Shigetoh Miyachi

Ultrahigh-cell-density culture of a marine green alga Chlorococcum littorale in a flat-plate photobioreactor

Abstract To test the feasibility of CO2 remediation by microalgal photosynthesis, a modified type of flat-plate photobioreactor [Hu et al. (1996) Biotechnol Bioeng 51:51–60] has been designed for cultivation of a high-CO2-tolerant unicellular green alga Chlorococcum littorale. The modified reactor has a narrow light path in which intensive turbulent flow is provided by streaming compressed air through perforated tubing into the culture suspension. The length of the reactor light path was optimized for the productivity of biomass. The interrelationship between cell density and productivity, as affected by incident light intensity, was quantitatively assessed. Cellular ultrastructural and biochemical changes in response to ultrahigh cell density were investigated. The potential of biomass production under extremely high CO2 concentrations was also evaluated. By growing C. littorale cells in this reactor, a CO2 fixation rate of 16.7u2009g CO2 l−1 24 h−1 (or 200.4u2009g CO2 m−2 24u2009h−1) could readily be sustained at a light intensity of 2000u2009μmolu2009m−2u2009s−1 at 25u2009°C, and an ultrahigh cell density of well over 80u2009g l−1 could be maintained by daily replacing the culture medium.

Ethanol production by dark fermentation in the marine green alga, Chlorococcum littorale

Abstract Dark fermentation in the marine green alga, Chlorococcum littorale, was investigated with emphasis on ethanol production. Under dark anaerobic conditions, 27% of cellular starch was consumed within 24 h at 25°C, the cellular starch decomposition being accelerated at higher temperatures. Ethanol, acetate, hydrogen and carbon dioxide were obtained as fermentation products. The maximum productivity of ethanol was 450 μmol/g-dry wt. at 30°C. The fermentation pathway for cellular starch was proposed from the yields of the end-products and the determined enzyme activities. Ethanol was formed from pyruvate by pyruvate decarboxylase and alcohol dehydrogenase. the change in fermentation pattern that varied with cell concentration in the reaction vials suggested that the hydrogen partial pressure affected the consumption mode of reducing equivalents under dark fermentation. Ethanol productivity was improved by adding methyl viologen, while hydrogen production decreased.

Isolation and characterization of biliprotein aggregates from Acaryochloris marina, a Prochloron-like prokaryote containing mainly chlorophyll d

Phycobiliprotein aggregates were isolated from the prokaryote Acaryochloris marina, containing chlorophyll d as major pigment. In the electron microscope the biliprotein aggregates appear as rod‐shaped structures of 26.0×11.3 nm, composed of four ring‐shaped subunits 5.8 nm thick and 11.7 nm in diameter. Spectral data indicate that the aggregates contain two types of biliproteins: phycocyanin and an allophycocyanin‐type pigment, with very efficient energy transfer from the phycocyanin‐ to allophycocyanin‐type constituent. The chromophore‐binding polypeptides of the pigments have apparent molecular masses of 16.2 and 17.4 kDa. They crossreact with antibodies against phycocyanin and allophycocyanin from a red alga.

Historical perspective on microalgal and cyanobacterial acclimation to low- and extremely high-CO2 conditions

Reports in the 1970s from several laboratories revealed that the affinity of photosynthetic machinery for dissolved inorganic carbon (DIC) was greatly increased when unicellular green microalgae were transferred from high to low-CO2 conditions. This increase was due to the induction of carbonic anhydrase (CA) and the active transport of CO2 and/or HCO3− which increased the internal DIC concentration. The feature is referred to as the ‘CO2-concentrating mechanism (CCM)’. It was revealed that CA facilitates the supply of DIC from outside to inside the algal cells. It was also found that the active species of DIC absorbed by the algal cells and chloroplasts were CO2 and/or HCO3−, depending on the species. In the 1990s, gene technology started to throw light on the molecular aspects of CCM and identified the genes involved. The identification of the active HCO3− transporter, of the molecules functioning for the energization of cyanobacteria and of CAs with different cellular localizations in eukaryotes are examples of such successes. The first X-ray structural analysis of CA in a photosynthetic organism was carried out with a red alga. The results showed that the red alga possessed a homodimeric β-type of CA composed of two internally repeating structures. An increase in the CO2 concentration to several percent results in the loss of CCM and any further increase is often disadvantageous to cellular growth. It has recently been found that some microalgae and cyanobacteria can grow rapidly even under CO2 concentrations higher than 40%. Studies on the mechanism underlying the resistance to extremely high CO2 concentrations have indicated that only algae that can adopt the state transition in favor of PS I could adapt to and survive under such conditions. It was concluded that extra ATP produced by enhanced PS I cyclic electron flow is used as an energy source of H+-transport in extremely high-CO2 conditions. This same state transition has also been observed when high-CO2 cells were transferred to low CO2 conditions, indicating that ATP produced by cyclic electron transfer was necessary to accumulate DIC in low-CO2 conditions.

ACARYOCHLORIS MARINA GEN. ET SP. NOV. (CYANOBACTERIA), AN OXYGENIC PHOTOSYNTHETIC PROKARYOTE CONTAINING CHL D AS A MAJOR PIGMENT1

The phylogenetic position of an oxygenic photosynthetic prokaryote containing chl d as a major pigment, which have been tentatively named “Acaryochloris marina,” was analyzed using small subunit rDNA sequences. Phylogenetic relationships inferred among A. marina, selected strains from the Cyanobacteria, and plastids showed that A. marina was within the cyanobacterial radiation. The A. marina lineage diverged independently from other phylogenetic subgroups of the Cyanobacteria. No organism was found to be identical or related closely to A. marina by a similarity search and phylogenetic analysis. Based on these results, in addition to the reported characteristics of the cell morphology, pigment composition, and photosynthesis, a new taxon, Acaryochloris marina Miyashita et Chihara gen. et sp. nov., is formally proposed for the oxy‐genic photosynthetic prokaryote.

FLUORESCENCE PROPERTIES OF CHLOROPHYLL D-DOMINATING PROKARYOTIC ALGA, ACARYOCHLORIS MARINA : STUDIES USING TIME-RESOLVED FLUORESCENCE SPECTROSCOPY ON INTACT CELLS

Antenna components and the primary electron donor of the photosystem (PS) II in the Chlorophyll (Chl) d-dominating prokaryote, Acaryochloris marina, were studied using time-resolved fluorescence spectroscopy in the ps time range. By selective excitation of Chl a or Chl d, differences in fluorescence properties were clearly resolved. At physiological temperature, energy transfer was confirmed by a red shift of emission maximum among PS II antenna components, and the equilibrium of energy distribution among Chl a and Chl d was established within 30 ps. A fluorescence component that can be assigned to delayed fluorescence (DF) was observed at 10 ns after the excitation; however, it was not necessarily resolved by the decay kinetics. At -196 degrees C, a red shift of emission maximum was reproduced but the equilibrium of energy distribution was not detected. DF was resolved in the wavelength region corresponding to Chl a by spectra and by decay kinetics. The lifetime of the DF was estimated to be approx. 15 ns, and the peaks were located at 681 and 695 nm, significantly shorter wavelengths than those of Chl d. These findings strongly suggest that an origin of DF is Chl a, and Chl a is most probably the primary electron donor in the PS II reaction center (RC). These results indicate that the constitution of PS II RC in this alga is essentially identical to that of other oxygenic photosynthetic organisms.

EFFECT OF EXTREMELY HIGH-CO2 STRESS ON ENERGY DISTRIBUTION BETWEEN PHOTOSYSTEM I AND PHOTOSYSTEM II IN A 'HIGH-CO2' TOLERANT GREEN ALGA, CHLOROCOCCUM LITTORALE AND THE INTOLERANT GREEN ALGA STICHOCOCCUS BACILLARIS

Abstract A green alga, Chlorococcum littorale , has a tolerance to extremely high-CO 2 conditions (Kodama et al., J. Marine Biotech. 1 (1993) 21–25). In order to elucidate the mechanism underlying the resistance to such high CO 2 levels, we compared the changes in excitation energy ditribution between photosystem I (PS 1) and photosystem II (PS II) by 77 K fluorescence in cells of the high CO 2 -resistant C. littorale and the non-resistant Stichococcus bacillaris . Immediately after the cell are transferred from air to 40% CO 2 , the F 714 / F 687 ratio derived from 77 K fluorescence increases in C. littorale cells, suggesting an increase of transition from state 1 to state 2. During this period, more than 80% of plastoquinone A is in the reduced form and the activity of PS I increass. Eventually the F 714 / F 687 ratio, the concentration of reduced plastoquinone A and PS I activity decrease. However, no significant increase of F 714 / F 687 ratio is observed after the transfer of S. bacillaris cells from air to 40% CO 2 . The level of reduced plastoquinone A in S. bacillaris gradually increases and the activity of PS I does not show a large change. During the transient period, the level of the D1 protein is approximately constant in C. littorale cells, but is lowered in S. bacillaris . These results suggest that, under extremely high-CO 2 conditions, PS II is protected from photoinhibition by control of the state transition in C. littorale cells, whereas such a protection mechanism does not function in the alga S. bacillaris , non-resistant to CO 2 .

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.

Outdoor culture of a cyanobacterium with a vertical flat-plate photobioreactor : effects on productivity of the reactor orientation, distance setting between the plates, and culture temperature

Abstract The ability of a photobioreactor to fix CO2 was evaluated with the thermophilic cyanobacterium, Synechocystis aquatilis SI-2. The reactor consisted of three to five flat plates of transparent acrylic plastic standing upright and in parallel and giving a 0.015-m light path. The reactor was 0.8u2009m high and 1u2009m long with 9u2009l working volume. The effects of the orientation of the vertical bioreactor, distance between the plates, and culture temperature on the productivity of biomass were investigated during the summer of 1998 in Kamaishi (39°N, 142°E), Japan. When the illuminated surface reactor was placed in an east–west-facing orientation, the biomass productivity was roughly 1.4-fold higher than that obtained in a north–south-facing orientation, because the former received more solar energy than the latter. The productivity based on the overall land area was the same for plates set either 0.25u2009m or 0.5u2009m apart. However, the volumetric productivity of the reactor in which the plates were set 0.25u2009m apart was lower than that when the plates were set 0.5u2009m apart, since the former plates received relatively lower solar irradiation because of severe mutual shading. When the culture temperature was maintained in its optimal range (37–43u2009°C), the productivity was 50% greater than that obtained in a culture at ambient temperature (20–44u2009°C). The biomass productivity and CO2 fixation rate were investigated under various experimental conditions. The maximum rate of 53u2009g CO2 m−2u2009day−1 was achieved in the temperature-regulated culture with the reactor set in an east–west-facing orientation, the distance between plates being 0.25u2009m.

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO2

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO2 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% CO2 (5% CO2-grown cells) could grow in 40% CO2 without any lag period. When air-grown cells were transferred to 40% CO2, the quantum efficiency of PS II (ΦII) decreased greatly, while no decrease in ΦII was apparent when the 5% CO2-grown cells were transferred to 40% CO2. In contrast to air-grown cells, 5% CO2-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO2-grown cells to air, photosynthetic susceptibility to 40% CO2 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% CO2. The intracellular pH value (pHi) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO2 for 1–2 h, but no such decrease in pHi was apparent in the presence of EZA. Both air- and 5% CO2-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO2 concentrations, grew in 40% CO2 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 CO2. No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells.