Kazumasa Hirata

Biological elimination of nitric oxide and carbon dioxide from flue gas by marine microalga NOA-113 cultivated in a long tubular photobioreactor

Nitric oxide (NO) and carbon dioxide (CO2) were simultaneously eliminated from a model flue gas using a marine microalga, strain NOA-113, cultivated in a long tubular photobioreactor. About 40 mg of NO and 3.5 g of CO2 were eliminated per day in a 4-l reactor column with aeration of 300 ppm (v/v) NO and 15% (v/v) CO2 in N2 at a rate of 150 ml/min. This reactor system is thought to be suitable for evaluating NO elimination by microalgae. The effects of NO concentration, gas flow rate, and light conditions on NO elimination were investigated using this system.

Enhancement of tolerance to heavy metals and oxidative stress in Dunaliella tertiolecta by Zn-induced phytochelatin synthesis

The synthesis of phytochelatins (PCs) in a marine alga, Dunalliela tertiolecta, is strongly induced by Zn. Pretreatment of the cells with Zn enhances the tolerance toward toxic heavy metals such as Cd, Hg, Cu, Pb, and arsenate. Moreover, the pretreatment also increases the tolerance toward oxidative stress caused by hydrogen peroxide or paraquat. In vitro analysis shows that PC is a stronger scavenger of hydrogen peroxide and superoxide radical than glutathione. These results suggest that PCs inducibly synthesized by Zn treatment could play a role not only in detoxification of heavy metals but also in mitigation of oxidative stress.

High throughput and exhaustive analysis of diverse lipids by using supercritical fluid chromatography-mass spectrometry for metabolomics.

We have developed an analytical system that enables the simultaneous rapid analysis of lipids with varied structures and polarities through the use of supercritical fluid chromatography-mass spectrometry (SFC-MS). The separation conditions for SFC (column, modifier, back pressure, etc.) and the detection conditions for mass spectrometry (ionization method, parameters, etc.) were investigated to develop a simultaneous analytical method for lipid mixtures that included phospholipids, glycolipids, neutral lipids, and sphingolipids. When cyanopropylated silica gel-packed column was used for the separation, all lipids were successfully detected and the analysis time was less than 15 min. The use of an octadecylsilylated column resulted in separation, which was dependent on the differences in the unsaturation of the fatty acid side chains and isomer separation. This system is a powerful tool for studies on lipid metabolomics because it is useful not only as a fingerprinting method for the screening of diverse lipids but also for the detailed profiling of individual components.

Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes

Significance We found that individual cytochrome P450 (CYP) metabolism capacity and drug sensitivity could be predicted by examining them in the primary human hepatocytes–human induced pluripotent stem cells–hepatocyte-like cells (PHH-iPS-HLCs). We also confirmed that interindividual differences of CYP metabolism capacity and drug responsiveness that are due to the diversity of individual single nucleotide polymorphisms in the CYP gene could also be reproduced in the PHH-iPS-HLCs. These findings suggest that interindividual differences in drug metabolism capacity and drug response could be predicted by using HLCs differentiated from human iPS cells. We believe that iPS-HLCs would be a powerful technology not only for accurate and efficient drug development, but also for personalized drug therapy. Interindividual differences in hepatic metabolism, which are mainly due to genetic polymorphism in its gene, have a large influence on individual drug efficacy and adverse reaction. Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells have the potential to predict interindividual differences in drug metabolism capacity and drug response. However, it remains uncertain whether human iPSC-derived HLCs can reproduce the interindividual difference in hepatic metabolism and drug response. We found that cytochrome P450 (CYP) metabolism capacity and drug responsiveness of the primary human hepatocytes (PHH)-iPS-HLCs were highly correlated with those of PHHs, suggesting that the PHH-iPS-HLCs retained donor-specific CYP metabolism capacity and drug responsiveness. We also demonstrated that the interindividual differences, which are due to the diversity of individual SNPs in the CYP gene, could also be reproduced in PHH-iPS-HLCs. We succeeded in establishing, to our knowledge, the first PHH-iPS-HLC panel that reflects the interindividual differences of hepatic drug-metabolizing capacity and drug responsiveness.

Enhanced Accumulation of Cd2+ by a Mesorhizobium sp. Transformed with a Gene from Arabidopsis thaliana Coding for Phytochelatin Synthase

ABSTRACT We expressed the Arabidopsis thaliana gene for phytochelatin synthase (PCSAt) in Mesorhizobium huakuii subsp. rengei B3, a microsymbiont of Astragalus sinicus, a legume used as manure. The PCSAt gene was expressed under the control of the nifH promoter, which regulates the nodule-specific expression of the nifH gene. The expression of the PCSAt gene was demonstrated in free-living cells under low-oxygen conditions. Phytochelatin synthase (PCS) was expressed and catalyzed the synthesis of phytochelatins [(γ-Glu-Cys)n-Gly; PCs] in strain B3. A range of PCs, with values of n from 2 to 7, was synthesized by cells that expressed the PCSAt gene, whereas no PCs were found in control cells that harbored the empty plasmid. The presence of CdCl2 activated PCS and induced the synthesis of substantial amounts of PCs. Cells that contained PCs accumulated 36 nmol of Cd2+/mg (dry weight) of cells. The expression of the PCSAt gene in M. huakuii subsp. rengei B3 increased the ability of cells to bind Cd2+ approximately 9- to 19-fold. The PCS protein was detected by immunostaining bacteroids of mature nodules of A. sinicus containing the PCSAt gene. When recombinant M. huakuii subsp. rengei B3 established the symbiotic relationship with A. sinicus, the symbionts increased Cd2+ accumulation in nodules 1.5-fold.

H2 production from algal biomass by a mixed culture of Rhodobium marinum A-501 and Lactobacillus amylovorus

To produce hydrogen from starch accumulated in an algal biomass, we used a mixed culture of the lactic acid bacterium, Lactobacillus amylovorus, and the photosynthetic bacterium, Rhodobium marinum A-501. In this system L. amylovorus, which possesses amylase activity, utilized algal starch for lactic acid production, and R. marinum A-501 produced hydrogen in the presence of light using lactic acid as an electron donor. Algal starch accumulated in the marine green alga Dunaliella tertiolecta, and the freshwater green alga Chlamydomonas reinhardtii, was more suitable for lactic acid fermentation by L. amylovorus than an authentic starch sample. Consequently, the yields of hydrogen obtained from starch contained in D. tertiolecta and C. reinhardtii were 61% and 52%, respectively, in the mixed culture of L. amylovorus and R. marinum A-501. These values were markedly superior to those obtained using a mixed culture of Vibrio fluvialis T-522 and R. marinum A-501 described previously. The yield and production rate of hydrogen by R. marinum A-501 from the lactic acid fermentates were higher than from authentic lactic acid, suggesting that the fermentates contain a factor(s) which promotes H2 production by this bacterium.

Uptake pathway and continuous removal of nitric oxide from flue gas using microalgae

Abstract Nitric oxide (NO), a major constituent of NOx in fossil fuel flue gas, can be removed by the microalga, Dunaliella tertiolecta, in a bubble-column-type bioreactor. The uptake pathway of NO was investigated, and it was found that little NO was oxidized in the medium before its uptake by algal cells and that NO mostly permeated directly into the cells by diffusion based on the mass balance of nitrogen and the change in nitrate and nitrite concentration in the medium in batch culture. For further application of this system, it is necessary to remove NO over a long duration, and the stability of NO removal is important. NO removal rate of about 50–60% could be maintained stably for 15 days in continuous culture under the light condition. Because the consumption of nitrate was reduced by the amount of taken NO, NO rather than nitrate is preferentially utilized as a nitrogen source for cell growth. Therefore, this algal system is useful for continuous NO removal and production of algal biomass using NO as a nitrogen source.

Removal of hazardous phenols by microalgae under photoautotrophic conditions

Various algae were screened for their ability to decrease the concentration of 2,4-dinitrophenol (DNP), as a model compound of hazardous phenols, under photoautotrophic conditions. Chlorella fusca var. vacuolata and Anabaena variabilis grew well and showed high DNP removal ability over the concentration range of 5 to 40 microM. Their abilities to remove various phenols were investigated. More than 90% of 40 microM o- and m-nitrophenol and DNP was removed during the cultivation period of 5 d. o-, p-Chlorophenol and 2,4-dichlorophenol could be removed, but not to a significant extent. C. fusca also removed 85% of bisphenol A, suspected to be an endocrine disrupter. It was found that microalgae would be applicable to the removal of hazardous phenols without the addition of any organic carbon sources.

Characteristics of Biological NOx Removal from Flue Gas in a Dunaliella tertiolecta Culture System

Abstract A system for the biological removal of NOx from fuel flue gas was investigated using the unicellular microalga Dunaliella tertiolecta . When nitric oxide (NO), the main component of NOx in flue gas, was supplied to the algal culture in a bioreactor with a 2-m column in the light at concentrations ranging from 25 to 500 ppm, about 65% of the NO was removed. Under these conditions, cell growth was not affected by the concentration of the NO supplied, and about 1.6% O 2 was constantly evolved by photosynthesis. About 30% of the NO was removed by the medium without cells at 2% O 2 , in which case the NO was probably photochemically oxidized by Fe 3+ present in the medium. In cell cultures without Fe 3+ , however, 65% NO removal was achieved. In the dark, on the other hand, the rate of NO removal was governed by the amount of O 2 supplied in the inlet gas, i.e. , achievement of a NO removal rate similar to that achieved in the light required the presence of O 2 at 2% or more, and NO removal did not occur without the supply of O 2 . It is thus clear that both algal cells and O 2 are essential in the reactor system. NO removal is assumed to proceed as follows: NO in the gas is first dissolved in the aqueous phase, after which it is oxidized and assimilated by the algal cells. The results of investigations under various culture conditions indicate that the dissolution of NO in the aqueous phase is the rate-limiting step in this reactor system.

Highly sensitive and accurate profiling of carotenoids by supercritical fluid chromatography coupled with mass spectrometry

We attempted to establish a high-speed and high-resolution profiling method for a carotenoid mixture as a highly selective and highly sensitive detection method; the analysis was carried out by supercritical fluid chromatography (SFC) coupled with mass spectrometry (MS). When an octadecyl-bonded silica (ODS) particle-packed column was used for separation, seven carotenoids including structural isomers were successfully separated within 15 min. This result indicated not only improved separation but also improved throughput compared to the separation and throughput in RP-HPLC. The use of a monolithic ODS column resulted in additional improvement in both the resolution and the throughput; the analysis time was reduced to 4 min by increasing the flow rate. Furthermore, carotenoids in biological samples containing the complex matrices were separated effectively by using several monolithic columns whose back pressure was very low. The mass spectrometer allowed us to perform a more sensitive analysis than UV detection; the detection limit of each carotenoid was 50 pg or below. This is the first report of carotenoid analysis carried out by SFC-MS. The profiling method developed in this study will be a powerful tool for carrying out accurate profiling of biological samples.