amino acid residues (aa); 48 kDa] is a salt-tolerant enzyme. Our previous study determined the structure of its major 42-kDa fragment. Here, using new crystallization conditions, we determined the structures of the intact enzyme in the presence and absence of its product L-glutamate and its activator Tris, which activates the enzyme by sixfold. With the exception of a 'lid' part (26-29 aa) and a few other short stretches, the structures were all very similar over the entire polypeptide chain. However, the presence of the ligands significantly reduced the length of the disordered regions: 41 aa in the unliganded structure (N), 21 aa for L-glutamate (G), 8 aa for Tris (T) and 6 aa for both L-glutamate and Tris (TG). L-glutamate was identified in both the G and TG structures, whereas Tris was only identified in the TG structure. Comparison of the glutamate-binding site between Mglu and salt-labile glutaminase (YbgJ) from Bacillus subtilis showed significantly smaller structural changes of the protein part in Mglu. A comparison of the substrate-binding pocket of Mglu, which is highly specific for L-glutamine, with that of Erwinia carotovora asparaginase, which has substrates other than L-glutamine, shows that Mglu has a larger substrate-binding pocket that prevents the binding of L-asparagine with proper interactions.

A very long chain polyunsaturated hydrocarbon, hentriacontanonaene (C31:9), was detected in an eicosapentaenoic acid (EPA)-producing marine bacterium, which was isolated from the mid-latitude seashore of Hokkaido, Japan, and was tentatively identified as mesophilic Shewanella sp. strain osh08 from 16S rRNA gene sequencing. The geometry and position of the double bonds in this compound were determined physicochemically to be all cis at positions 3, 6, 9, 12, 15, 19, 22, 25, and 28. Although C31:9 was detected in all of the seven EPA- or/and docosahexaenoic acid-producing bacteria tested, an EPA-deficient mutant (strain IK-1Δ8) of one of these bacteria had no C31:9. Strain IK-1Δ8 had defects in the pfaD gene, one of the five pfa genes responsible for the biosynthesis of EPA. Although Escherichia coli DH5α does not produce EPA or DHA inherently, cells transformed with the pfa genes responsible for the biosynthesis of EPA and DHA produced EPA and DHA, respectively, but not C31:9. These results suggest that the Pfa protein complex is involved in the biosynthesis of C31:9 and that pfa genes must not be the only genes responsible for the formation of C31:9. In this report, we determined for the first time the molecular structure of the C31:9 and discuss the possible biosynthetic pathways of this compound.

Indigo-reducing, obligately alkaliphilic strains A11T, F11 and F12 were isolated from indigo fermentation liquor obtained from Tokushima Prefecture, Shikoku, Japan. The isolates grew at pH 9.0-12.3, but not at pH 7.0-8.0. The optimum pH range for growth was 9.5-11.5. They were Gram-negative, facultatively anaerobic, rod-shaped strains with peritrichous flagella. The isolates grew in 0-14 % (w/v) NaCl, with optimum growth at 1-11 %. They grew at temperatures of 15-35 degrees C with optimum growth at around 20-30 degrees C. dl-Lactate was the major end product from d-glucose. No quinones were detected. The peptidoglycan type was A4 alpha, l-Lys (l-Orn)-d-Asp. The major cellular fatty acids were C16 : 0, C16 : 17c and C18 : 19c. The DNA G+C contents were 47.0-47.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that the isolates belong to the genus Alkalibacterium. DNA-DNA hybridization revealed low relatedness values between the isolates and the three phylogenetically most closely related species, Alkalibacterium olivapovliticus, Alkalibacterium psychrotolerans and Alkalibacterium iburiense (

The effects of H2O2 under low- and high-aeration-level conditions on growth and catalase activity in Exiguobacterium oxidotolerans T-2-2T were investigated. Continuous addition of 5-200 mM H2O2 to the culture medium from the mid-exponential growth phase enhanced the growth of the strain under the low-aeration-level condition, whereas the addition of 5-50 mM H2O2 decreased intracellular specific catalase activity and extracellular total catalases activity. The detection of extracellular catalase by the cells and the decrease in intracellular specific catalase activity and extracellular total catalase activity under the high-aeration-level condition account for the stimulation of growth by the introduced H2O2 and the decrease in catalase activities induced by O(2) from H2O2 in the medium. On the other hand, the addition of H2O2 to the medium prior to the initiation of growth inhibited the growth but increased the specific activity of intracellular catalase in the stationary growth phase. Strain T-2-2T grew when 10 mM H2O2 was added to the medium prior to growth. However, the growth was completely inhibited by the catalase inhibitor 3-amino-1,2,4-triazole (3-AT). The continuous addition of H2O2 at an appropriate concentration from prior to the initiation of growth to the stationary growth phase under the low-aeration-level condition resulted in higher intracellular specific catalase activity and cell growth rate than single H2O2 addition prior to growth.

A soluble class I cytochrome c of an alkaliphile was purified and characterized, and its primary structure was determined. This is the first example of a soluble class I cytochrome c in alkaliphiles. Cells the alkaliphilic gram-negative bacterium Pseudomonas alcaliphila AL15-21(T) grown at pH 10 had a soluble cytochrome c content that was more than twofold that of strain AL15-21(T) cells grown at pH 7 under air-limited conditions. Cytochrome c-552, a soluble cytochrome c with a low molecular weight, was purified from strain AL15-21(T) cells grown at pH 10 under air-limited conditions. Cytochrome c-552 had a molecular mass of 7.5 kDa and exhibited an almost fully reduced state in the resting form, which exhibited absorption maxima at wavelengths of 552, 523 and 417 nm. In the oxidized state, it exhibited an absorption maximum at 412 nm when it was oxidized by ferricyanide, its isoelectric point (pI) was 4.3 and it contained one heme c as a prosthetic group. Cytochrome c-552 was autoreduced at pH 10, and the autoreduction was reproducible. On the other hand, the autoreduction of cytochrome c-552 was not observed at pH 7.0. When pH was increased from 7.0 to 8.3, its midpoint redox potentials (E(m) values) increased from +228 mV to +276 mV as determined by redox titrations, and from +217 mV to +275 mV as determined by cyclic voltammetric measurements. The amino acid sequence deduced by cytochrome c-552 gene analysis revealed that the sequence consists of 96 residues, including 19 residues as an amino-terminal signal peptide. A phylogenetic tree based on amino acid sequence indicated that the protein belongs to group 4, cytochrome c(5) in class I cytochrome c.

Cellular eicosapentaenoic acid (EPA) makes up approximately 3% of total fatty acids in Escherichia coli DH5alpha, a strain that carries EPA biosynthesis genes (pEPADelta1). EPA was increased to 12% of total fatty acids when the host cell co-expressed the vector pGBM3::sa1(vktA), which carried the high-performance catalase gene, vktA. Where this vector was co-expressed, the transformant accumulated a large amount of VktA protein. However, the EPA production of cells carrying the vector, that included the insert lacking almost the entire vktA gene, was approximately 6%. This suggests that the retention of a large DNA insert in the vector and the accumulation of the resulting protein, but not the catalytic activity of VktA catalase, would potentially be able to increase the content of EPA.

Anaerobic degradation of lignin-derived aromatics is an important metabolism for carbon and nutrient cycles in soil environments. Although there are some studies on degradation of lignin-derived aromatics by nitrate- and sulfate-reducing bacteria, knowledge on their degradation under methanogenic conditions are quite limited. In this study, methanogenic microbial communities were enriched from rice paddy field soil with lignin-derived methoxylated monoaromatics (vanillate and syringate) and their degradation intermediates (protocatechuate, catechol and gallate) as the sole carbon and energy sources. Archaeal community analysis disclosed that both aceticlastic (Methanosarcina sp.) and hydrogenotrophic (Methanoculleus sp. and Methanocella sp.) methanogens dominated in all of the enrichments. Bacterial community analysis revealed the dominance of acetogenic bacteria (Sporomusa spp.) only in the enrichments on the methoxylated aromatics, suggesting that Sporomusa spp. initially convert vanillate and syringate into protocatechuate and gallate, respectively, with acetogenesis via O-demethylation. As the putative ring-cleavage microbes, bacteria within the phylum Firmicutes were dominantly detected from all of the enrichments, while the dominant phylotypes were not identical between enrichments on vanillate/protocatechuate/catechol (family Peptococcaceae bacteria) and on syringate/gallate (family Ruminococcaceae bacteria). This study demonstrates the importance of cooperation among acetogens, ring-cleaving fermenters/syntrophs and aceticlastic/hydrogenotrophic methanogens for degradation of lignin-derived aromatics under methanogenic conditions.

A novel marine bacterium, designated strain 4k5T, 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 4k5Tis a member of the genusPsychrobacter. Strain 4k5Texhibited the closely phylogenetic affinity toPsychrobacter pacificensisIFO 16270T(99.4 % 16S rRNA gene sequence similarity),P. piscatoriiT-3-2T(97.7 %),P. nivimaris88/2-7T(97.7 %),P. celerSW-238T(97.7 %),P. aestuariiSC35T(97.6 %) andP. vallisCMS39T(97.6 %). DNA–DNA hybridization between strain 4k5TandP. pacificensisNBRC 103191T,P. piscatoriiJCM 15603T.P. nivimarisDSM 16093T,P. celerJCM 12601T,P. aestuariiJCM 16343TandP. vallisDSM 15337Twas 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 namePsychrobacter oceanisp. nov. is proposed. The type strain is 4k5T( = JCM 30235T = NCIMB 14948T).

A catalase that exhibits a high level of activity and a rapid reaction with organic peroxides has been purified from Exiguobacterium oxidotolerans T-2-2T (EKTA catalase). The amino acid sequence of EKTA catalase revealed that it is a novel clade 1 catalase. Amino acid residues in the active site around the protoheme are conserved in the primary structure of EKTA catalase. Although the general interactions of molecules larger than hydrogen peroxide with catalases are strongly inhibited because of the selection role of long and narrow channels in the substrate reaching the active site, the formation rate of reactive intermediates (compound I) in the reaction of EKTA catalase with peracetic acid is 77 times higher than that of bovine liver catalase (BLC) and 1200 times higher than that of Micrococcus luteus catalase (MLC). The crystal structure of EKTA catalase has been determined and refined to 2.4 A resolution. The main channel structure of EKTA catalase is different from those of BLC and MLC. The rate constant of compound I formation in catalases decreased with an increase in the molecular size of the substrate. For EKTA catalase with a larger bottleneck 15 A from the iron (entrance of narrow channel) in the main channel, a lower rate of reduction in compound I formation rate with an increase in the molecular size of substrates was found. The increase in the rate constant of compound I formation in these catalases was directly proportional to the increase in the size of the bottleneck in the main channel when molecules of substrates larger than H2O2, such as organic peroxides, are used in the reaction. The results indicate that the size of the bottleneck in the main channel in catalase is an important factor in defining the rate of compound I formation corresponding to the molecular size of the substrates, and this was demonstrated. The Leu149-Ile180 and Asp109-Met167 combinations at the entrance of the narrow channel in EKTA catalase determine the size of the bottleneck, and each atom-to-atom distance for the combination of residues was larger than those of corresponding combinations of amino acid residues in BLC and MLC. The combination of these four amino acids is quite specific in EKTA catalase as compared with the combinations in other catalases in the gene database (compared with more than 432 catalase genes in the database).

Two novel exopolysaccharide-producing bacteria, strains S18K6T and S18K5, were isolated from Pacific Ocean sediment. The isolates were Gram-negative, motile, strictly aerobic chemoheterotrophic bacteria. The DNA G+C contents of strains S18K6T and S18K5 were 44.8 and 46.3 mol%, respectively. DNA–DNA relatedness between the two strains was 70 %. Major fatty acids were hexadecanoic acid (C16 : 0), hexadecenoic acid (C16 : 1 ω7c) and octadecenoic acid (C18 : 1 ω7c). 16S rRNA gene sequence, chemotaxonomic and morphological data indicated that these strains clearly belonged to the genus Glaciecola. Based on phenotypic properties and DNA–DNA hybridization data, strains S18K6T and S18K5 are considered to represent a novel species of the genus Glaciecola, for which the name Glaciecola chathamensis sp. nov. is proposed. The type strain is S18K6T (=JCM 13645T=NCIMB 14146T).

Degradation of n-alkanes in diesel oil by Pseudomonas aeruginosa strain WatG (WatG) was verified in soil microcosms. The total petroleum hydrocarbon (TPH) degradation level in two bioaugmentation samples was 51% and 46% for 1 week in unsterilized and sterilized soil microcosms, respectively. The TPH degradation in the biostimulation was of control level (15%). The TPH degradation in aeration-limited samples was clearly reduced when compared with that in aeration-unlimited ones under both sterilized and unsterilized conditions. Addition of WatG into soil microcosms was accompanied by dirhamnolipid production only in the presence of diesel oil. These findings suggest that degradation of n-alkanes in diesel oil in soil microcosms would be facilitated by bioaugmentation of WatG, with production of dirhamnolipid, and also by participation of biostimulated indigenous soil bacteria.

A novel mesophilic, chemolithoautotrophic, sulfur-oxidizing bacterium, designated strain SO07T, was isolated from a microaerobic waste-water biofilm. Chemolithoautotrophic growth was observed with elemental sulfur, sulfide and thiosulfate as sole electron donors and oxygen as electron acceptor. Anaerobic and heterotrophic growth were not observed. Nitrate was not used as a terminal electron acceptor. The optimum pH and temperature for growth were pH 7·5 and 30 °C, respectively. The major isoprenoid quinone was Q-8. The DNA G+C content of strain SO07T was 47·1 mol%. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that strain SO07T formed a monophyletic group in the γ-Proteobacteria with only 89 % similarity to members of the genus Halothiobacillus, its nearest phylogenetic neighbours. In addition, the isolate differed from members of the genus Halothiobacillus in its requirement for and tolerance of NaCl; strain SO07T was unable to grow in NaCl concentrations of more than 180 mM. On the basis of phylogenetic, chemotaxonomic and physiological data, it is proposed that isolate SO07T (=JCM 12417T=ATCC BAA-1033T) represents the type strain of a novel species in a new genus, Thiovirga sulfuroxydans gen. nov., sp. nov.

Bacteria possessing high capacity to degrade gasoline, kerosene, diesel oil, and lubricating oil were screened from several areas of Hokkaido, Japan. Among isolates, two strains, WatG and HokM, which were identified as new strains of Pseudomonas aeruginosa and Serratia marcescens species, respectively, showed relatively high capacity and wide spectrum to degrade the hydrocarbons in gasoline, kerosene, diesel, and lubricating oil. About 90-95% of excess amount of total diesel oil and kerosene added to mineral salts media as a sole carbon source could be degraded by WatG within 2 and 3 weeks, respectively. The same amount of lubricating oil was 60% degraded within 2 weeks. Strain HokM was more capable than WatG in degrading aromatic compounds in gasoline. This strain could also degrade kerosene, diesel, and lubricating oil with a capacity of 50-60%. Thus, these two isolates have potential to be useful for bioremediation of sites highly contaminated with petroleum hydrocarbons.

A psychrotolerant, obligately alkaliphilic bacterium, IDR2-2T, which is able to reduce indigo dye was isolated from a fermented polygonum indigo (Polygonum tinctorium Lour.) produced in Date, Hokkaido, using a traditional Japanese method. The isolate grew at pH 9–12 but not at pH 7–8. It was a Gram-positive, facultatively anaerobic, straight rod-shaped bacterium with peritrichous flagella. The isolate grew in 0–17 % (w/v) NaCl but not at NaCl concentrations higher than 18 % (w/v). Its major cellular fatty acids were C14 : 0, C16 : 0, C16 : 19c and C18 : 19c, and its DNA G+C content was 40·6 mol%. dl-lactic acid was the major end-product from d-glucose. No quinones could be detected. The peptidoglycan type was A4β, Orn–d-Glu. A phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain IDR2-2T is a member of the genus Alkalibacterium. DNA–DNA hybridization revealed low relatedness (less than 25 %) between the isolate and two phylogenetically related strains, Alkalibacterium olivapovliticus and Marinilactibacillus psychrotolerans. On the basis of phenotypic characteristics, phylogenetic data and DNA–DNA relatedness data, the isolate merits classification as a novel species, for which the name Alkalibacterium psychrotolerans sp. nov. is proposed. The type strain is IDR2-2T (=JCM 12281T=NCIMB 13981T).

A novel strictly aerobic, heterotrophic, mesophilic bacterium, strain Mb8(T), was isolated from soil in Sapporo City, Hokkaido, Japan. The G+C content of strain Mb8(T) was 66.0 mol%. It had mycolic acids with 44-52 carbon atoms and C16 : 0 and C18 : 1 (9) as the major fatty acids. The major isoprenoid quinone was MK-8(H(2)). The cell wall contained meso-diaminopimelic acid, arabinose and galactose. 16S rDNA, chemotaxonomic and morphological data indicated that this strain clearly belonged to the genus RHODOCOCCUS: Based on phenotypic properties and DNA-DNA hybridization data, strain Mb8(T) (=JCM 11308(T)=NCIMB 13903(T)) has been assigned to the genus Rhodococcus as the type strain of Rhodococcus tukisamuensis sp. nov.

A bacterium, Ikeda-3, that was isolated from the wastewater of a winery was found to produce floating and precipitating exopolysaccharides (EPSs) when grown aerobically in a medium containing sucrose as the sole carbon source. The concentrations of floating and precipitating EPSs were approximately 2.7 g/l and 0.6 g/l, respectively, in a mini-jar fermentor after incubations of the bacterium at 15 degrees C for 5 d in a synthetic medium containing 1% sucrose, 0.01% CaCO3, 0.05% MgSO4, 0.05% K2HPO4, 0.0001% Na2MoO4, 0.05% peptone and 0.82% bicine. A component analysis of two types of EPS suggested that they represent a novel type of sphingan composed of glucose, rhamnose, mannose and mannuroic acid. Glucoronic acid, which is commonly found in sphingans, was absent. From physiological, biochemical and chemotaxonomic characterization, phylogenetic analysis and DNA-DNA relatedness, Ikeda-3 was identified as Novosphingobium rosa. To our knowledge the production of EPS by N. rosa is reported here for the first time.

Catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1 T , which was isolated from an environment exposed to H 2 O 2 and exhibited high catalase activity, was purified and characterized, and its localization in the cell was determined. Its molecular mass was 230 kDa, and the molecule consisted of four identical subunits. The enzyme, which was not apparently reduced by dithionite, showed a Soret peak at 406 nm in a resting state. The catalytic activity was 527,500 U · mg of protein −1 under standard reaction conditions at 40°C, 1.5 and 4.3 times faster, respectively, than those of the Micrococcus luteus and bovine catalases examined under the same reaction conditions, and showed a broad optimum pH range (pH 6 to 10). The catalase from strain S-1 T is located not only in the cytoplasmic space but also in the periplasmic space. There is little difference in the activation energy for the activity between strain S-1 T catalase and M. luteus and bovine liver catalases. The thermoinstability of the activity of the former catalase were significantly higher than those of the latter catalases. The thermoinstability suggests that the catalase from strain S-1 T should be categorized as a psychrophilic enzyme. Although the catalase from strain S-1 T is classified as a mammal type catalase, it exhibits the unique enzymatic properties of high intensity of enzymatic activity and thermoinstability. The results obtained suggest that these unique properties of the enzyme are in accordance with the environmental conditions under which the microorganism lives.

A water-insoluble, extracellular polysaccharide was isolated from the culture medium of the snow mold fungus, Microdochium nivale, that had been cultivated in potato/dextrose broth. The polysaccharide consisted of glucose only. Its Fourier transform infrared spectrum showed a beta configuration of the C1 position of glucose. Linkage analysis of the polysaccharide showed that it had a linear structure of β-(1→4)-linked glucose. The polysaccharide was therefore identified as cellulose. This is the first report of extracellular cellulose occurring in fungi.

Two strictly aerobic, heterotrophic and mesophilic new organisms, strains Kitami A1 and Kitami C2T, were isolated from the wastewater of a sugar-beet factory in Kitami City, Hokkaido, Japan. In batch cultures, these organisms produced both insoluble and soluble exopolysaccharides (EPSs) utilizing sucrose as the sole carbon source. The G + C contents of the strains Kitami C2T and Kitami A1 were 69.2 mol%. Both strains had anteiso-C15:0 acid, anteiso-C17:0 acid and iso-C16:0 as major components. The major isoprenoid quinones from these strains included menaquinone-11 and menaquinone-12. Physiological and biochemical characterization, phylogenetic analysis and DNA-DNA relatedness indicated that these two organisms are new species of the genus Microbacterium, for which the name Microbacterium kitamiense is proposed. The type strain of M. kitamiense is strain Kitami C2T (= JCM 10270T).

デンプン質の糖化能とL-乳酸発酵能を合わせ持つLactobacillus amylophilusを活用することにより, バレイショデンプン液化物から直接発酵で乳酸を製造することができた.また, 発酵原料としては, 精製バレイショデンプンよりもバレイショが好ましく, バレイショに含まれるタンパク質などの成分が発酵の促進に寄与していると推定された.培地栄養源として, 高価なペプトンや酵母エキスの代わりに水産廃棄物であるイカゴロあるいはホタテウロの有効性が確認され, 特にイカゴロ培地の効果が大きく, 菌の糖化能が活性化される結果と考えられた.

The cytochrome content was estimated in a wide range of alkaliphilic Bacillus strains with DNA G + C contents from 34.4 to 44.3%. Among the strains tested, four facultative alkaliphiles, whose G + C contents were around 36%, and one obligate alkaliphile, with a G + C content of 41.4%, possessed relatively high amounts of total cytochrome (more than 0.3 nmol · mg cells −1 ). These five alkaliphiles contained high amounts of cytochromes b and c compared with the other strains tested, but their cytochrome a contents were not noticeably different. All the tested alkaliphilic strains had much higher amounts of cytochrome than B. subtilis (neutrophile), except for two facultative alkaliphiles with G + C contents around 40%. These results suggest that various bioenergetic strategies are used by alkaliphilic Bacillus for adaptation to a high alkaline environment, and that cytochromes b and c have a bioenergetically very important role in certain kinds of alkaliphiles.

Fermentation of L-(+)-lactic acid from soluble starch by Lactobacillus amylophilus was studied. The bacterium produced about 30 g of L-(+)-lactic acid from 50 g of soluble starch when the pH of the culture was ranging from pH 5 to pH 6.8 at 28°C. 53.4 g of L-(+)-lactic acid was produced when 100 g of starch was added in the medium. The fermentation procedures will reduce the cost of complete hydrolysis of starch to glucose prior to fermentation.

Heterologous gene expression is a widely used and vital biotechnology in basic and applied biology research fields. In particular, this technology (bioengineering ) is emerging as a useful tool in fields of applied biology, such as medical, pharmaceutical, and agricultural sciences including food science. With this technology, animals, fishes, plants, and eukaryotic and prokaryotic microorganisms can be used as host organisms for transformation, and various types of vectors have been developed and have become commercially available. It is only about 40 years since a heterologous cloned DNA was expressed in Escherichia coli cells (Annie et al., 1974; Old & Primrose, 1989). Development of various types of gene transfer systems has assisted the widespread use of gene engineering technologies in all research fields.

A novel facultatively alkaliphilic bacterium that grows on a chemically defined medium containing n-alkanes as the sole carbon source was isolated from soil. The isolate was obligately aerobic, non-motile, gram-positive, and formed metachromatic granules. It was not acidfast and did not form endospores. The cell wall contained meso-diaminopimelic acid, arabinose, and galactose; the glycan moiety of the cell wall contained acetyl residues. The bacterium was catalase-positive, oxidasenegative, and the G+C content of DNA was 70.8 mol%. According to these tests, the isolate was assigned to the genus Corynebacterium. The bacterium grew well between pH 6.2 to 10.2 and the doubling time in this pH range was 4–6 h. For the growth of the isolate, added Na+ in the culture medium stimulated growth, but was not indispensable at both pH 7.2 and pH 10.2. In addition to hydrocarbons, the isolate was able to grow on a chemically defined medium containing acetate, glucose, or fructose as the sole carbon source. Analysis of reduced minus oxidized difference spectra of whole cells showed that the bacterium only possessed less than one tenth the amount of total cytochromes as compared with Bacillus alcalophilus. The above results sugest that the bacterium has characteristics different than those of the alkaliphilic Bacillus previously described.

Cytochrome aco3 of Bacillus YN-2000 was purified by an improved procedure and its molecular features and catalytic activity were extensively studied. The enzyme molecule was composed of three subunits with M(r)s of 50,000, 41,000, and 22,000, and contains 1 molecule each of cytochrome a, cytochrome c, and cytochrome o3 in the minimal structural unit (M(r), 113,000). The 41,000 subunit (subunit II) contains heme c. The EPR, optical, and resonance Raman spectra of the oxidized enzyme demonstrated the presence of CuA whose coordination environment bore close resemblance to that of the aa3-type cytochrome c oxidase. Resonance Raman studies demonstrated that the cytochrome a moiety was similar to that of an aa3-type oxidase and also that the cytochrome o3 contained a five-coordinated high-spin heme with histidine as an axial ligand. The Fe-CO stretching mode of the cytochrome o3.CO complex was observed at 520 cm-1, which is the same frequency as that of cytochrome aa3.CO. The oxygen consumption activity of cytochrome aco3 was measured using several kinds of cytochromes c as the electron mediators. The reaction between cytochrome aco3 and eucaryotic cytochromes c was completely inhibited by poly-L-lysine. In contrast, poly-L-lysine was indispensable for sufficient reaction between the oxidase and Bacillus YN-2000 cytochrome c-553, the physiological electron donor. The combined results on the structure and enzymatic properties suggest that the cytochrome aco3 is very similar to cytochrome caa3 except that the cytochrome aco3 has cytochrome o3 in place of cytochrome a3 and the cytochrome c component has a very low redox midpoint potential (95 mV).(ABSTRACT TRUNCATED AT 250 WORDS)

Two novel thermophilic micro-organisms, designated YMO81T and YMO722T, were isolated from a high-temperature compost (internal temperature >95 °C). The isolates were able to grow at 80 °C in a nutrient broth and in a synthetic medium. Cells were aerobic, Gram-negative rods (0.3×4.0 μm). Spore formation was not observed. Strain YMO81T grew at 83 °C and pH 6.9–8.9 and grew optimally at 78 °C and pH 7.5 with 2 % NaCl. For growth in a synthetic minimal medium at 70 °C, the vitamins biotin, folic acid and thiamine and the amino acids glutamine and methionine were essential for growth of both strains; at 80 °C, strain YMO81T also required histidine, isoleucine, leucine, lysine, phenylalanine, serine, tryptophan and valine. Cellular fatty acids of the isolates comprised mainly iso-C17 : 0 and anteiso-C17 : 0. The DNA G+C contents of strains YMO81T and YMO722T were 70 and 64 mol%, respectively. When the 16S rRNA gene sequences of the isolates were compared with those of other bacteria, highest similarity was observed with Planifilum yunnanense LA5T (90 % 16S rRNA gene sequence similarity). DNA–DNA relatedness between strain YMO722T and strain YMO81T was 55 %. N 4-Aminopropylspermine was identified as a major polyamine, which suggested that the isolates were distinct from other related taxa. On the basis of phylogenetic, phenotypic and chemotaxonomic analyses, we propose a new genus, Calditerricola gen. nov., and two novel species, the type species Calditerricola satsumensis sp. nov., with type strain YMO81T (=ATCC BAA-1462T =JCM 14719T =DSM 45223T), and Calditerricola yamamurae sp. nov., with type strain YMO722T (=ATCC BAA-1461T =JCM 14720T =DSM 45224T).

A Gram-negative, non-motile, psychrotolerant bacterium exhibiting high catalase activity, designated strain T-3-2T, 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 °C and in the presence of 0–12 % NaCl. The predominant isoprenoid quinone was ubiquinone-8 (Q-8), and C16 : 1 ω9c and C18 : 1 ω9c were the predominant cellular fatty acids. The DNA G+C content of strain T-3-2T was 43.9 mol%. 16S rRNA gene sequence phylogeny suggested that strain T-3-2T 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-3-2T (=JCM 15603T =NCIMB 14510T).

The catalase gene of Psychrobacter sp. T-3 was cloned, and the gene product (PktA) was overexpressed in Escherichia coli. The specific activity of the purified PktA was slightly lower than that of the native purified enzyme obtained from Psychrobacter sp. T-3. Spectrophotometric measurements of the purified enzymes suggested that the recombinant PktA contains a mixture of heme b and d, although the native enzyme contains the sole heme b. An addition of the heme precursor 5-aminolevulinic acid (ALA) to the medium increased the heme b content of the recombinant PktA, and the resulting enzyme showed higher specific activity than the native enzyme. This is the first report that shows the heme content of overproduced catalase altered by the host cell growth conditions.

Catalase was purified to an electrophoretically homogeneous state from the facultative alkalophilic bacterium, Bacillus YN-2000, and some of its properties were studied. Its molecular weight was 282,000 and its molecule was composed of four identical subunits. The enzyme contained two protoheme molecules per tetramer. The enzyme showed an absorption spectrum of typical high-spin ferric heme with a peak at 406 nm in the oxidized form and peaks at 440, 559, and 592 nm in the reduced form. In contrast to the typical catalases, the enzyme was reduced with sodium dithionite, like peroxidases. The enzyme showed an appreciable peroxidase activity in addition to high catalase activity. The amino acid composition of Bacillus YN-2000 catalase was very similar to those of catalase from Neurospora crassa and peroxidase from Halobacterium halobium. The catalase content in the soluble fraction from the bacterium was higher with the cells grown at pH 10 than with the cells grown at lower pHs (pH 7-9).

A novel aco-type cytochrome-c oxidase was highly purified from the facultative alkalophilic bacterium, Bacillus YN-2000, grown at pH 10. The enzyme contained 9.0 nmol heme a/mg protein. It contained 1.23 mol of protoheme, 1.06 mol of heme c, 2.0 g atoms of copper, 2.5 g atoms of iron, and 1.8 g atoms of magnesium per mol of heme a. The enzyme molecule seemed to be composed of two subunits with Mrs of 52,000 and 41,600. On the basis of these results, the enzyme seemed to contain one molecule each of heme a, protoheme, and heme c per minimal structural unit (Mr, 93,600). Only protoheme among the three kinds of hemes in the enzyme reacted with CO and CN-. Heme a did not react with CO; cytochrome a3 did not seem to be present in the enzyme. The enzyme oxidized 314 mol of horse ferrocytochrome c per heme a per sec at pH 6.5 and the catalytic activity was 50% inhibited by 7.65 microM KCN. The enzymatic activity was found to be optimal at pH 6.0.

Three indigo-reducing obligately alkaliphilic strains, M3T, 41A and 41C, were isolated. The isolates grew at pH 9–12, but not at pH 7–8. They were Gram-positive, facultatively anaerobic, straight rod-shaped strains with peritrichous flagella. The isolates grew in 0–14 % (w/v) NaCl, with optimum growth at 3–13 %. They grew at temperatures between 10 and 45 °C, with optimum growth at around 30–37 °C. They did not hydrolyse starch or gelatin.dl-lactate was the major end-product fromd-glucose. No quinones could be detected. The peptidoglycan type was A4β, Orn–d-Asp. The major cellular fatty acids were C16 : 0, C16 : 17cand C18 : 19c. The DNA G+C content was 42·6–43·2 mol%. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that the isolates belong to the genusAlkalibacterium. DNA–DNA hybridization revealed low similarity (less than 16 %) of the isolates with respect to the two closest phylogenetically related strains,Alkalibacterium olivapovliticusandAlkalibacterium psychrotolerans. On the basis of phenotypic and chemotaxonomic characteristics, phylogenetic data and DNA–DNA relatedness, the isolates merit classification as a novel species of the genusAlkalibacterium, for which the nameAlkalibacterium iburienseis proposed. The type strain is M3T(=JCM 12662T=NCIMB 14024T).

A novel, oil-degrading bacterium (strain T1) was isolated from a hot spring in Hokkaido, Japan. It efficiently degrades different types of fats and oils, including edible oil waste. When grown in a mineral salt medium containing 1% triacylglycerol (as salad oil), hydrolysis products were 1,3- and 1,2-diacylglycerols, monoacylglycerol, and free fatty acid. However, these products were almost completely consumed during cultivation at 30 degrees C for 5 days, indicating that extracellular lipase acts randomly at different sn-positions of acylglycerols and that strain T1 has a high capacity to utilize free fatty acids. Secreted lipase activity was induced by salad oil and oleic acid. This strain was a Gram-negative straight rod shaped, aerobic, with a polar flagellum, capable of growing in temperature ranges between 15 degrees C and 55 degrees C. The 16S rRNA gene sequence analysis and DNA-DNA hybridization revealed it as a new strain of Pseudomonas aeruginosa. The type strain was T1.

A possible mechanism of resistance to hydrogen peroxide (H 2 O 2 ) in Vibrio rumoiensis , isolated from the H 2 O 2 -rich drain pool of a fish processing plant, was examined. When V. rumoiensis cells were inoculated into medium containing either 5 mM or no H 2 O 2 , they grew in similar manners. A spontaneous mutant strain, S-4, derived from V. rumoiensis and lacking catalase activity did not grow at all in the presence of 5 mM H 2 O 2 . These results suggest that catalase is inevitably involved in the resistance and survival of V. rumoiensis in the presence of H 2 O 2 . Catalase activity was constitutively present in V. rumoiensis cells grown in the absence of H 2 O 2 , and its occurrence was dependent on the age of the cells, a characteristic which is observed for the HP II-type catalase of Escherichia coli . The presence of the HP II-type catalase in V. rumoiensis cells was evidenced by partial sequencing of the gene encoding the HP II-type catalase from this organism. A notable difference between V. rumoiensis and E. coli is that catalase is accumulated at very high levels (∼2% of the total soluble proteins) in V. rumoiensis , in contrast to the case for E. coli . When V. rumoiensis cells which had been exposed to 5 mM H 2 O 2 were centrifuged, most intracellular proteins, including catalase, were recovered in the medium. On the other hand, when V. rumoiensis cells were grown on plates containing various concentrations of H 2 O 2 , individual cells had a colony-forming ability inferior to those of E. coli , Bacillus subtilis , and Vibrio parahaemolyticus . Thus, it is suggested that when V. rumoiensis cells are exposed to high concentrations of H 2 O 2 , most cells will immediately be broken by H 2 O 2 . In addition, the cells which have had little or no damage will start to grow in a medium where almost all H 2 O 2 has been decomposed by the catalase released from broken cells.

A bacterium Tomakomai B-1 that was isolated from the activated sludge of a treatment process for wastewater of the pulp industry was found to produce soluble and insoluble exopolysaccharides (EPSs) when grown aerobically in a medium containing sucrose as the sole carbon source. The sugar component of soluble EPSs consisted of rhamnose, mannose, galactose, glucose, mannuronic acid, galacturonic acid and glucuronic acid, and that of insoluble EPSs consisted of rhamnose, mannose, galactose, glucose, mannuronic acid and glucuronic acid. The concentrations of insoluble and soluble EPS were approximately 1.63 and 2.58 g/l, respectively, by Erlenmeyer flask culture at 20 degrees C for 3 d with a synthetic medium containing 1% sucrose, 0.05% CaCO(3), 0.05% MgSO(4), 0.1% K(2)HPO(4), 0.0001% Na(2)MoO(4), 0.01% urea, 1.07% MES and small amounts of trace metals. Tomakomai B-1 was identified as Rahnella aquatilis. To our knowledge the production of EPSs by R. Aquatilis is reported here for the first time.