Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-activated cell sorting.

Euglena gracilis, a microalgal species of unicellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (ß-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions-a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich E. gracilis with fluorescence-activated cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intracellular lipids with BODIPY(505/515), and FACS-based isolation of top 0.5% lipid-rich E. gracilis cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich E. gracilis mutant strain, named B1ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel.

Production of a thermal stress resistant mutant Euglena gracilis strain using Fe-ion beam irradiation.

Euglena gracilis is a common phytoplankton species, which also has motile flagellate characteristics. Recent research and development has enabled the industrial use of E. gracilis and selective breeding of this species is expected to further expand its application. However, the production of E. gracilis nuclear mutants is difficult because of the robustness of its genome. To establish an efficient mutation induction procedure for E. gracilis, we employed Fe-ion beam irradiation in the RIKEN RI beam factory. A decrease in the survival rate was observed with the increase in irradiation dose, and the upper limit used for E. gracilis selective breeding was around 50 Gy. For a practical trial of Fe-ion irradiation, we conducted a screening to isolate high-temperature-tolerant mutants. The screening yielded mutants that proliferated faster than the wild-type strain at 32 °C. Our results demonstrate the effectiveness of heavy-ion irradiation on E. gracilis selective breeding.

Selection and characterization of Euglena anabaena var. minor as a new candidate Euglena species for industrial application.

Euglena gracilis is a microalgae used as a model organism. Recently, mass cultivation of this species has been achieved for industrial applications. The genus Euglena includes more than 200 species that share common useful features, but the potential industrial applications of other Euglena species have not been evaluated. Thus, we conducted a pilot screening study to identify other species that proliferate at a sufficiently rapid rate to be used for mass cultivation; we found that Euglena anabaena var. minor had a rapid growth rate. In addition, its cells accumulated more than 40% weight of carbohydrate, most of which is considered to be a euglenoid specific type of beta-1-3-glucan, paramylon. Carbohydrate is stored in E. anabaena var. minor cells during normal culture, whereas E. gracilis requires nitrogen limitation to facilitate paramylon accumulation. These results suggest the potential industrial application of E. anabaena var. minor.

Spongitalea numazuensis gen. nov., sp. nov., a new marine bacterium of the family Flavobacteriaceae isolated from an unidentified marine sponge.

Polyphasic taxonomic investigation was conducted on a strictly aerobic, Gram-negative, non-motile, pale-yellow-pigmented, rod-shaped strain designated HJ24(T) which was isolated from an unidentified orange-colored marine sponge. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the novel isolate could be affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (88.6%) with Pseudozobellia thermophila KMM 3531(T). The novel isolate is phenotypically and physiologically different from other related genera. The DNA G+C contents were 41 mol%, MK-6 was the major menaquinone and iso-C15:0, iso-C15:1 and iso-C17:0 3-OH were the predominant fatty acids. On the basis of polyphasic taxonomic studies, it was concluded that the novel strain represents a new genus and a novel species of the family Flavobacteriaceae. We propose the name Spongitalea numazuensis gen. nov., sp. nov. for this strain; its type strain is HJ24(T) (= DSM 21243(T)).

Oral administration of paramylon, a beta-1,3-D-glucan isolated from Euglena gracilis Z inhibits development of atopic dermatitis-like skin lesions in NC/Nga mice.

Paramylon is a beta-1,3-D-glucan isolated from Euglena gracilis Z. This study was designed to evaluate the suppressive effects of the oral administration of paramylon on the development of atopic dermatitis (AD)-like skin lesions induced by repeated application of 2,4,6-trinitrochlorobenzene (TNCB) in sensitized NC/Nga mice. The effects of paramylon were assessed by measuring macroscopical and histopathological findings of skin, ear swelling, serum levels of total IgE, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) and IL-18 and IL-12 contents in the skin lesions. Oral administration of paramylon inhibited the development of AD-like skin lesions as exemplified by a significant decrease in dermatitis scores for the back, ear swelling and hypertrophy of the skin, infiltration of inflammatory cells in the skin, and serum IgE levels. Oral administration of paramylon reduced serum levels of both IL-4 and IFN-gamma and IL-18 and IL-12 contents in the skin lesions. Oral administration of paramylon did not cause weight loss, as was observed with prednisolone. These results suggest that paramylon inhibits the development of AD-like skin lesions in NC/Nga mice by suppressing both the T-helper (Th) 1 and Th 2 cell responses. Our results indicate that paramylon treatment could provide an effective alternative therapy for the management of AD.

Hepatoprotective effects of paramylon, a beta-1, 3-D-glucan isolated from Euglena gracilis Z, on acute liver injury induced by carbon tetrachloride in rats.

Paramylon is a beta-(1-3)-D-glucan isolated from Euglena gracilis Z. This study was designed to evaluate the protective effects of paramylon on liver injury induced by carbon tetrachloride (CCl(4)) in rats. Wistar stain male rats were orally administered paramylon (500, 1,000 and 2,000 mg/kg body weight) before treatment with a single intraperitoneal dose of 50% CCl(4) (2 ml/kg body weight). The rats were sacrificed 24 hr later, and blood samples were collected for assay of serum biochemical parameters. The livers were excised to evaluate the activity of antioxidant enzymes. Histopathological examination of the livers was also performed. The results showed that the treatment of paramylon prevented elevation of the serum levels of hepatic enzyme markers and inhibited fatty degeneration and hepatic necrosis induced by CCl(4). Pre-administration of paramylon reduced the liver apoptotic index. The treatment of paramylon recovered reductions of activity of hepatic superoxide dismutase, catalase and glutathione peroxidase induced by CCl(4). These results demonstrate that paramylon exhibits protective action on acute hepatic injury induced by CCl(4) via an antioxidative mechanism. To the best of our knowledge, this is the first report of a hepatoprotective effect based on the antioxidative action of paramylon.

Leptobacterium flavescens gen. nov., sp. nov., a marine member of the family Flavobacteriaceae, isolated from marine sponge and seawater.

Six obligately aerobic, Gram-negative, non-motile, pale-yellow-pigmented, rod-shaped bacterial strains, designated YM3-301(T), HG868, 04PA2 Co4-8B, 04PA2 Co4-99A, 04PA2 018SW-3 and 04PA2 018SW-18, were isolated from a marine sponge and seawater and were subjected to a polyphasic taxonomic investigation. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the novel isolates were affiliated with the family Flavobacteriaceae (phylum Bacteroidetes) and that they showed the highest sequence similarity (90.3-90.9 %) to members of the genus Leeuwenhoekiella and to Zhouia amylotica. The 16S rRNA gene sequence similarities with respect to members of other related genera were only 86.3-89.1 %. In contrast, the six isolates shared high levels of 16S rRNA gene sequence similarity (99.7-100 %) and DNA-DNA relatedness (72-94 %) with each other. The novel isolates were phenotypically and physiologically different from members of related genera. The G+C content of the DNA was 41.8-43.5 mol%, MK-6 was the major menaquinone and i15 : 0, i15 : 1 and i17 : 0 3-OH were the major fatty acids. On the basis of the data from the taxonomic studies, it was concluded that these six novel strains represent a novel genus and species of the family Flavobacteriaceae, for which the name Leptobacterium flavescens gen. nov., sp. nov. is proposed. The type strain of Leptobacterium flavescens is YM3-301(T) (=KCTC 22160(T)=MBIC 06275(T)=NBRC 104141(T)).