Hitoshi Miyasaka

Stably sustained hydrogen production by biophotolysis in natural day/night cycle

Our photobiological hydrogen production system was successfully scaled up to a pilot plant scale in the photosynthetic starch accumulation and the fermentative production of organic compounds from starch by Chlamydomonas MGA 161 and the hydrogen photoproduction by Rhodovulum sulfidophilum W-1S from organic compounds produced by Chlamydomonas MGA 161 in the natural day/night cycle. The conversion yield of organic compounds from starch of Chlamydomonas MGA 161 was 80 to 100 % of the theoretical yield, but the molar yield of hydrogen photoproduction of Rv. sulfidophilum W-1S was approximately 40 %. One main cause for the low hydrogen yield of Rv. sulfidophilum W-1S was a competition between the accumulation of intracellular Poly (3-hydroxybutyric acid), PHB, and the hydrogen production under light anaerobic conditions. The PHB accumulation was repressed and the hydrogen photoproduction was promoted by enhancing the nitrogenase activity of Rv. sulfidophilum W-1S.

Regulation of ferruginol and cryptotanshinone biosynthesis in cell suspension cultures of Salvia miltiorrhiza

Abstract Ferruginol was produced only in the lag phase in habituated cell suspension cultures of Salvia miltiorrhiza, while it was produced in both lag and stationary phases by 2,4-dichlorophenoxyacetic acid-requiring cultures. On addition of sucrose to habituated cultures, in the stationary phase, production of ferruginol was restored, indicating that habituated cultures still retained the capacity to produce ferruginol. A two-stage culture method, with normal medium and then medium without Fe-EDTA, for ferruginol production was established. This culture method was also found to be suitable for production of cryptotanshinone, a clinically active principle in this plant.

Excretion of glycerol by the marine Chlamydomonas sp. strain W-80 in high CO2 cultures☆

Abstract A Chlamydomonas sp. strain, which excretes glycerol as the major carbon compound, was isolated and designated as strain W-80. The glycerol production was much enhanced by a high concentration of CO2, and the final concentration of the product was measured at levels up to 24.9 mM (2.29 g/l).

Production of ferruginol by cell suspension cultures of Salvia miltiorrhiza

Abstract The time-course production of ferruginol, a diterpene, was examined during growth of Salvia miltiorrhiza in cell culture. Ferruginol was produced in the lag and stationary phases of growth and so production of this secondary metabolite was inversely related to active cell division. The effects of auxins and light on ferruginol production were also examined.

Production of cryptotanshinone and ferruginol by immobilized cultured cells of Salvia miltiorrhiza

Abstract The production of the diterpenes cryptotanshinone and ferruginol by immobilized cultured cells of Salvia miltiorrhiza was examined. Cryptotanshinone and ferruginol were produced continuously by the immobilized cells. Much of the cryptotanshinone was released into the medium, while most of the ferruginol was retained in the cells. The production of cryptotanshinone and ferruginol by the immobilized cells was about 39% and 61% of those by cell suspensions. Re-use of the immobilized cells for the production of these compounds was also examined.

Application of fluorescent protein-tagged trans factors and immobilized cis elements to monitoring of toxic metals based on in vitro protein-DNA interactions.

Environmental toxic metals cause serious global public health problems. On-site monitoring protects people from exposure to such harmful elements. In this study, the bacterial transcriptional switches were applied to monitoring of toxic metals. ArsR and CadC, trans factors of Escherichia coli and Staphylococcus aureus, were fused to GFP. The fusion proteins, ArsR-GFP and CadC-GFP, associated with cis elements, P(ars)-O(ars) and P(cad)-O(cad), respectively and dissociated from those upon recognition of As(III) or Pb/Cd. Cell lysates containing ArsR-GFP were pre-incubated with As(III) standard solutions for 15 min and loaded into P(ars)-O(ars)-immobilized microplate wells. Cell lysates containing CadC-GFP were pre-incubated with Pb or Cd solutions and loaded into P(cad)-O(cad)-immobilized wells. The cell lysates were incubated for 15 min and removed from the wells. Fluorescence intensity in the wells dose-dependently decreased in response to As(III) up to 200 μg/l or Pb/Cd up to 100 μg/l. Detection limits were 10 μg/l for As(III) 10 μg/l for Cd, and 20 μg/l for Pb with a microplate fluororeader, whereas 5.0 μg/l for As(III), 1.0 μg/l for Cd, and 10 μg/l for Pb with a handheld fluorometer. This method was available to detect Pb/Cd or As(III) in water containing soil extracts. This is the first demonstration of a simple and rapid fluorometry to detect analytes based on in vitro interaction between a cis element and a trans factor.

Influence of Sulfate-Reducing Bacteria on Outdoor Hydrogen Production by Photosynthetic Bacterium with Seawater

Abstract. The application of seawater for bacterial fermentative production is a cost-effective technology. Hydrogen production by marine photosynthetic bacterium with seawater failed to continue after more than 10 days, and was accompanied by the formation of hydrogen sulfide and a change in culture color from red to black. However, substrate consumption in the blackish culture was comparable to that in a hydrogen-producing culture. A decrease in hydrogen production occurred upon the addition of sodium sulfide at concentrations of 1.5 mM or higher. PCR analysis targeted at the 16S rDNA sequence selective for sulfate-reducing bacteria revealed the existence of sulfate-reducing bacteria in inoculation cultures of the phototrophic bacterium and medium for hydrogen production. Hence, the high sulfate concentration of seawater, the low oxidation-reduction potential under hydrogen-producing conditions, and the presence of electron donors such as acetate might promote the metabolic activities of sulfate-reducing bacteria, resulting in the deterioration of hydrogen production with seawater.

Effect of nutritional factors on cryptotanshinone and ferruginol production by cell suspension cultures of Salvia miltiorrhiza

Abstract The effects of the components of Murashige-Skoog (MS) medium on the production of the diterpenes cryptotanshinone and ferruginol in cell suspension cultures of Salvia miltiorrhiza were examined. Sucrose, a nitrogen source and thiamine were needed for the production of these compounds, and phosphate, MnSO 4 and kinetin showed slight beneficial effects. All the other components of MS medium were found to be either unnecessary or inhibitory for the production of these compounds, when tested separately. A revised medium for the production of cryptotanshinone, a clinically active principle in this plant, was designed.

Unusual Accumulation of Demethylspheroidene in Anaerobic-Phototrophic Growth of crtA-Deleted Mutants of Rhodovulum sulfidophilum

Rhodovulum sulfidophilum produces carotenoids in the spheroidene pathway. Spheroidene monooxygenase, CrtA, catalyzes the conversion of spheroidene to spheroidenone. crtA-deleted mutants of R. sulfidophilum did not produce spheroidenone and demethylspheroidenone. In these mutants, the ratio of demethylspheroidene to spheroidene increased with exposure to light. One mutant exhibiting a spheroidene-predominant phenotype did not grow under anaerobic-light conditions and was devoid of bacteriochlorophyll a, even under semiaerobic-light conditions There was no difference in the growth of the mutants under aerobic-dark conditions. These data suggest that demethylspheroidene is important for photosynthesis in R. sulfidophilum.

Enhancement of starch degradation by CO2 in a marine green alga, Chlamydomonas sp. MGA161

Abstract Algal starch photosynthetically accumulated in Chlamydomonas sp. MGA161 was metabolized to low-molecular-weight compounds—H 2 , ethanol, glycerol, and acetate—under an O 2 -free CO 2 atmosphere. When cells in the logarithmic growth phase was fermented under CO 2 -anaerobic conditions, starch degradation in the alga was enhanced to 1.4-fold that in N 2 -anaerobic fermentation. Due to the inhibition of hydrogenase under CO 2 , H 2 and acetate decreased, while ethanol and glycerol increased, when the atmosphere was changed from N 2 to CO 2 . Addition of CO, an inhibitor of hydrogenase, also stimulated starch degradation and changed the excretion pattern of the fermentation products.