Evaluation of a vertical flat-plate photobioreactor for outdoor biomass production and carbon dioxide bio-fixation: effects of reactor dimensions, irradiation and cell concentration on the biomass productivity and irradiation utilization efficiency.

Outdoor culture of the thermophilic cyanobacterium Synechocystis aquatilis SI-2 with a vertical flat-plate photobioreactor (VFPP) was studied during the period of January to August of 1999 in the northern region of Japan (Kamaishi, Iwate, 39 degrees N, 142 degrees E). The aim of this study was to investigate the CO2 fixation ability of the VFPP device under various irradiation conditions. An average biomass productivity of over 30 g m(-2) day(-1), which corresponds to a CO2 fixation rate of 50 g m(-2) day(-1), was achieved during this period with a 192-l scale culture. The effects on biomass productivity of the light path, height of the reactor, cell concentration and irradiation were also investigated. Variation of the optimal cell concentration to achieve the highest productivity for outdoor operation is discussed. A cell concentration of 1-2 g l(-1) was found to be most suitable for the irradiation range of 1-12 MJ m(-2) day(-1) under the experimental conditions used.

Characterization of ascorbate peroxidases from unicellular red alga Galdieria partita.

Galdieria partita, a unicellular red alga isolated from acidic hot springs and tolerant to sulfur dioxide, has at least two ascorbate peroxidase (APX) isozymes. This was the first report to demonstrate that two isozymes of APX are found in algal cells. Two isozymes were separated from each other at the hydrophobic chromatography step of purification and named APX-A and APX-B after the elution order in the chromatography. APX-B accounted for 85% of the total activity. Both isozymes were purified. APXs from Galdieria were monomers whose molecular weights were about 28,000, similar to stromal APX of higher plants. APX-A cross-reacted with monoclonal antibody raised against APX of Euglena gracilis in immunoblotting, but APX-B did not, although the antibody can recognize all other APXs tested. The amino-terminal sequences of APX-A and -B from Galdieria had some homology with each other but little homology with those from other sources. Their Km values for ascorbate and hydrogen peroxide were comparable with those of APX from higher plants. Unlike the green algal enzymes, the donor specificities of Galdieria APXs were as high as those of plant chloroplastic APX. On the contrary, these APXs reduced tertiary-butyl hydroperoxide as an electron acceptor as APXs from Euglena and freshwater Chlamydomonas do. The inhibition of APX-A and -B by cyanide and azide, and characteristics of their light absorbance spectra indicated that they were heme peroxidases.

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO(2).

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO(2) concentrations (>20%), active photosynthesis resumed after a lag period which lasted for 1-4 days. In contrast, C. littorale cells which had been grown in 5% CO(2) (5% CO(2)-grown cells) could grow in 40% CO(2) without any lag period. When air-grown cells were transferred to 40% CO(2), the quantum efficiency of PS II (Phi(II)) decreased greatly, while no decrease in Phi(II) was apparent when the 5% CO(2)-grown cells were transferred to 40% CO(2). In contrast to air-grown cells, 5% CO(2)-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO(2)-grown cells to air, photosynthetic susceptibility to 40% CO(2) was induced. This change was associated with the induction of CA. In addition, neither suppression of photosynthesis nor arrest of growth was apparent when ethoxyzolamide (EZA), a membrane-permeable inhibitor of CA, had been added before transferring air-grown cells of C. littorale to 40% CO(2). The intracellular pH value (pH(i)) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO(2) for 1-2 h, but no such decrease in pH(i) was apparent in the presence of EZA. Both air- and 5% CO(2)-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO(2) concentrations, grew in 40% CO(2) without any lag period. The activity of CA was much lower in air-grown cells of this alga than those in air-grown C. littorale cells. These results prompt us to conclude that intracellular CA caused intracellular acidification and hence inhibition of photosynthetic carbon fixation when air-grown C. littorale cells were exposed to excess concentrations of CO(2). No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells.

[Clinical symptoms and characteristic MR spectroscopic findings in Pelizaeus-Merzbacher disease].

Clinical symptoms and MR spectroscopic findings were studied on 4 cases of Pelizaeus-Merzbacher disease including 1 autopsy case. Common symptoms were severe mental retardation and spastic tetraplegia. These cases had nystagmus, and one had involuntary athetotic movement. Genetical diagnosis revealed in 2 cases, duplication of proteolipid protein (PLP) and deletion in 1, whereas one case had no abnormality of PLP gene. MRI indicated the reversal of signal intensities on T1- and T2-weighed images, a characteristic finding of PMD MR spectroscopy demonstrated a pattern of NAA in 3 cases. This was specific to PMD because other white matter diseases show a decrease in NAA. In conclusion, MRS was useful to differentiate PMD from other white matter diseases.

Molecular structure, localization and function of biliproteins in the chlorophyll a/d containing oxygenic photosynthetic prokaryote Acaryochloris marina.

We investigated the localization, structure and function of the biliproteins of the oxygenic photosynthetic prokaryote Acaryochloris marina, the sole organism known to date that contains chlorophyll d as the predominant photosynthetic pigment. The biliproteins were isolated by means of sucrose gradient centrifugation, ion exchange and gel filtration chromatography. Up to six biliprotein subunits in a molecular mass range of 15.5-18.4 kDa were found that cross-reacted with antibodies raised against phycocyanin or allophycocyanin from a red alga. N-Terminal sequences of the alpha- and beta-subunits of phycocyanin showed high homogeneity to those of cyanobacteria and red algae, but not to those of cryptomonads. As shown by electron microscopy, the native biliprotein aggregates are organized as rod-shaped structures and located on the cytoplasmic side of the thylakoid membranes predominantly in unstacked thylakoid regions. Biochemical and spectroscopic analysis revealed that they consist of four hexameric units, some of which are composed of phycocyanin alone, others of phycocyanin together with allophycocyanin. Spectroscopic analysis of isolated photosynthetic reaction center complexes demonstrated that the biliproteins are physically attached to the photosystem II complexes, transferring light energy to the photosystem II reaction center chlorophyll d with high efficiency.

Purification and characterization of hydrogenase from the marine green alga, Chlorococcum littorale.

Hydrogenase from the marine green alga, Chlorococcum littorale, was purified 1485-fold, resulting in a specific activity for hydrogen evolution of 75.7 micromol/min/mg of protein at 25 degrees C, using reduced methyl viologen as an electron donor. The K(m) value for methyl viologen was 0.5 mM. The purity of the enzyme was judged by native PAGE. The molecular weight was estimated to be 55 kDa by SDS-PAGE, and 57 kDa by gel filtration. The optimum temperature and pH value for hydrogen evolution were 50 degrees C and 7.5, respectively. The partially purified hydrogenase catalyzed hydrogen evolution from ferredoxin that had been isolated from the same cells, but not from NADH or NADPH. The K(m) value for ferredoxin was 0.68 microM. The enzyme was extremely oxygen sensitive, losing over 95% of its activity upon exposure to air within minutes, even at 4 degrees C. Two peptide fragments were obtained from the hydrogenase protein digested enzymatically, and their amino acid sequences were determined. No significant homology was found to any other known sequences of hydrogenases.

Rubisco activase transcript (rca) abundance increases when the marine unicellular green alga Chlorococcum littorale is grown under high-CO2 stress.

cDNA and the corresponding genomic DNA region encoding Rubisco activase were isolated from the unicellular green alga Chlorococcum littorale. The deduced amino acid sequence encoded by the cDNA was 403 amino acids long and exhibited important homology with those of other known Rubisco activases. Its N-terminal sequence was similar to the chloroplastic transit peptides in Chlamydomonas reinhardtii. The mature protein had a predicted molecular mass of 42 kDa. Five introns were located inside the genomic gene encoding Rubisco activase (rca). Genomic Southern blots indicated that two copies of the rca gene were present in the genome of C. littorale. The level of rca messenger RNA increased when cells of C. littorale were subjected to high-CO2 stress (i.e. grown under at least 20% CO2). Hsp70 heat-shock protein was also induced under high-CO2 conditions and, as expected, was also induced at 35 degrees C. The rca gene, in contrast, was not induced at 35 degrees C, indicating that this gene was induced in response to the high CO2 concentration and not to general stress. A search of the promoter-binding proteins by a gel retardation assay showed that, under the high-CO2 conditions, a protein(s) which was probably an activator of the rca transcription was synthesized.

A photosystem I reaction center driven by chlorophyll d in oxygenic photosynthesis.

A far-red type of oxygenic photosynthesis was discovered in Acaryochloris marina, a recently found marine prokaryote that produces an atypical pigment chlorophyll d (Chl d). The purified photosystem I reaction center complex of A. marina contained 180 Chl d per 1 Chl a with PsaA-F, -L, -K, and two extra polypeptides. Laser excitation induced absorption changes of reaction center Chl d that was named P740 after its peak wavelength. A midpoint oxidation reduction potential of P740 was determined to be +335 mV. P740 uses light of significantly low quantum energy (740 nm = 1.68 eV) but generates a reducing power almost equivalent to that produced by a special pair of Chl a (P700) that absorbs red light at 700 nm (1.77 eV) in photosystem I of plants and cyanobacteria. The oxygenic photosynthesis based on Chl d might either be an acclimation to the far-red light environments or an evolutionary intermediate between the red-absorbing oxygenic and the far-red absorbing anoxygenic photosynthesis that uses bacteriochlorophylls.

Cloning and characterization of high-CO2-specific cDNAs from a marine microalga, Chlorococcum littorale, and effect of CO2 concentration and iron deficiency on the gene expression.

Two cDNA clones exclusively induced under an extremely high-CO2 concentration (20%) were isolated from Chlorococcum littorale by differential screening and named HCR (high-CO2 response) 1 and 2, respectively. The amino acid sequence of the protein encoded by HCR2 exhibited homology to the gp91-phox protein, a critical component of a human phagocyte oxidoreductase, and to the yeast ferric reductases, Saccharomyces cerevisiae FRE1 and FRE2 and Schizosaccharomyces pombe Frp1. The induction of both HCR mRNAs required extremely high-CO2 conditions and iron deficiency, being suppressed under air conditions and by iron sufficiency, suggesting that the expression of these two HCR genes required extremely high-CO2 conditions and iron deficiency in combination. The HCR2 protein was detected in the membrane fractions of cells grown under conditions which would favor the induction of HCR2-mRNA and the protein level was lowered when the cells were transferred from iron deficient to 10 microM FeSO4 conditions (with 20% CO2).

Growth kinetics of Chinese hamster ovary cells in a compact loop bioreactor. 3. Selection and characterization of an anchorage-independent subline and medium improvement.

Four sublines of Chinese hamster ovary (CHO) cells were selected or cloned on a 10% fetal calf serum supplemented MEM-alpha medium. Three of them were monolayer cultures and could proliferate by 2000 times a week (mu = 1.1 d 1) in T-flasks. The other subline, S1, could grow in suspension even in static T-flask cultures. The stability in chromosome number of these cell lines was investigated. By evaluating the kinetic growth parameters, i.e. the specific rates of growth, glucose consumption and lactic acid production, and the yields of cells and lactic acid from glucose, the S1 cells were considered to be the most suitable subline for the bioreactor suspension culture. The S1 cells reached the greatest maximum of cell concentration among all cell lines tested because of their efficient glucose utilization. Observed nutrient limitations in the S1 cell culture was overcome by modification of the medium composition, that is addition of 10 mg l-1 hypoxanthine, 1 mg l-1 FeSO4.7H2O, and 0.1 mg l-1 sodium putrescine, elimination of glutamine, supplementation of 6 mM asparagine and double amount of isoleucine, leucine, methionine and vitamins other than ascorbic acid, cyanocobalamin and biotin, increase of NaHCO3 concentration from 26 to 40 mM, and finally decrease of NaCl concentration from 122 to 100 mM. With this modified medium, 7.2 X 10(6) ml-1 of the maximum cell concentration was observed in a glucose fed-batch culture, the cell concentration which was twice as much as in batch cultures with the original medium.

Utilization and stability of vitamins in serum-containing and serum-free media in CHO cell culture.

The concentrations of four vitamins, ascorbic acid, nicotinamide, choline and thiamine were evaluated in the culture supernatant of Chinese hamster ovary (CHO) cells. The media used were alpha-modified Eagle's minimum essential medium (MEM-alpha) supplemented with 10% fetal calf serum, and a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (DME/F12), containing neither serum nor protein. The reference experiment without cells revealed instability of ascorbic acid and thiamine. Moreover, a significant amount of each vitamin decreased in the culture supernatant. The possibility of growth limitation by vitamin depletion is strongly suggested.

Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor: 1. Effects of physical and chemical environments.

Chinese hamster ovary (CHO) cells were cultivated in a compact loop bioreactor using MEM-alpha medium supplemented with 10% fetal calf serum. Effects of physical and chemical environments, i.e., pH in the medium, stirring speed of impellers, temperature and partial pressure of oxygen (pO2) upon growth of suspended cells in the bioreactor were determined in batch cultures. Growth behavior was characterized by specific rates of growth (mu), glucose consumption (qG) and lactate production (qL), and the yield coefficients (cell yield from glucose, YX/G, and lactate yield from glucose, YL/G). An effect of medium osmolality was also evaluated with T-flask monolayer cultivation. The best growth was observed at pH 7.6, 37 degrees C, 400 rpm, 50-100% saturation with oxygen and 320 mOsmol kg-1. Corresponding to the previous work with a human melanoma cell line, the sophisticated cultivation and process control systems have been improved for CHO cells.

Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor. 2. Effects of medium components and waste products.

Effects of biochemical factors, i.e., medium components and metabolic byproducts, on growth of Chinese hamster ovary (CHO) cells were investigated. Glucose and ammonia were found to inhibit the growth. Kinetic analysis gave the inhibition constants, 0.14 g l-1 for ammonia and 5.0 g l-1 for glucose. Since glutamine was unstable and was a main source of ammonia, precise studies on glutamine degradation and ammonia formation process were done. By evaluating the spontaneous reactions, net glutamine utilization and net ammonia production by the cells could be estimated. It became evident that asparagine could support the growth of CHO cells as a stable substitute for glutamine. Then, a glucose fed-batch culture was grown on a glutamine free and asparagine supplemented medium. Because of (1) low glucose concentration, but (2) no glucose limitation and (3) low ammonia accumulation, the maximum total cell concentration reached 3.4 x 10(6) ml-1, which was 1.8 times greater than that in the control experiment (initial 1.15 g l-1 glucose and 0.29 g l-1 glutamine, and no glucose feed).