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Two Gram-stain-negative, terminal endospore-forming, rod-shaped and aerotolerant bacterial strains designated D1-1T and B3 were isolated from soil samples of an organic paddy in Japan. Strain D1-1T grew at 15–37 °C, pH 5.0–7.3, and with up to 0.5 % (w/v) NaCl. Phylogenetic analysis of the 16S rRNA gene revealed that strain D1-1T belonged to the genus Clostridium and was closely related to Clostridium zeae CSC2T (99.7 % sequence similarity), Clostridium fungisolvens TW1T (99.7 %) and Clostridium manihotivorum CT4T (99.3 %). Strains D1-1T and B3 were whole-genome sequenced and indistinguishable, with an average nucleotide identity value of 99.7 %. The average nucleotide identity (below 91.1 %) and digital DNA–DNA hybridization (below 43.6 %) values between the two novel isolates and their corresponding relatives showed that strains D1-1T and B3 could be readily distinguished from their closely related species. A novel Clostridium species, Clostridium folliculivorans sp. nov., with type strain D1-1T (=MAFF 212477T=DSM 113523T), is proposed based on genotypic and phenotypic data.

In this study, we isolated a novel strain of lactic acid bacteria, AF129

Two Gram-stain-positive, rod-shaped, non-motile, non-spore-forming, catalase-negative bacteria, designated strains SG162T and NK01, were isolated from Japanese rice grain silage and total mixed ration silage, respectively. They were initially identified as Lactobacillus buchneri based on the 16S rRNA gene sequence similarities. However, the two strains were separated into a distinct clade from L. buchneri DSM 20057T (=JCM 1115T) through whole-genome sequence-based characterization, forming an infraspecific subgroup together with strains CD034 and S42, whose genomic sequences were available in the public sequence database. Strains within the subgroup shared 99.4–99.7 % average nucleotide identity (ANI) and 97.5–99.0 % digital DNA–DNA hybridization (dDDH) with each other, albeit 96.9–97.0 % ANI and 76.0–76.6 % dDDH against DSM 20057T. Strains SG162T and NK01 could utilize more substrates as sole carbon sources than DSM 20057T, potentially owing to the abundance of genes involved in carbon metabolism, especially the Entner–Doudoroff pathway. The inability of γ-aminobutyric acid (GABA) production was evidenced by the lack of glutamate decarboxylase and glutamate/GABA antiporter genes in the new subgroup strains. Strain SG162T grew at 10–45 °C (optimum, 30 °C), pH 3.5–8.0, and 0–8 % (w/v) NaCl. Its genomic DNA G+C content was 44.1 mol%. The predominant fatty acids were C16 : 0, C19 : 0 cyclo ω8c, and summed feature 8. On the basis of the polyphasic characterization findings, strains SG162T and NK01 represent a novel subspecies of L. buchneri , for which the name Lactobacillus buchneri subsp. silagei subsp. nov. is proposed. The type strain is SG162T (=JCM 32599T=DSM 107969T), and strains CD034 and S42 are also transferred to L. buchneri subsp. silagei.

Clostridium diolis shares high similarity based on 16S rRNA gene sequences and fatty acid composition with Clostridium beijerinckii . In this study, the taxonomic status of C. diolis was clarified using genomic and phenotypic approaches. High similarity was detected among C. diolis DSM 15410T, C. beijerinckii DSM 791T and NCTC 13035T, showing average nucleotide identity on blast and in silico DNA–DNA hybridization values over 97 and 85 %, respectively. Results of investigations for substrate utilization and enzyme activity displayed no striking differences between C. diolis DSM 15410T and C. beijerinckii JCM 1390T. Based on the results, we propose the reclassification of Clostridium diolis as a later heterotypic synonym of Clostridium beijerinckii . The type strain is ATCC 25752T (=CIP 104308T=DSM 791T=JCM 1390T=LMG 5716T=NCTC 13035T).

In Japan, during a screening of lactic acid bacteria in spent mushroom substrates, an unknown bacterium was isolated and could not be assigned to any known species. Strain YK48GT is Gram-stain-positive, rod-shaped, non-motile, non-spore-forming and catalase-negative. The isolate grew in 0–4 % (w/v) NaCl, at 15–37 °C (optimum, 30 °C) and at pH 4.0–8.0 (optimum, pH 6.0). The genomic DNA G+C content of strain YK48GT was 42.5 mol%. Based on its 16S rRNA gene sequence, strain YK48GT represented a member of the genus Lentilactobacillus and showed the highest pairwise similarity to Lentilactobacillus rapi DSM 19907T (97.86 %). Phylogenetic analyses based on amino acid sequences of 466 shared protein-encoding genes also revealed that the strain was phylogenetically positioned in the genus Lentilactobacillus but did not suggest an affiliation with previously described species. The average nucleotide identity and digital DNA–DNA hybridization values between strain YK48GT and the type strains of phylogenetically related species were 72.2–76.6% and 19.0–21.2 %, respectively, indicating that strain YK48GT represents a novel species within the genus Lentilactobacillus . Phenotypic data further confirmed the differentiation of strain YK48GT from other members of the genus Lentilactobacillus . According to the results of the polyphasic characterization presented in this study, strain YK48GT represents a novel species of the genus Lentilactobacillus , for which the name Lentilactobacillus fungorum sp. nov. is proposed. The type strain is YK48GT (=JCM 32598T=DSM 107968T).

A rod-shaped, Gram-stain-negative, strictly anaerobic, catalase-negative and endospore-forming bacterial strain CSC2T was isolated from corn silage preserved in Tochigi, Japan. The strain CSC2T grew at 15–40 °C, at pH 5.0–7.7 and with up to 0.5 % (w/v) NaCl. The main cellular fatty acids were C14 : 0, C16 : 0 and C16 : 0 dimethyl acetal. The cellular polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, lysophosphatidylethanolamine, phosphatidylserine, lysophosphatidylcholine and two unidentified polar lipids. Phylogenetic analysis of the 16S rRNA gene showed that strain CSC2T was a member of the genus Clostridium and closely related to Clostridium polyendosporum DSM 57272T (95.6 % gene sequence similarity) and Clostridium fallax ATCC 19400T (95.3 %). The genomic DNA G+C content of strain CSC2T was 31.1 mol% (whole genome analysis). The average nucleotide identity based on blast and digital DNA–DNA hybridization values between strain CSC2T and the type strains of phylogenetically related species were below 71 and 24 %, respectively. On the basis of the genotypic, phenotypic and chemotaxonomic characteristics, it is proposed to designate strain CSC2T as representing Clostridium zeae sp. nov. The type strain is CSC2T (=MAFF212476T=JCM 33766T=DSM 111242T).

The taxonomic status of the species

The taxonomic status of the species Clostridium methoxybenzovorans was assessed. The 16S rRNA gene sequence, whole-genome sequence and phenotypic characterizations suggested that the type strain deposited in the American Type Culture Collection ( C. methoxybenzovorans ATCC 700855T) is a member of the species Eubacterium callanderi . Hence, C. methoxybenzovorans ATCC 700855T cannot be used as a reference for taxonomic study. The type strain deposited in the German Collection of Microorganism and Cell Cultures GmbH (DSM 12182T) is no longer listed in its online catalogue. Also, both the 16S rRNA gene and the whole-genome sequences of the original strain SR3T showed high sequence identity with those of Lacrimispora indolis (recently reclassified from Clostridium indolis ) as the most closely related species. Analysis of the two genomes showed average nucleotide identity based on blast and digital DNA–DNA hybridization values of 98.3 and 87.9 %, respectively. Based on these results, C. methoxybenzovorans SR3T was considered to be a member of L. indolis .

The taxonomic status of Paenibacillus thermophilus was analyzed using genomic and phenotypic approaches. The results of RNA polymerase beta subunit gene sequence comparisons indicated that two type strains of P. thermophilus (DSM 24746T and JCM 17693T) and Paenibacillus macerans ATCC 8244T shared 100 % sequence similarity. By whole-genome sequence comparison, their average nucleotide identity values were over 99.3 %. Investigation of substrate utilization, enzyme activities and cellular fatty acid patterns displayed no striking differences between P. thermophilus JCM 17693T and P. macerans JCM 2500T. On the basis of these results, we propose that the name Paenibacillus thermophilus is a later heterotypic synonym of Paenibacillus macerans .

During a study on the biodiversity of bacteria that inhabit woody biomass, we isolated a strain coded B40T from hardwood bark used as a compost ingredient in Japan. The strain, characterized as B40T, is a Gram-stain-positive, rod-shaped, non-motile, non-spore-forming and catalase-negative bacterium. This novel isolate showed growth at 30–50 °C, at pH 3.5–7.5 and in the presence of up to 4 % (w/v) NaCl. Its major fatty acids include C16:0, C18:1 ω9c and summed feature 8. The genomic DNA G+C content of strain B40T is 42.2 mol%. Results of 16S rRNA gene sequence-based phylogenetic analysis indicated that strain B40T belongs to the genus Lactobacillus and the closest neighbours of strain B40T are Lactobacillus gigeriorum 202T (95.7 %), Lactobacillus pasteurii CRBIP 24.76T (95.6 %), Lactobacillus psittaci DSM 15354T (95.4 %), Lactobacillus fornicalis TV1018T (95.4 %) and Lactobacillus jensenii ATCC 25258T (95.2 %). The amino acid sequence-based phylogenetic analyses of 489 shared protein-encoding genes showed that the strain forms a phylogenetically independent lineage in the genus Lactobacillus but could not be assigned to any known species. Strain B40T has an average nucleotide identify of Lactobacillus species. Differential genomic, phenotypic and chemotaxonomic properties, in addition to phylogenetic analyses, indicated that strain B40T represents a novel species of the genus Lactobacillus , for which the name Lactobacillus corticis sp. nov. is proposed. The strain type is B40T (=JCM 32597T=DSM 107967T).

Three strains, JCM 5343T, JCM 5344 and JCM 1130, currently identified as Lactobacillus gasseri , were investigated using a polyphasic taxonomic approach. Although these strains shared high 16S rRNA gene sequence similarities with L. gasseri ATCC 33323T (99.9 %), they formed a clade clearly distinct from ATCC 33323T based on whole-genome relatedness. The average nucleotide identity and in silico DNA–DNA hybridization values of these three strains compared to L. gasseri ATCC 33323T were 93.4–93.7 and 53.1–54.1 %, respectively, and both were less than the widely accepted threshold to distinguish two species (95 and 70 %, respectively). The three strains were Gram-stain positive, non-motile, non-spore-forming, catalase-negative and rod-shaped bacteria. They grew at 25–45 °C and in the presence of 2.0 % (w/v) NaCl. The major fatty acids of the three strains were C16 : 0 and C18 : 1 ω9c. Based on phylogenetic analyses of the phenylalanyl-tRNA synthase alpha subunit and RNA polymerase alpha subunit genes, and on phenotypic and chemotaxonomic characteristics, strains JCM 5343T, JCM 5344 and JCM 1130 represent a novel species distinct from L. gasseri , for which we propose the name Lactobacillus paragasseri sp. nov. In addition, a large portion of genomes currently labelled as L. gasseri in the public sequence database should be reclassified as L. paragasseri based on whole-genome relatedness.

A Gram-stain-variable, strictly anaerobic, rod-shaped, catalase-negative and endospore-forming bacterial strain, designated MJC39T, was isolated from grass silage preserved in Hokkaido, Japan. Growth occurred at 20–42 °C, pH 5.0–7.0 and NaCl concentrations up to 2 % (w/v). The isolated strain MJC39T produced butyric acid in peptone yeast extract medium with glucose. The DNA G+C content of strain MJC39T was 34.4±0.2 mol%. The major cellular fatty acids (>10 %) were C14 : 0, C16 : 0 and summed feature 3 (including C16 : 1ω7c/C16 : 1ω6c). No respiratory quinones were detected. The polar lipids of strain MJC39T were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, one unidentified lipid, one unidentified aminolipid, two unidentified glycolipids, one unidentified phospholipid, one unidentified aminoglycolipid and one unidentified phosphoaminoglycolipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MJC39T was a member of the genus Clostridium and is closely related to Clostridium tyrobutyricum JCM 11008T (95.8 % similarity) and Clostridium algifaecis MB9-7T (95.5 % similarity). Based on the genotypic, phenotypic and chemotaxonomic characteristics, strain MJC39T represents a novel species of the genus Clostridium , for which the name Clostridium pabulibutyricum sp. nov. is proposed. The type strain is MJC39T (=JCM 31506T=DSM 103944T).

Three Gram-stain positive, non-motile, non-spore-forming, catalase-negative and rod-shaped bacterial strains (IWT5T, IWT25T and IWT140), isolated from silage, were investigated by using a polyphasic taxonomic approach. Strains IWT5T and IWT25T grew at 10-37 °C and 30-37 °C, and at pH 4.0-7.5 and 4.0-7.0, respectively. The G+C contents of genomic DNA of strains IWT5T and IWT25T were 43.2 and 44.4 mol%, respectively. Strains IWT5T and IWT25T contained C16 : 0, C18 : 1 ω9c and summed feature 7 (unknown 18.846/C19 : 1 ω6c/C19 : 0cyclo ω10c) as the major fatty acids. Strain IWT5T was most closely related to the type strains of Lactobacillus mixtipabuli (99.9 % 16S rRNA gene sequence similarity) and Lactobacillus silagei (99.5 %). For IWT25T, the 16S rRNA gene sequence similarities with the closely related neighbour type strains L. mixtipabuli and L. silagei were 99.5 and 99.5 %, respectively. The 16S rRNA gene sequence similarities among the three novel isolates were 99.5-99.9 %. The average nucleotide identities of strains IWT5T and IWT25T to other neighbours of the genus Lactobacillus were less than 82 % and the genomes of IWT25T and IWT140 shared 97.3 % average nucleotide identity, demonstrating that the three strains were allocated to two different novel species of the genus Lactobacillus. Together with multilocus sequence analysis, phenotypic and chemotaxonomic characteristics, strains IWT5T (=JCM 31144T=DSM 102973T) and IWT25T (=JCM 31145T=DSM 102974T) are proposed as the type strains of novel species of the genus Lactobacillus, with the names Lactobacillus silagincola sp. nov. and Lactobacillus pentosiphilus sp. nov., respectively.

Whole-genome sequencing was performed for Lactobacillus parakefiri JCM 8573T to confirm its hitherto controversial taxonomic position. Here, we report its first reliable reference genome. Genome-wide metrics, such as average nucleotide identity and digital DNA-DNA hybridization, and phylogenomic analysis based on multiple genes supported its taxonomic status as a distinct species in the genus Lactobacillus. The availability of a reliable genome sequence will aid future investigations on the industrial applications of L. parakefiri in functional foods such as kefir grains.

A taxonomic study of a Gram-stain-positive, rod-shaped, non-motile, non-spore-forming, catalase-negative bacterium, strain YK43T, isolated from spent mushroom substrates stored in Nagano, Japan was performed. Growth was detected at 15–45 °C, pH 5.0–8.5, and 0–10 % (w/v) NaCl. The genomic DNA G+C content of strain YK43T was 43.6 mol%. The predominant fatty acids were C16 : 0, C18 : 1 ω9c and summed feature 8. Based on 16S rRNA gene sequence analysis, the type strains of Lactobacillus acidipiscis (sequence similarity, 97.6 %) and Lactobacillus pobuzihii (97.4 %) were most closely related to YK43T. The average nucleotide identities were 74.1 % between strain YK43T and L. acidipiscis DSM 15836T and 74.0 % between YK43T and L. pobuzihii E100301T. Based on a multilocus sequence analysis, comparative genomic analysis and a range of phenotypic and chemotaxonomic characteristics, strain YK43T represents a novel species of the genus Lactobacillus , for which the name Lactobacillus salitolerans sp. nov. is proposed. The type strain is YK43T (=JCM 31331T = DSM 103433T).

To identify the best ensiling process and moisture content for rice grain silage, we determined the effects of additive (i.e. no additive, glucose additive or lactic acid bacteria [LAB] inoculation), crushing process (non-crushing or crushing) and moisture control (none [13.6%], 20.0, 27.5, 35.0, 42.5 or 50.0% moisture content [in fresh matter, FM]) on rough rice and brown rice. Forage rice harvested at the fully ripe stage was ensiled in small-scale experiments, and the silage was stored for 60 days. Regardless of the use of additives or LAB inoculation, the pH values tended to decrease with increasing moisture content. LAB inoculation and crushing resulted in low pH values, low butyric acid contents, low ethanol contents and high lactic acid contents, and the lactic acid content increased to more than 2.0% in FM at moisture contents of 27.5% and higher. The parameters of fermentation quality (pH, lactic acid, butyric acid, volatile basic nitrogen and ethanol content) were affected by not only the main effect of additive, crushing process and moisture control but also two-way or three-way interaction effects. LAB inoculation and crushing improved the lactic acid fermentation under lower moisture conditions, that is, 27.5% moisture content. In both rough rice and brown rice, we suggest that the appropriate pretreatment is the combined use of LAB inoculation, crushing process and controlling 27.5% moisture content.

Whole-genome sequencing was performed for Lactobacillus parakefiri JCM 8573T to confirm its hitherto controversial taxonomic position. Here, we report its first reliable reference genome. Genome-wide metrics, such as average nucleotide identity and digital DNA-DNA hybridization, and phylogenomic analysis based on multiple genes supported its taxonomic status as a distinct species in the genus Lactobacillus. The availability of a reliable genome sequence will aid future investigations on the industrial applications of L. parakefiri in functional foods such as kefir grains.

Paddy rice has been of particular interest as a forage crop in Japan. In this study, the isolated strains TO1000, TO1001, TO1002, and TO1003 were characterized by phenotypic and genotypic approaches. These strains were identified as Lactobacillus plantarum subsp. plantarum by species-specific PCR. Phenotypic characteristics varied among different strains of the same subspecies, and the strains represented unique and diverse phenotypes related to fermentation factors, such as carbohydrate assimilation and range of pH and temperature allowing growth. PCR analysis revealed that the patterns of presence/absence of known plantaricin genes differed in a strain-specific manner. Using these strains as inoculants for preparation of whole crop paddy rice silage, fermentation quality was significantly improved, as shown by lower pH, higher lactic acid content, and inhibition of the growth of undesirable microorganisms such as molds, coliform bacteria, and clostridia, after 30 and 60 days of storage, with effectiveness differing from strain to strain. These observations suggest that suitable candidates for bacterial inoculants in silage preparation should be screened at the strain level. Strain TO1002 may be useful for producing silage inoculants for the production of well-preserved whole crop paddy rice silage.

In 2006, the distribution of bur cucumber (Sicyos angulatus L.) along the river bank of the Abukuma River, Japan, was examined. Twenty-six populations were found in the area surrounding the upper reach of the river and they were limited to dairy land. On the basis of polymerase chain reaction–restriction fragment length polymorphism analysis for four of the six polymorphic loci that were detected in 16 non-coding regions of the chloroplast DNA from 300 samples, five multilocus haplotypes (A–E) were identified. Despite its smaller size, the surrounding area's population showed a higher genetic diversity at the haplotype level than did the riverbank population. The haplotype composition of the surrounding area's population was similar to that of the riverbank population in the upper-stream region. The distribution of haplotype D was biased towards the upper reach of the river, possibly because of migration from the surrounding area's population. The distribution of haplotype E was biased towards the middle-to-lower reaches of the river and was associated with the existence of source populations other than the surrounding area's population. The results also showed that the estimated seed flow from another dairy land could transport seeds into the middle reach of the river at a high frequency, suggesting that dairy land is largely responsible for the development of the riverbank populations. The eradication of dairyland populations should be given priority in order to prevent future migration along the river bank and to protect the existing endangered wetland species from invasive S. angulatus.

We estimated the prevalence of mycotoxin-producing Fusarium fungi and the levels of fumonisin in corn (maize; Zea mays L.) grown and harvested in Tochigi, Japan, in 2009. Fusarium fujikuroi was the species most frequently isolated from ear samples collected after the yellow-ripe stage, and these isolates produced fumonisin in incubation tests on corn grain medium. Other Fusarium species were also isolated. The concentration of fumonisin in corn grown in the field increased abruptly after the dough-ripe stage and kept increasing until the full-ripe stage. The mean concentrations of fumonisin B1 and B2 each exceeded 2000 μg kg–1 fresh matter at the full-ripe stage, and almost all of the fumonisin was found in ears. Fungi capable of producing fumonisin were consistently isolated from ears sampled after the dough-ripe stage; we conclude that these fungi produced fumonisin in ears. Although fumonisin-producing fungi were isolated also from stems and leaves after the dough-ripe stage, concentrations of fumonisin in these tissues were very low and did not increase over time. It appears that fumonisin-producing fungi such as F. fujikuroi, F. incarnatum and F. verticillioides were present in the plants from the dough-ripe stage to full-ripe stage and the conditions for fumonisin production became more favorable in ears as the plants matured.

We assessed fumonisin production during the ensiling of rice grain. Rice grain was harvested at the full-ripe stage and prepared as rough rice, crushed rough rice, brown rice or crushed brown rice. Each material was ensiled under six conditions: (1) no fungus, anaerobic; (2) no fungus, aerobic; (3) water added, anaerobic; (4) water and fumonisin-producing fungus added, anaerobic; (5) water and fumonisin-producing fungus added, aerobic; or (6) fumonisin-producing fungus added to autoclaved material, aerobic. After 40 days of ensilage, we analyzed the silage fermentative quality and fumonisin concentration. The fermentative quality of all materials was good in treatments (3) and (4) (pH 6.5). The fumonisin concentration was low in all materials in treatments (1) to (4), slightly increased in the three materials other than rough rice in treatment (5), and enormously increased in all materials in treatment (6). The results indicate that the fumonisin-producing fungus does not produce fumonisin in anaerobic conditions. It is important that an anaerobic condition be maintained during ensiling in order to reduce the fumonisin content in rice grain silage.

In order to understand the relationship between lactic acid bacteria (LAB) species and silage fermentation, a total of 65 LAB strains isolated from mixed pasture of timothy (Phleum pratense L.) and orchardgrass (Dactylis glomerata L.), and its badly preserved silages were subjected to phenotypic and genetic analysis. According to these analyses, the isolates were divided into 13 groups, including Enterococcus gallinarum, Lactobacillus acidipiscis, L. coryniformis subsp. coryniformis, L. coryniformis subsp. torquens, L. curvatus, L. paraplantarum, L. plantarum subsp. argentoratensis, L. plantarum subsp. plantarum, L. sakei subsp. carnosus, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Leuconostoc pseudomesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus, Weissella hellenica, Weissella paramesenteroides and Carnobacterium divergens. This is the first report to document that C. divergens, L. acidipiscis, L. sakei subsp. carnosus, L. garvieae, phenotypically novel L. lactis subsp. cremoris, E. gallinarum and W. hellenica are present in vegetative forage crops. L. plantarum group strains were most frequently isolated from the badly preserved silages. Some isolates showed a wide range of growth preferences for carbohydrate utilization, optimal growth pH and temperature in vitro, indicating that they have a high growth potential. These results are useful in understanding the diversity of LAB associated with decayed silage of timothy and orchardgrass.

Twenty-three lactic acid bacteria (LAB) isolated from three cultivars (Akiaoba, Nagahahikari and Tachiwase) of Italian ryegrass (Lolium multiflorum Lam.) silage were precisely characterized by a combination of phenotypic tests, genotypic 16S ribosomal DNA sequencing and rapid PCR-based analyses, focusing on their useful phenotypes for silage preparation as inoculants. We successfully identified both at the species and subspecies levels: phenotypically novel Lactococcus lactis subsp. lactis, Lactobacillus brevis, Lactobacillus coryniformis subsp. torquens, Lactobacillus curvatus, Lactobacillus plantarum subsp. plantarum, Lactobacillus sakei subsp. carnosus, Leuconostoc mesenteroides subsp. dextranicum and Pediococcus parvulus. This is the first report to elucidate the presence of Lactobacillus coryniformis ssp. torquens and Leuconostoc mesenteroides subsp. dextranicum in Italian ryegrass silages. Physiological and biochemical tests revealed that phenotypic characteristics are different among the different strains of the same species and subspecies, and that the isolates show unique and diverse phenotypes related to fermentation factors, such as available carbohydrates, optimal growth pH and temperature. These results suggest that, for various well-preserved silage preparations, the isolates may be useful in producing novel inoculants corresponding to their optimally climatic and ecological niches.

We assessed fumonisin production during the ensiling of rice grain. Rice grain was harvested at the full-ripe stage and prepared as rough rice, crushed rough rice, brown rice or crushed brown rice. Each material was ensiled under six conditions: (1) no fungus, anaerobic; (2) no fungus, aerobic; (3) water added, anaerobic; (4) water and fumonisin-producing fungus added, anaerobic; (5) water and fumonisin-producing fungus added, aerobic; or (6) fumonisin-producing fungus added to autoclaved material, aerobic. After 40 days of ensilage, we analyzed the silage fermentative quality and fumonisin concentration. The fermentative quality of all materials was good in treatments (3) and (4) (pH 4), reasonable in treatment (5) (pH = 5∼6) and unacceptable in treatments (1) and (2) (pH 6.5). The fumonisin concentration was low in all materials in treatments (1) to (4), slightly increased in the three materials other than rough rice in treatment (5), and enormously increased in all materials in treatment (6). The results indicate that the fumonisin-producing fungus does not produce fumonisin in anaerobic conditions. It is important that an anaerobic condition be maintained during ensiling in order to reduce the fumonisin content in rice grain silage.

Twenty-three lactic acid bacteria (LAB) isolated from three cultivars (Akiaoba, Nagahahikari and Tachiwase) of Italian ryegrass (Lolium multiflorum Lam.) silage were precisely characterized by a combination of phenotypic tests, genotypic 16S ribosomal DNA sequencing and rapid PCR-based analyses, focusing on their useful phenotypes for silage preparation as inoculants. We successfully identified both at the species and subspecies levels: phenotypically novel Lactococcus lactis subsp. lactis, Lactobacillus brevis, Lactobacillus coryniformis subsp. torquens, Lactobacillus curvatus, Lactobacillus plantarum subsp. plantarum, Lactobacillus sakei subsp. carnosus, Leuconostoc mesenteroides subsp. dextranicum and Pediococcus parvulus. This is the first report to elucidate the presence of Lactobacillus coryniformis ssp. torquens and Leuconostoc mesenteroides subsp. dextranicum in Italian ryegrass silages. Physiological and biochemical tests revealed that phenotypic characteristics are different among the different strains of the same species and subspecies, and that the isolates show unique and diverse phenotypes related to fermentation factors, such as available carbohydrates, optimal growth pH and temperature. These results suggest that, for various well-preserved silage preparations, the isolates may be useful in producing novel inoculants corresponding to their optimally climatic and ecological niches.

NAD(P)H-model compounds that have a sulfur or oxygen substituent in the molecule have been synthesized and kinetics for the reduction of both substituted and unsubstituted α,α,α-trifluoroacetophenones with these model compounds are studied. Reduction with the heteroatom-containing model compounds proceeds at a slower rate than the reduction with a model without any heteroatom substituents. Furthermore, the dependency of the kinetic isotope effect on the electron-deficiency of the substrate differs. Kinetic parameters reveal that the retardation caused by the heteroatom is due to the loss of entropy in the initial electron-transfer process. The enthalpic term favors the reduction with the heteroatom-containing model.

Kinetics on the reduction with an NAD(P)H model bearing a divalent sulfur atom in the N-alkyl substituent has been studied. The sulfur atom retards the electron-transfer process.

1-Propyl-1,4-dihydronicotinamide (PNAH), 1-(3-phenylpropyl)-1,4-dihydronicotinamide (1a), 1-(2-phenylthioethyl)-1,4-dihydronicotinamide (1b), and 1-(4-phenylthiobutyl)-1,4-dihydronicotinamide (1c) and their 4-deuterated analogs are synthesized as NAD(P)H-models and subjected to the reduction of m-nitro-α,α,α-trifluoroacetophenone in the presence or absence of magnesium ion in acetonitrile. Kinetics at 323 K have been studied. Kinetic isotope effect is small (2.33) only when 1a or 1b is employed for the reduction in the presence of magnesium ion, whereas it appears large (≈3.2) under other conditions. The result has been interpreted in terms of steric interference by a proximate phenyl group on the approach of the substrate to form a binary (in the absence of magnesium ion) or ternary (in the presence of magnesium ion) complexes. Thermodynamic parameters for the formation of complexes between the models and magnesium ion have been studied. It has been elucidated that enthalpy of complexation is small for 1...

Effect of water on catalytic activity of magnesim ion in the reduction with an NAD(P)H-model has been studied. It is concluded that small amount of water does not affect the catalytic activity.