Nobuo Kaku

Bacteroides graminisolvens sp. nov., a xylanolytic anaerobe isolated from a methanogenic reactor treating cattle waste.

A strictly anaerobic bacterial strain, designated XDT-1T, was isolated from plant residue from a methanogenic reactor treating waste from cattle farms. Cells of the strain were Gram-negative, non-motile, non-spore-forming rods. Haemin was required for growth. The strain utilized xylan as well as various sugars including arabinose, xylose, glucose, mannose, cellobiose, raffinose, starch and pectin. The strain produced acetate, propionate and succinate from saccharides in the presence of haemin. The optimum pH for growth was approximately 7.2 and the optimum growth temperature was 30-35 degrees C. The strain was sensitive to bile. The major cellular fatty acids of the strain were anteiso-C15:0 and iso-C17:0 3-OH, MK-10(H0) was the major respiratory quinone and the genomic DNA G+C content was 38.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences placed the strain in the phylum Bacteroidetes. The closest phylogenetic neighbour of strain XDT-1T was Bacteroides ovatus NCTC 11153T, with a 16S rRNA gene sequence similarity of 94.2%. On the basis of data from the phylogenetic, physiological and chemotaxonomic analyses, strain XDT-1T represents a novel species of the genus Bacteroides, for which the name Bacteroides graminisolvens sp. nov. is proposed. The type strain is XDT-1T (=JCM 15093T=DSM 19988T).

Bacteroides propionicifaciens sp. nov., isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms.

Two strictly anaerobic bacterial strains (SV434(T) and S562) were isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms in Japan. They had identical 16S rRNA gene sequences and showed almost the same phenotypic properties. The cells of both strains were Gram-negative, non-motile, non-spore-forming rods; extraordinarily long rods often occurred. Remarkable stimulation of growth occurred with the addition of haemin and cobalamin (vitamin B(12)) to the medium. The supplementary cobalamin and haemin could be replaced if autoclaved and clarified sludge fluid obtained from the reactor was added. Both strains utilized a range of growth substrates, including arabinose, fructose, galactose, glucose, mannose, cellobiose, maltose, glycogen, starch, dextrin, amygdalin, lactate and pyruvate. Both strains produced acetate and propionate with a small amount of succinate from these substrates in the presence of haemin and cobalamin. Both strains were slightly alkaliphilic, having a pH optimum at about 7.9. The temperature range for growth was 5-35 degrees C, the optimum being 30 degrees C. The NaCl concentration range for growth was 0-4 % (w/v). Catalase activity was not detected in cells cultivated without haemin, whereas cells cultivated with haemin usually had the enzyme activity. Oxidase and nitrate-reducing activities were not detected. Aesculin was hydrolysed, but gelatin was not hydrolysed. Both strains were sensitive to bile acids. The major cellular fatty acids of both strains were anteiso-C(15 : 0) and iso-C(15 : 0). Menaquinones MK-8(H(0)) and MK-9(H(0)) were the major respiratory quinones and the genomic DNA G+C contents were 46.2-47.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequences placed both strains in the phylum Bacteroidetes. Bacteroides coprosuis (isolated from swine-manure storage pits) was the species most closely related to both strains (95.9 % 16S rRNA gene sequence similarity to the type strain). On the basis of the phylogenetic, physiological and chemotaxonomic analyses, strains SV434(T) and S562 represent a novel species of the genus Bacteroides, for which the name Bacteroides propionicifaciens sp. nov. is proposed. The type strain is SV434(T) (=JCM 14649(T) =DSM 19291(T)).

Clostridium sufflavum sp. nov., isolated from a methanogenic reactor treating cattle waste.

A strictly anaerobic, mesophilic, cellulolytic bacterial strain, designated CDT-1(T), was isolated from rice-straw residue from a methanogenic reactor treating waste from cattle farms. The isolation was performed using enrichment culture with filter paper as a substrate. Cells stained Gram-negative, but reacted Gram-positively in the KOH test. Cells were slightly curved rods and were motile by means of peritrichous flagella. The strain produced yellow pigment when grown on filter-paper fragments. Although spore formation was not confirmed microscopically, thermotolerant cells were produced when the strain was grown on filter paper. The optimum temperature for growth was 33 degrees C and the optimum pH was 7.4. Oxidase, catalase and nitrate-reducing activities were absent. The strain utilized xylose, fructose, glucose, cellobiose, xylooligosaccharide, cellulose (filter-paper fragments and ball-milled filter paper) and xylan. The major fermentation products were acetate, ethanol, H(2) and CO(2). The major cellular fatty acids were iso-C(15 : 0), iso-C(14 : 0) and C(16 : 0) DMA. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The genomic DNA G+C content was 40.7 mol%. On the basis of 16S rRNA gene sequence similarities, strain CDT-1(T) could be placed in cluster III of the genus Clostridium, being closely related to type strains of Clostridium hungatei (96.6 % sequence similarity), Clostridium termitidis (96.2 %) and Clostridium papyrosolvens (96.1 %). On the basis of the cellular, physiological and phylogenetic differences between CDT-1(T) and its close relatives, this strain represents a novel species of the genus Clostridium, for which the name Clostridium sufflavum sp. nov. is proposed. The type strain is CDT-1(T) (=JCM 14807(T)=DSM 19573(T)).

Methanogenic activities on rice roots and plant residue and their contributions to methanogenesis in wetland rice field soil.

Changes in potential of methanogenic activities of three fractions (soil, living rice roots and plant residue) in wetland rice field soil during the cropping season of rice were determined by incubating them anaerobically in diluent in tubes. The soil fraction was prepared by removing various materials such as rice roots and plant residue mixed in core soil samples of the plow layer. Rice roots and plant residue (mainly rice straw plowed into soil) collected were washed in anoxic diluent to remove adhering soil and cut to pieces before the incubation. The methanogenic activity of the soil fraction was rather low from mid-May, when rice seedlings were transplanted, until late-June. Thereafter, the activity rapidly increased and at early-July it reached the maximum level of the activity in the year. The high activity was kept for about one month and then, during the period of intermittent irrigation, it dropped rapidly to much lower levels. The overall pattern of changes in the activity of the rice roots fraction resembled to that of the soil fraction, however, the activity on a dry weight basis was much higher than that of the soil fraction on almost all the days of determination. For the plant residue fraction, the pattern of seasonal changes was much different from those of the other two fractions and the activity increased soon after the start of flooding of the field. At the early-cropping season, the activity on a dry weight basis was much higher (up to 150 times) than that of the soil fraction of the same day. Changes in the weights of the three fractions in the rice field were measured by using hill plots, each of which was applied with rice straw and transplanted with rice seedlings. From the changes in the methanogenic activities and the weights of the three fractions in the field, the seasonal variations in the contribution of methanogenic activity of each fraction to the total methanogenic activity in the soil ecosystem were estimated. It was shown that plant residue should significantly enhance the methanogenic activity in the first half of the cropping season, and in contrast, rice roots should enhance it in the latter half.

Anaerosphaera aminiphila gen. nov., sp. nov., a glutamate-degrading, Gram-positive anaerobic coccus isolated from a methanogenic reactor treating cattle waste

A strictly anaerobic, mesophilic and aminolytic strain (WN036(T)) was isolated from a methanogenic reactor treating waste from cattle farms. Cells were Gram-positive cocci, often occurred in pairs and were non-motile. Although spore formation was not confirmed by microscopic observation of cells, the strain produced thermotolerant cells. The optimum temperature for growth was 35-37 degrees C and the optimum pH was 6.7. Oxidase, catalase and nitrate-reducing activities were negative. The novel strain did not ferment carbohydrates and grew in PY medium without additional substrates. The strain utilized l-glutamate, l-glutamine, l-histidine and l-arginine as growth substrates. Major fermentation products were acetate and butyrate with a small amount of propionate. The genomic DNA G+C content was 32.5 mol%. The major cellular fatty acids were C(17 : 1)omega8, C(18 : 1)omega7 DMA and C(16 : 0). The diagnostic diamino acid of the cell-wall peptidoglycan was lysine. Glutamic acid, glycine, alanine and aspartic acid were also detected in the cell-wall peptidoglycan. On the basis of 16S rRNA gene sequences, the most closely related species to strain WN036(T) were Peptoniphilus asaccharolyticus ATCC 14965(T) (89.8 %) and Peptoniphilus indolicus ATCC 29427(T) (89.6 %). Based on the differences in the phenotypic and phylogenetic characteristics of strain WN036(T) compared with those of closely related species, a novel genus and species, Anaerosphaera aminiphila gen. nov., sp. nov., is proposed. The type strain is WN036(T) (=JCM 15094(T)=DSM 21120(T)).

Fermentative l-(+)-lactic acid production from non-sterilized rice washing drainage containing rice bran by a newly isolated lactic acid bacteria without any additions of nutrients

A newly isolated lactic acid producing bacterium Lactobacillus rhamnosus M-23, from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. The maximum lactate yield attained was 59 g/l with a productivity of 1.23 g/l/h and a product optical purity of 95% corresponding to a conversion of 0.85 g of lactic acid per gram of sugar equivalent.

Changes and recovery of soil bacterial communities influenced by biological soil disinfestation as compared with chloropicrin-treatment

Soil bacterial composition, as influenced by biological soil disinfestation (BSD) associated with biomass incorporation was investigated to observe the effects of the treatment on the changes and recovery of the microbial community in a commercial greenhouse setting. Chloropicrin (CP) was also used for soil disinfestation to compare with the effects of BSD. The fusarium wilt disease incidence of spinach cultivated in the BSD- and CP-treated plots was reduced as compared with that in the untreated control plots, showing effectiveness of both methods to suppress the disease. The clone library analyses based on 16S rRNA gene sequences showed that members of the Firmicutes became dominant in the soil bacterial community after the BSD-treatment. Clone groups related to the species in the class Clostridia, such as Clostridium saccharobutylicum, Clostridium tetanomorphum, Clostridium cylindrosporum, Oxobacter pfennigii, etc., as well as Bacillus niacini in the class Bacilli were recognized as the most dominant members in the community. For the CP-treated soil, clones affiliated with the Bacilli related to acid-tolerant or thermophilic bacteria such as Tuberibacillus calidus, Sporolactobacillus laevolacticus, Pullulanibacillus naganoensis, Alicyclobacillus pomorum, etc. were detected as the major groups. The clone library analysis for the soil samples collected after spinach cultivation revealed that most of bacterial groups present in the original soil belonging to the phyla Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes, Planctomycetes, TM7, etc. were recovered in the BSD-treated soil. For the CP-treated soil, the recovery of the bacterial groups belonging to the above phyla was also noted, but some major clone groups recognized in the original soil did not recover fully.

Development of anaerobic bacterial community consisted of diverse clostridial species during biological soil disinfestation amended with plant biomass

Biological soil disinfestation (BSD) using plant biomass incorporation is an effective method and a good alternative to chemical fumigants for controlling soil-borne plant pathogens. In this study the bacterial communities in pot soil treated with three different BSD conditions (without plant biomass and with Brassica juncea L. plants or bran of wheat, Triticum aestivum L.) were analyzed using mainly molecular techniques. Earlier dropping of redox potential of both biomass-treated soils indicated rapid development of anaerobic conditions in the soil. The population of Fusarium oxysporum F. pathogen incorporated in the soil at the start was decreased considerably during the treatment, and the number of culturable anaerobic bacteria increased in both biomass-treated soils. Rather high concentrations of acetate and butyrate were detected from the biomass-treated soils. The polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis for the V3 region of 16S rRNA gene sequences revealed that the profiles of both biomass-treated soils were initially represented by similar and dominant groups, many of which were closely related to the species in the classes Clostridia and Bacilli of the phylum Firmicutes. Based on the clone library analysis, the control soil samples showed diverse bacterial groups with a few number of anaerobic clones. In contrast, for both biomass-treated libraries, clones belonging to the class Clostridia, a strictly anaerobic spore-forming bacterial group, appeared exceedingly dominant. The clostridial group detected was composed of phylogenetically diverse members, and it seemed likely that the diverse clostridial species were responsible for suppression of pathogens by making various compounds including volatile fatty acids and other compounds during anaerobic decomposition of plant biomass.

Propioniciclava tarda gen. nov., sp. nov., isolated from a methanogenic reactor treating waste from cattle farms.

Two facultatively anaerobic bacterial strains, designated WR061(T) and WR054, were isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms in Japan. The two strains were phylogenetically positioned close to one another and had almost the same phenotypic properties. Cells were Gram-reaction-positive, non-motile, non-spore-forming, irregular rods. Cobalamin (vitamin B₁₂) was required for growth. The strains utilized various carbohydrates, including hexoses and disaccharides, and produced acetate and propionate from these carbohydrates. Pentoses and polysaccharides were not utilized. They grew at 20-37 °C (optimum 35 °C) and pH 5.3-8.0 (optimum pH 6.8-7.5). Catalase and nitrate-reducing activities were detected. Aesculin was hydrolysed. The major cellular fatty acids were anteiso-C₁₅:₀ and C₁₅:₀ DMA, the major respiratory quinone was menaquinone MK-9(H₄) and the genomic DNA G+C content was 69.3-69.5  mol%. The diagnostic diamino acid in the peptidoglycan was meso-diaminopimelic acid. Phylogenetic analysis based on 16S rRNA gene sequences placed the strains in the phylum Actinobacteria. Both strains were remotely related to the species in the family Propionibacteriaceae and Propionibacterium propionicum JCM 5830(T) was the most closely related type strain with a sequence similarity of 91.6 %. Based on phylogenetic, physiological and chemotaxonomic analyses, the two novel strains together represent a novel species of a new genus, for which the name Propioniciclava tarda gen. nov., sp. nov. is proposed. The type strain is WR061(T) ( = JCM 15804(T)  = DSM 22130(T)).

Bacteroides paurosaccharolyticus sp. nov., isolated from a methanogenic reactor treating waste from cattle farms.

A strictly anaerobic bacterial strain (WK042(T)) was isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms in Japan. Cells were Gram-staining-negative, non-motile, non-spore-forming rods. Growth was stimulated well by haemin, and was enhanced by cobalamin (vitamin B(12)). Strain WK042(T) utilized arabinose, xylose, glucose, mannose and aesculin as preferred substrates. Maltose, dextrin, glycogen, starch and pectin were also utilized, although growth on these substrates was much slower. The strain produced acetate, propionate and succinate from these saccharides. The strain was slightly alkaliphilic, with optimum growth at pH 7.7. The temperature range for growth was 10-40 °C, the optimum being 35 °C. The strain was sensitive to bile. The major cellular fatty acids were anteiso-C(15 : 0), iso-C(17 : 0) 3-OH and C(15 : 0). Menaquinone 11 (MK-11) was the major respiratory quinone and the genomic DNA G+C content was 41.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences placed the strain in the phylum Bacteroidetes. Strain WK042(T) was related distantly to the type strains of species in the cluster including Bacteroides massiliensis, Bacteroides vulgatus and Bacteroides dorei (91-92 % 16S rRNA gene sequence similarity). Based on data from the present phylogenetic, physiological and chemotaxonomic analyses, strain WK042(T) is considered to represent a novel species of the genus Bacteroides, for which the name Bacteroides paurosaccharolyticus sp. nov. is proposed. The type strain is WK042(T) (=JCM 15092(T) =DSM 21004(T)).