Naoto Urano

Sphingomonas cloacae sp. nov., a nonylphenol-degrading bacterium isolated from wastewater of a sewage-treatment plant in Tokyo

A nonylphenol-assimilating bacterium isolated at a sewage-treatment plant in Tokyo was studied phenotypically, genotypically and phylogenetically. Analysis of the 16S rDNA sequence, the G+C content of the DNA (63 mol%) and the isoprenoid quinone composition, as well as the presence of sphingoglycolipid and the whole-cell fatty acid profile, revealed that the isolate is a member of the genus Sphingomonas. However, the sequence similarity of the 16S rDNA with that of known Sphingomonas spp. was found to be at most 96%, implying that the isolate is distinctive. Furthermore, the results of DNA-DNA hybridization experiments and its physiological characteristics clearly indicated that the isolate represents a new Sphingomonas species, for which the name Sphingomonas cloacae is proposed; strain S-3T (= JCM 10874T = IAM 14885) is the type strain.

Hydrogen production from glucose by immobilized growing cells ofClostridium butyricum

SummaryClostridium butyricum was immobilized in a porous carrier (acetylcellulose filter) with agar. Addition of peptone to the reaction mixture increased the hydrogen productivity from glucose. The number of cells in the agaracetylcellulose increased during incubation in the medium containing glucose and peptone, and the immobilized growing cells converted 45% of the glucose to hydrogen. Riboflavin enhanced the hydrogen productivity and the lactate produced by the native cells decreased remarkably. Therefore, the immobilized whole cells incubated with riboflavin were employed for repeated hydrogen production in the medium containing glucose and peptone. The hydrogen productivity of the immobilized cells increased markedly after repeated use, and the immobilized cells produced hydrogen in stoichiometric amounts from glucose.

Effect of osmotic stabilizers on protoplast generation of Chlorella ellipsoidea yellow/white color mutants

The effect of osmotic stabilizers on protoplast formation of yellow/white color mutants of Chlorella ellipsoidea was studied. The UF-1 strain, one of the mutants, had the same growth rate as the parent C. ellipsoidea at 4.02 microE/m2/s and no reversion to normal cells at 0-26.8 microE/m2/s. Therefore, protoplast formation from UF-1 was carried out, and it was found that the yield of protoplasts was 93% in the medium containing 1.0 M NaCl as an osmotic stabilizer.

Cloning and expression of the hydrogenase gene from Clostridium butyricum in Escherichia coli

Hydrogenase gene from Clostridium butyricum was cloned in Escherichia coli HK16 (Hyd−) using pBR322 and PstI. The plasmid, pCBH1, containing hydrogenase gene was 7.3 MDa and pCBH1 had 5 PstI‐DNA fragments (3.9, 2.6, 0.7, 0.03–0.04, <0.02 MDa, respectively). The hydrogenase activity of HK16 (pCBH1) was about 3.1–3.5‐times as high as those of the present strains, such as C. butyricum and E. coli C600 (Hyd+).

Molecular phylogeny and phenotypic variation in the heterotrophic green algal genus Prototheca (Trebouxiophyceae, chlorophyta)

Species of the heterotrophic green microalgal genus Prototheca and related taxa were phylogenetically analyzed based on the nuclear small subunit (SSU) and the 5′ end of the large subunit (LSU) rRNA gene (rDNA) sequences. We propose restricting the genus Prototheca to the four species: P. moriformis Krüger, P. stagnora (Cooke) Pore, P. ulmea Pore, and P. zopfii Krüger. The main diagnostic feature of these taxa is the absence of growth on trehalose.Of these, it was suggested that P. moriformis should be merged into P. zopfii; P. moriformis and three varieties of P. zopfii constituted a paraphyletic assemblage with estimated short evolutionary distances. The trehalose‐assimilating strains (Prototheca wickerhamii Tubaki et Soneda strains and Auxenochlorella protothecoides (Krüger) Kalina et Punčochářová SAG 211‐7a), together with an invertebrate pathogen Helicosporidium sp., diverged before the radiation of the four species of Prototheca in the SSU rDNA and composite (SSU rDNA plus LSU rDNA) analyses. Comparison between the results from physiological data in this work (fermentative pattern) and those described earlier (growth requirements) lead us to propose a hypothesis that the phenotypic variation, which did not represent diagnostic characters for species delimitation, may reflect the history of genetic diversification within the genus Prototheca as inferred from rDNA sequence characters.

Optimization of heterotrophic culture conditions for n-alkane utilization and phylogenetic position based on the 18S rDNA sequence of a thermotolerant Prototheca zopfii strain

This study reports on the optimization of the culture conditions for a thermotolerant eukaryotic algal strain, Prototheca zopfii RND16, which can effectively degrade and assimilate n-alkanes at elevated temperatures. RND16 was able to grow on 1% (v/v) n-alkanes (C14-C17) at temperatures up to 38 degrees C. This ability differs from a previous finding that P. zopfii did not grow on hydrocarbons under temperature conditions above 25 degrees C. Increasing the temperature from 25 degrees C to 30-35 degrees C resulted in an increase in the rate of n-alkanes consumption during growth of RND16 in quiescent culture. In shaking culture at 35 degrees C, RND16 removed a 1% n-alkanes mixture almost completely at the basal medium salinity within 8 d. However, an appreciable decrease in the extent of hydrocarbon utilization was observed with increasing salinity and substrate concentration in the medium. The slow consumption of the n-alkanes in the early stage of degradation at 25 degrees C was improved by supplementing 0.1% glucose. A comparative study on the nuclear small subunit rDNA (18S rDNA) sequences of three strains within the genus Prototheca revealed that both P. zopfii ATCC 30253, which utilize n-alkanes at room temperature and P. moriformis ATCC 50081, which does not assimilate n-alkanes, are closely related to RND16.

Conversion of a non-flocculent brewer's yeast to flocculent ones by electrofusion: 1. Identification and characterization of the fusants by pulsed field gel electrophoresis

Abstract Conversion of non-flocculent brewers yeasts to flocculent ones was carried out by electrofusion using Saccharomyces cerevisiae (FLO5) , and eight of the fusants obtained were characterized by pulsed field gel electrophoresis. Flocculation activity of fusants was various and intermediate between that of the parent cells [Grade 0: a brewers yeast, Grade 1: F(18) and F(39), Grade 2: F(26), F(59), F(100) and F(107), Grade 3: F(74), Grade 4: F(80), Grade 5: S. cerevisiae ]. The electrophoresis showed that DNA patterns of all fusants were closely related to those of a brewers yeast except for some differences. Inclination of chromosomal DNA in the fusants to the brewers yeast was F(18) > F(100), F(107), F(59) > F(39) > F(26) > F(74) > F(80) and there was a good correlation with the results of flocculation activities in the fusants.

Occurrence of fibrillar collagen with structure of (α1)2α2 in the test of sea urchin Asthenosoma ijimai

Test collagen from the sea urchin Asthenosoma ijimai was disaggregated into collagen fibrils in a neutral salt solution containing β-mercaptoethanol, solubilized by limited pepsin digestion and isolated by selective precipitation with NaCl. The test collagen was mostly rendered soluble and found to consist of two distinct α-chains, α1 and α2, which could be isolated by CM-cellulose chromatography followed by Sepharose CLAB gel filtration. Each of the α-chains had a molecular mass of about 100 kDa and the α1- and α2-chains existed in a molar ratio of about 2:1, indicating the occurrence of (α1), α2 heterotrimers. This was confirmed by amino acid analyses of the isolated a-chains. From these results, the fibrillar collagen of sea urchin test appears to share common characteristics, such as precipitation properties with NaCl, chromatographic and electrophoretic behaviours and subunit composition with vertebrate type-1 collagen. This type of fibrillar collagen was also found in other species of sea urchins.

Conversion of a non-flocculent brewer's yeast to flocculent ones by electrofusion: 2. Small-scale brewing by fusants

Abstract A non-flocculent brewers yeast was converted to flocculent ones by electrofusion using Saccharomyces cerevisiae (FL05) , and the fusants were characterized from chromosomal DNA (Urano et al., 1993). In this study, small-scale brewing (2 1) by the fusants was carried out and DNA patterns compared with microbial quality. The brewing reaction rate, number of floating and sedimenting yeasts in the fusants were varied and intermediate between those of parent strains. Physiological activities of the fusants, from viability to ploidy, were also investigated after brewing. With some exceptions, the fusants showed a good relationship between DNA patterns and brewing and F(100) was closely related to a commercial strain of brewers yeast.

Synergetic effects of cell immobilization in polyurethane foam and use of thermotolerant strain on degradation of mixed hydrocarbon substrate by Prototheca zopfii

The synergetic effects of an immobilized cell system in an oleophilic polyurethane foam (PUF) and use of a thermotolerant strain on degradation of hydrocarbons by colorless green microalga Prototheca zopfii are reported. Two strains of P. zopfii, i.e. thermotolerant RND 16 and non-thermotolerant ATCC30253, were immobilized in PUF to compare their abilities to biodegrade a mixed hydrocarbon substrate (MHS) containing aliphatic and polycyclic aromatic hydrocarbons (PAHs), as a function of temperature. The thermotolerant strain RND 16 degraded MHS at 35°C, while ATCC30253 did not degrade hydrocarbons at temperatures higher than 30°C. Immobilization of P. zopfii in PUF resulted in shortened lag for growth-associated biodegradation of n-alkanes in MHS, the effect of which was most significant in cultures of RND 16 at 25°C. Nevertheless, the decrease in the amount of degraded PAHs was caused by PUF immobilization and the level of this decrease was marked in the cultures of RND 16, in which rate and extent of n-alkane degradation were higher than for ATCC30253.