Shin Hirayama

CO2 fixation and ethanol production with microalgal photosynthesis and intracellular anaerobic fermentation

Microalgae were screened from seawater. More than 250 strains were isolated, and some of the isolated strains and two strains from culture collections were tested to examine ethanol productivity. Some strains had high growth rate of 20–30 g dry biomass/m2/day and high starch content of more than 20% (dry base). A strain Chlorella vulgaris (IAM C-534) had a high starch content of 37%. Starch was extracted from the cells of the Chlorella, saccharified and fermented with yeasts; 65% of the ethanol-conversion rate was obtained as compared to the theoretical rate from starch. The algal starch proved to be a good source for ethanol production using the conventional process. As an example of another type of ethanol production process, intracellular starch fermentation under dark and anaerobic conditions was examined. All of the tested strains showed intracellular starch degradation and ethanol production, but the levels of ethanol production were significantly different from each other. Higher ethanol productions were obtained with Chlamydomonas reinhardtii (UTEX2247) and Sak-1 isolated from seawater. These showed a maximum ethanol concentration of 1 (w/w)%. The characteristics of intracellular ethanol production were examined with the Chlamydomonas. These results indicate that intracellular ethanol production is simpler and less energy intensive than the conventional ethanol-fermentation process.

Detection of Hydroxyl Radical in Intact Cells of Chlorella Vulgaris

Using ESR with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin-trapping reagent, we measured the levels of free radical species generated from living cells of Chlorella vulgaris var. vulgails (IAM C-534). To investigate the production of free radicals in the living Chlorella vulgaris cells, the influence of DMPO toward the intact cells of the Chlorella vulgaris using the O2 evolution rate was first studied as a guide. Since the O2 evolution rate was not changed by DMPO, it was judged that DMPO has no toxicity toward the intact cells of Chlorella vulgaris. Only hydroxyl radicals (.OH) were detected as the DMPO-OH adduct in the suspension of intact cells of Chlorella vulgaris irradiated with visible light. Moreover, since production of .OH was inhibited by some hydroxyl radical scavengers such as KI and ethanol, production of .OH was proved to be due to hydroxyl radicals. It was also clear that the intensity of .OH increased with increasing irradiation intensity of visible light. Therefore, it was suggested that .OH might be one of the photoinhibition factors of the intact Chlorella vulgaris cells in severe light conditions.

Effect of hydroxyl radical on intact microalgal photosynthesis

Abstract Photosynthetic CO 2 fixation with microalgae for new energy and chemical sources is one of the potential method to mitigate CO 2 emission. To gain the more productivities for CO 2 mitigation, the enhancement of photosynthetic productivity is required. We focused on the active oxygen that is supposed to be produced in algal cells and causes harmful effects on photosynthesis under high irradiation that is the case of outdoor cultivation circumstances. In this report, we have challenged to detect the active oxygens in microalgal cells, and then the results were described. The levels of free radical species generated in the living cells of Chlorella vulgaris var. vulgaris (IAM C-534) were detected with electron spin resonance (ESR) spectrometer with 5,5 dimethyl-1-pyrroline N-oxide (DMPO) as a spin-trapping reagent. Before detecting the free radicals in the living Chlorella cells, the influence of DMPO concentration toward the photosynthetic growth rate of the cells was measured. Since the growth rate was not influenced by up to 130 mM DMPO, the DMPO concentration was adjusted to 90 mM during the measurement. Only one DMPO adduct, which is assigned as the hydroxyl radical (DMPO-OH; aN=1.49mT, aH=1.49mT) was detected in the solution of intact cells of Chlorella vulgaris irradiated with visible light (1). Moreover, the production of DMPO-OH adducts was inhibited by some hydroxyl radical ( − OH) scavengers such as KI and ethanol. It has been estimated that − OH is one of the photoinhibition factors of the photosynthetic organisms. But, − OH has never been detected in vivo. In this report, − OH was detected in vivo, and the − OH was increased according to the light intensity.

Production of optically pure D-lactic acid by Nannochlorum sp. 26A4.

Microalgae were screened from seawater for greenhouse gas CO2 fixation and d-lactic acid production by self-fermentation and tested for their growth rate, starch content, and conversion rate from starch into d-lactic acid. More than 300 strains were isolated, and some of them were found to have suitable properties for this purpose. One of the best strains, Nannochlorum, sp. 26A4, which was isolated from Sakito Island, had a starch content of 40% (dry weight), and a conversion rate from consumed starch into d-lactic acid of 70% in the dark under anaerobic conditions. The produced d-lactic acid showed a high optical purity compared with the conventional one. The proposed new d-lactic acid production system using Nannochlorum sp. 26A4 should also be an effective technology for greenhouse gas CO2 fixation and/or conversion into industrial raw materials.

Evaluation of Active Oxygen Effect on Photosynthesis of Chlorella vulgaris

The relationship between O2 and an active oxygen scavenging system in Chlorella vulgaris var.vulgaris (IAM C-534) was investigated. When Chlorella vulgaris was exposed to 2% O2, only traces of active oxygen scavenging enzymes were found. When the Chlorella vulgaris was treated with 20% or 50% O2, it was shown that the level of enzyme activity increased as the O2 concentration increased. An increase in enzyme activity was not found in any specific enzyme but in all of the enzymes, but the level of glutathione and ascorbate remained the same in all the cases. In addition, the photosynthetic efficiency also decreased as the concentration of O2 was increased. These results suggest that an O2 enriched environment can lead to an increase in the production of active oxygen species such as O2.- and H2O2 and to a decrease in the photosynthetic efficiency in Chlorella vulgaris. The hydroxyl radical (.OH) was detected directly in the Chlorella vulgaris suspension with a spin trapping reagent. It was also clear that the increase in the .OH intensity as the visible light intensity increased was unrelated to the O2 concentration. It was suggested that the conditions for producting .OH and the other active oxygen species were different, and that two types of oxygen stress should exist in the Chlorella vulgaris.

Functional sulfur amino acid production and seawater remediation system by sterile Ulva sp. (Chlorophyta).

Sterile Ulva, which is a macroalga, has the potential to grow stably; therefore, this seaweed is expected to be an efficient resource of functional food containing various nutrients such as sulfur amino acids, proteins, carbohydrates, and minerals. Ulva lactuca was selected from the “Marine Park” in Tokyo Bay, and its growth rate (g-dry/[m2·d]) was measured using model reactors located on the land or on the surface of the sea at Yokohama. The growth rate of U. lactuca was recorded to be approx 20 g-dry/(m2·d), which is estimated to be 10 times greater than that in a natural field in the Marine Park. In addition, this growth rate was higher than that of conventional crops such as corn and rice on a farm or paddy. These data led us to newly design and propose a floating type of labor-efficient U. lactuca production system. d-Cysteinolic acid, which is included in U. lactuca as a major sulfur amino acid, inhibited the Fenton reaction, resulting in suppression of hydroxyl radical production and singlet oxygen. Addition of the sulfur amino acid (1µM) to HepG2 cells markedly decreased the intracellular triglyceride level. Hence, this proposed facility also has the potential for industrial production of a valuable resource for the primary prevention of lifestyle-related diseases using enriched or eutrophied seawater.