Electron microscopy of the marine microalga Dunaliella tertiolecta exposed to triphenyltin

Chemostat-grown cells of the chlorophyte Dunaliella tertiolecta (Butcher) exposed to triphenyltin were examined using transmission electron microscopy. Following a 1-h exposure to 21 and 84μM triphenyltin, mitochondria underwent structural damage and the thylakoid membranes of a small proportion of cells spread from the usual compact arrangement. Prolonging the exposure time resulted in significant cell lysis in cultures exposed to 84μM triphenyltin. Received 05 May 1997/ Accepted in revised form 28 January 1998     

Photoacclimation of Dunaliella tertiolecta (Chlorophyceae) Under Fluctuating Irradiance

We investigated photoacclimation of Dunaliella tertiolecta (Butcher) in irradiance (I) regimes simulating mixed layer conditions of turbid estuarine waters or lakes. D. tertiolecta was exposed to a range of fixed I regimes to establish baseline physiology-I relationships that were compared with subsequent photoacclimation to a simulated mixed layer. Measured indices of photoacclimation included cellular pigmentation, chlorophyll variable fluorescence, and effective photosystem 2 antenna size. While D. tertiolecta grown under fluctuating I maintained division rates comparable to cells grown at high I, the cells exhibited characteristics of photoacclimation consistent with cells grown under a stable regimes at irradiances considerably lower than the average I of the simulated mixed layer.     

Mitochondrial NDP kinase from Dunaliella tertiolecta (Chlorophyceae, Chlorophyta)

A cDNA (DtNDK1) encoding a nucleoside diphosphate (NDP) kinase with a putative mitochondrial targeting signal sequence was previously isolated from the halo‐tolerant green alga Dunaliella tertiolecta. When expressed in Saccharomyces cerevisiae, the processed DtNDK1 enzyme was specifically localized to mitochondria. The present study reports several biochemical characteristics of the mitochondrial NDP kinase from D. tertiolecta. The mature protein was expressed as either N‐ or C‐terminal hexahistidine‐tagged protein and purified to homogeneity by affinity chromato‐graphy. Native gel electrophoresis and sedimentation velocity in sucrose density gradients showed that the active enzyme consisted of a hexamer. The enzyme, with a pH optimum of 7, showed the highest specificity to dCDP (K_m= 50 μmol/L) and the highest turnover towards the synthesis of UTP (up to 140‐fold higher). The present study also provides evidence that purified DtNDK1 proteins are capable of transferring a phosphate group to another protein.     

Isolation of disproportionating enzyme from halotolerant microalga Dunaliella parva (Volvocales, Chlorophyceae)

Starch metabolism in Dunaliella parva Lerche is regulated by the osmolarity of the surrounding solute. Two isozymes showing amylolytic activity were obtained after purification by gel filtration chromatography. The isozymes show disproportionating activity (D‐enzyme) that is specific for malto‐oligosaccharides as substrate. Properties of the D‐enzyme in D. parva are similar to those in higher plants. The activity of the D‐enzyme is also found in various Dunaliella and Chlamydomonas, indicating that the D‐enzyme is also important in the starch metabolism in algae.     

Antifouling potential of the marine microalga Dunaliella salina

Marine organisms have usually been viewed as sources of environmentally friendly compounds with antifouling activity. We performed a series of operations to investigate the antifouling potential of the marine microalga Dunaliella salina. For the ethyl acetate crude extract, the antialgal activity was significant, and the EC₅₀ value against Skeletonema costatum was 58.9 μg ml^?1. The isolated purified extract was tested for antifouling activity, the EC ₅₀ value against S. costatum was 21.2 μg ml^?1, and the LC₅₀ against Balanus amphitrite larvae was 18.8 μg ml^?1. Subsequently, both UHR–TOF–MS and GC–MS were used for the structural elucidation of the compounds, and a series of unsaturated and saturated 16- and 18-carbon fatty acids were detected. The data suggested that the fatty acid extracts from D. salina possess high antifouling activity, and could be used as substitutes for potent, toxic antifouling compounds.     

Photosynthetic electron transport in Dunaliella tertiolecta (Chlorophyceae) measured by fast repetition rate fluorometry: relation to carbon assimilation

A comparison of photosynthesis-irradiance response curves (P–Eresponse curves) obtained through fast repetition rate (FRR)fluorometry and radiocarbon (¹⁴C) tracer method was made inthe chlorophyte, Dunaliella tertiolecta, grown under differentirradiance conditions. In FRR-based P–E response curveexperiments, actinic light provided by white light-emittingdiodes (LEDs) was increased gradually from 0 to 1500 µmolquanta m^–2 s^–1 and the rate of photosyntheticelectron transport was determined at each light level. Short-termexperiments (20 min) of ¹⁴C-based P–E response curvewere carried out with an improved photosynthetron, which containswhite LEDs as the light source. Irrespective of growth irradiance,the ratios of FRR to ¹⁴C-based initial slopes were almost uniform.The ratios of FRR- to ¹⁴C-based maximum rates were 25–36%higher than those of FRR- to ¹⁴C-based initial slopes. The relationshipbetween electron transport and carbon assimilation was non-linearwith increasing discrepancy towards high actinic light. Thisnon-linear relationship between FRR- and ¹⁴C-based estimatesis primarily due to the effect of physiological processes stimulatedat high levels of light, such as cyclic electron flow and theMehler reaction. The results of this study indicate that theFRR fluorometry can be used as a good indicator of photosyntheticrates from low to middle light levels, but becomes increasinglyquestionable as the maximum photosynthetic rate is approached.The degree to which this relationship is further affected bynutrient-status warrants investigation.     

Vitamin E (alpha-tocopherol) production by the marine microalgae Dunaliella tertiolecta and Tetraselmis suecica in batch cultivation

Batch experiments with Dunaliella tertiolecta and Tetraselmis suecica were performed to investigate alpha-tocopherol (alpha-T) production in time, in order to assess the effect of light availability per cell on the production of this antioxidant. In D. tertiolecta alpha-T content increased during growth, in other words, as the cell density increased and the light availability per cell decreased. In T. suecica the pattern was different: alpha-T content was highest during the exponential phase, decreased significantly during the linear phase and increased again towards the end of the cultivation. Chlorophyll (chl-a) content of T. suecica cells decreased after the exponential phase, instead of the expected increase typically observed in shade-adapted cells, suggesting that the culture was nutrient limited. An experiment with extra nutrients showed that chl-a and alpha-T content increased significantly during both the linear and the stationary phase when compared with values in nutrient-deficient conditions. No correlation between alpha-T and chl-a was observed. Our results indicate that diminished light availability does not limit alpha-T production in D. tertiolecta and T. suecica, opening up the possibility of using high cell density, light-limited cultures for the production of this commercially interesting compound.     

Renewal rate and nutrient concentration as tools to modify productivity and biochemical composition of cyclostat cultures of the marine microalga Dunaliella tertiolecta

A factorial experimental design with two nutrient concentrations (2 and 4 mmol Nl^–1 in the form of NaNO₃) and five rates of daily renewal of the cultures (10%, 20%, 30%, 40% and 50%) was carried out in cyclostat, light/dark-synchronized cultures of the marine microalga Dunaliella tertiolecta Butcher. Steady-state cellular density was a linear function inversely proportional to renewal rate. Maximal cellular productivity, 3 × 10⁹ cells1^–1 day^–1, equivalent to 0.24 g1^–1 day^–1 dry weight and 0.17 g1^–1 day^–1 organic weight, was found with renewal rates of 20%–30% and 4mmol N1^–1, but maximal protein productivity, 0.066 g1^–1 day^–1, was obtained with a renewal rate of 40% for both nutrient concentrations. The protein content ranged between 30% and 70% of the organic fraction depending on the culture conditions. Carbohydrates were the only fraction accumulating in response to nutrient stress, ranging from 57% to 10% of the organic fraction, meanwhile the lipid content was increased by increase of nutrient availability. Under non-nitrogen-limited conditions the C:N ratio stabilized around 5.2–5.3 and the protein content of the organic fraction around 70%, but the cell nitrogen quota decreased under these conditions with increasing renewal rates, owing to the lower organic content of cells obtained with high growth rates. The high capacity for changing the biochemical composition, demonstrated for D. tertiolecta in the cyclostat system, has interesting implications for the management of continuous cultures of microalgae and its applications in biotechnological processes.     

Culture of the marine phytoplankter,Dunaliella tertiolecta,with light-dark cycles

Summary Dunaliella tertiolecta was grown in continuous culture, maintained by a single daily dilution to a constant cell concentration, with photoperiods of duration 3 to 18 hours. Illumination was provided with filtered tungsten light having a maximum intensity at 580–590 m. Average intensity at the culture surface was maintained at 0.05 cal/cm²xmin. Temperature was regulated at 20° C. Daily cell production and the fraction of cells dividing daily are reported for each of the photoperiods. Cyclic diurnal variations were noted in cell pigment content, cell volume and photosynthetic rate. Corresponding variations in cell carbon/chlorophyll a ratios were small and may be unimportant for the estimation of biomass from chlorophyll a in field work. An equation for calculating daily growth rate in continuous light, from light absorption by cell chlorophyll a, derived previously, was modified to include the influence of providing light in discrete photoperiods.Supported by U. S. Atomic Energy Commission, Contract AT(11-1)-34 Project 108.     

Growth light level and photon absorption by cells of Chlamydomonas rheinhardii,Dunaliella tertiolecta (Chlorophyceae,Volvocales), Scenedesmus obliquus (Chlorophyceae,Chlorococcales) and Euglena viridis (Euglenophyceae,Euglenales)

Reductions in the growth light level (40 to 6 μmol m^-2 s^-1) resulted in increases in chlorophyll and protein per cell for all of the species examined. Only Dunaliella tertiolecta exhibited a reduction in chlorophyll a:b ratio with decreases in the photon flux density. However, the specific absorption coefficient (ā? _i ) normalized to chlorphyll a (ā? _a remained invariant for all of the microalgae studied. Constant values for the specific absorption coefficient normalized to the total pigment content (ā? _a+b ) were also found for the species Chlamydomonas rheinhardii, Euglena viridis and Scenedesmus obliquus. In contrast ā? _a+b for D. tertiolecta decreased with a reduction in light level due to an increase in the proportion of chlorophyll b. Differences in ā? _i were related to cell size and pigment content and possible reasons for the constancy of ā? _a discussed. Increases in the absorption cross sections (¯sQ _a ) were also found at reduced light levels due to an increase in the absorptance per cell (α_cell). The lower α_cell for D. tertiolecta, compared with C. rheinhardii was exactly compensated for by a larger light-capturing area. Although the increase in α_cell does not compensate for the reduction in the incident light level, it does reduce this range by half on an absorbed light basis.     

Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta

Production of biofuel from algae is dependent on the microalgal biomass production rate and lipid content. Both biomass production and lipid accumulation are limited by several factors, of which nutrients play a key role. In this research, the marine microalgae Dunaliella tertiolecta was used as a model organism and a profile of its nutritional requirements was determined. Inorganic phosphate PO4(3-) and trace elements: cobalt (Co2+), iron (Fe3+), molybdenum (Mo2+) and manganese (Mn2+) were identified as required for algae optimum growth. Inorganic nitrogen in the form of nitrate NO3- instead of ammonium (NH4+) was required for maximal biomass production. Lipids accumulated under nitrogen starvation growth condition and this was time-dependent. Results of this research can be applied to maximize production of microalgal lipids in optimally designed photobioreactors.     

Nigericin insensitive post-illumination reduction in fluorescence yield in Dunaliella tertiolecta (chlorophyte)

Cells of the green alga Dunaliella tertiolecta grown in a light/dark cycle were exposed to high light for about 15 min. In light, energy-dependent quenching reduced fluorescence emission and decreased PS II efficiency. Within 3 minutes after darkening fluorescence quenching largely relaxed. However, PS II fluorescence emission decreased again after further darkening. F_o and F_m decreased to the same relative extent and the PS II efficiency was not reduced. This Reduction in Fluorescence yield in Darkness, termed RFD for the purpose of this paper, lasted about 20 min. The deepoxidation state of xanthophylls remained unchanged during and after the 15-min exposure to high light. We show that RFD is insensitive to the uncoupler nigericin and thus unrelated to energy-dependent quenching. RFD correlated with a reduction of the PQ pool after darkening and low levels of far red or blue light (430 nm more than 460 nm) prevented RFD. This is in contrast to observations in higher plants, where a post-illumination reduction of the PQ pool causes and increase in F_o (Groom et al. (1993) Photosynth Res 36: 205–215). Changes in the adenylate energy charge were not correlated with RFD. Antimycin A and cyanide, both inhibitors of the PQ-oxidase, caused an increase in RFD whereas SHAM, an inhibitor of the chloroplastic glycolate-quinone oxidoreductase, caused a decrease. Low CO₂ concentrations, known to increase the oxygenase activity of Rubisco and to generate glycolate and P-glycolate in light, caused an increase in RFD. We propose that accumulated glycolate and P-glycolate reduce the PQ pool in darkness, leading to the formation of RFD. During RFD, 77 K fluorescence emission from PS II was more reduced than that from PS I, thus resembling a state I, state II transition. However, the reduction in fluorescence yield during RFD is much larger than the reduction previously attributed to state transitions and it is unclear whether RFD and state transitions are identical. The formation and relaxation of RFD increased with higher temperatures and the extent of RFD was largest at the growth temperature (25°C). RFD has to be taken into account when fluorescence is measured after darkening as it may be mistaken for energy-dependent quenching.Abbreviations F_o fluorescence, measured when PS II traps are open - F_o difference between F_o and F_o - F_m fluorescence, measured when PS II traps are temporarily closed - F_m difference between F_m and F_m - FR far red - PFD photosynthetically active photon flux density - PQ plastoquinone - RFD reduction in fluorescence in darkness - SHAM salicylhydroxamic acid - Q_A primary quinone acceptor of PS II     

Long-term hyposaline and hypersaline stresses produce distinct antioxidant responses in the marine alga Dunaliella tertiolecta

Tolerance to salinity stress in higher plants correlates to levels of antioxidant enzymes and/or substrates. Do hyperosmotic and hypoosmotic stress induce antioxidant responses in salt tolerant algae, and if so, are these responses the same for both excess and minimal salinity? To answer these questions, cultures of the marine alga Dunaliella tertiolecta (Chlorophyta) were grown in seven salinities covering a 60-fold range from 0.05 to 3.0 mol/L NaCl. Long-term effects of salinity on growth and antioxidant parameters were determined. Growth rates were reduced at the salinity extremes (0.05 mol/L NaCl and 3 mol/L NaCl) indicating the cultures were stressed. The levels of six antioxidant enzymes and three antioxidant substrates were quantified at these growth salinities. Compared to growth at optimum salinities (i.e. 0.2-0.5 mol/L NaCl), high salinities produced a 260% increase in monodehydroascorbate reductase, a doubling of ascorbate peroxidase activity and a three-fold increase in the rate of dark respiration. Cells acclimated to low growth salinities (hyposaline stress, i.e. < 0.2 mol/L NaCl) showed major increases in glutathione and alpha-tocopherol coupled with decreases in Fv/Fm ratios and in total and reduced ascorbate compared to moderate and high external salinities. Cell volumes remained unchanged, except at the lowest salinity where they doubled. Catalase, superoxide dismutase, dehydroascorbate reductase and glutathione reductase activities were not altered by extreme salinities. The involvement of oxidative stress at both salinity extremes is implied by the alterations in antioxidant enzymes and substrates, but the specific changes are very different between hypo and hypersaline stresses.     

Photosynthetic carbon partitioning and lipid production in the oleaginous microalga Pseudochlorococcum sp. (Chlorophyceae) under nitrogen-limited conditions

Photosynthetic carbon partitioning into starch and neutral lipid was investigated in the oleaginous green microalga Pseudochlorococcum sp. When grown under low light and nitrogen-replete conditions, the algal cells possessed a basal level of starch. When grown under high light and nitrogen-limited conditions, starch synthesis was transiently up-regulated. After nitrogen depletion, starch content decreased while neutral lipid rapidly increased to 52.1% of cell dry weight, with a maximum neutral lipid productivity of 0.35 g L⁻¹ D⁻¹. These results suggest that Pseudochlorococcum used starch as a primary carbon and energy storage product. As nitrogen was depleted for an extended period of time, cells shift the carbon partitioning into neutral lipid as a secondary storage product. Partial inhibition of starch synthesis and degradation enzymes resulted in a decrease in neutral lipid content, indicating that conversion of starch to neutral lipid may contribute to overall neutral lipid accumulation. Biotechnological application of Pseudochlorococcum sp. as a production strain for biofuel was assessed.     

Long-term outdoor growth and lipid productivity of Tetraselmis suecica, Dunaliella tertiolecta and Chlorella sp (Chlorophyta) in bag photobioreactors

There has been considerable interest on cultivation of green microalgae (Chlorophyta) as a source of lipid that can alternatively be converted to biodiesel. The ideal microalga characteristics are that it must grow well even under high cell density and under varying outdoor environmental conditions and be able to have a high biomass productivity and contain a high oil content (~25–30 %). The main advantage of Chlorophyta is that their fatty acid profile is suitable for biodiesel conversion. Tetraselmis suecica CS-187 and Chlorella sp. were grown semi-continuously in bag photobioreactors (120 L, W?×?L?=?40?×?380 cm) over a period of 11 months in Melbourne, Victoria, Australia. Monthly biomass productivity of T. suecica CS-187 and Chlorella sp. was strongly correlated to available solar irradiance. The total dry weight productivity of T. suecica and Chlorella sp. was 110 and 140 mg L^?1 d^?1, respectively, with minimum 25 % lipid content for both strains. Both strains were able to tolerate a wide range of shear produced by mixing. Operating cultures at lower cell density resulted in increasing specific growth rates of T. suecica and Chlorella sp. but did not affect their overall biomass productivity. On the other hand, self shading sets the upper limit of operational maximum cell density. Several attempts in cultivating Dunaliella tertiolecta CS-175 under the same climatic conditions were unsuccessful.     

Regulation of phytochelatin synthesis by zinc and cadmium in marine green alga,Dunaliella tertiolecta

Although Cd(2+) is a more effective inducer of phytochelatin (PC) synthesis than Zn(2+) in higher plants, we have observed greater induction of PC synthesis by Zn(2+) than Cd(2+) in the marine green alga, Dunaliella tertiolecta. To elucidate this unique regulation of PC synthesis by Zn(2+), we investigated the effects of Zn(2+) and Cd(2+) on the activities of both phytochelatin synthase (PC synthase) and enzymes in the GSH biosynthetic pathway. PC synthase was more strongly activated by Cd(2+) than by Zn(2+), but the difference was not very big. On the other hand, gamma-glutamylcysteine synthetase (gamma-ECS) and glutathione synthetase (GS) were activated by both heavy metals, but their activities were higher in Zn-treated cells than in Cd-treated cells. Dose-dependent stimulation of intracellular reactive oxygen species (ROS) production was observed with Zn(2+), but not Cd(2+) treatment. These results suggest that Zn(2+) strongly promotes the synthesis of GSH through indirect activation of gamma-ECS and GS by stimulating ROS generation. This acceleration of the flux rate for GSH synthesis might mainly contribute to high level PC synthesis.     

Genome Size Affects Fitness in the Eukaryotic Alga Dunaliella tertiolecta

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