Effect of nitrate-nitrogen limitation on photosynthesis of the diatom Phaeodactylum tricornutum Bohlin (Bacillariophyceae)

Abstract Nitrate limited growth of the diatom Phaeodactylum tricornutum in chemostat cultures produced marked changes in biochemical composition and a six-fold reduction in the specific growth rate. This was associated with a reduction in the carbon and chlorophyll a specific light saturated rates, with little effect on light limited photosynthesis. Variations in specific growth rate were quantitatively related to carbon specific net photosynthesis and maximum chlorophyll a specific light saturated rates were positively correlated with cell nitrogen contents. The correlation between nitrogen content and photosynthesis for P. tricornutum and the differential effect of nitrogen supply on the light response curve of photosynthesis is qualitatively and quantitatively similar to published results for terrestrial vascular plants. There was little change in the photon (quantum) yield of photosynthesis which was not significantly different from 0.125mol O₂ mol photon^-1 the theoretical upper limit based on the Z scheme, even under severe nitrate deficiency. The capacity to maintain a high photon yield under nitrate limitation is discussed in relation to the nitrogen requirements of the stromal and membrane components of the photosynthetic apparatus.     

Mixotrophic growth of Phaeodactylum tricornutum on glycerol: growth rate and fatty acid profile

Mixotrophic growth of the eicosapentaenoic acid (EPA)producing diatom Phaeodactylum tricornutum UTEX640 was carried out in 1-L batch cultures under anexternal irradiance of 165 mol photons m^-2s^-1 by supplementing the inorganic culture mediumwith glycerol. The effect on the growth and the fattyacid profile was studied for different initialglycerol concentrations (0–0.1 M). The optimalglycerol concentration was 0.1 M.A lag phase was observed at high glycerolconcentrations. The present study also shows thatsuccessive additions of glycerol at 0.1M concentrationand using ammonium chloride as a nitrogen sourceremarkably increased the maximum biomass concentration(16.2 g L^-1) and maximum biomass productivity(61.5 mg L^-1 h^-1). These values wererespectively 9 and 8-fold higher than in thephotoautotrophically grown control. The level ofsaponifiable lipids in mixotrophically cultured cellswas significantly higher than in photoautotrophicallycultured cells and increased with the glycerolconcentration in the medium. The concentration ofstorage lipids, saturated and monounsaturated fattyacids, were enhanced but the EPA content did notchange significantly. The EPA content was around 2.2%of biomass dry weight. The maximum EPA yield was33.5 mg L^-1 d^-1 and was obtained in aculture containing 0.1 M glycerol, supplementedperiodically by ammonium chloride. This productivitywas 10-fold higher than the EPA productivity obtainedunder mixotrophic conditions.     

Effects of agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum

The effect of mechanical agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum was investigated in aerated continuous cultures with and without the added shear protectant Pluronic F68. Damage to cells was quantified through a decrease in the steady state concentration of the biomass in the photobioreactor. For a given aeration rate, the steady state biomass concentration rose with increasing rate of mechanical agitation until an upper limit on agitation speed was reached. This maximum tolerable agitation speed depended on the microalgal species. Further increase in agitation speed caused a decline in the steady state concentration of the biomass. An impeller tip speed of >1.56 m s^–1 damaged P. tricornutum in aerated culture. In contrast, the damage threshold tip speed for P. cruentum was between 2.45 and 2.89 m s^–1. Mechanical agitation was not the direct cause of cell damage. Damage occurred because of the rupture of small gas bubbles at the surface of the culture, but mechanical agitation was instrumental in generating the bubbles that ultimately damaged the cells. Pluronic F68 protected the cells against damage and increased the steady state concentration of the biomass relative to operation without the additive. The protective effect of Pluronic was concentration-dependent over the concentration range of 0.01–0.10% w/v.     

Effects of Metal Combinations on the Production of Phytochelatins and Glutathione by the Marine Diatom Phaeodactylum tricornutum

Copper, Cd and Zn can be found at elevated concentrations in contaminated estuarine and coastal waters and have potential toxic effects on phytoplankton species. In this study, the effects of these metals on the intracellular production of the polypeptides phytochelatin and glutathione by the marine diatom Phaeodactylum tricornutum were examined in laboratory cultures. Single additions of Cu and Cd (0.4 μM Cu² and 0.45 μM Cd²⁺) to the culture medium induced the production of short-chained phytochelatins ((γ-Glu-Cys)_n-Gly where n = 2–5), whereas a single addition of Zn (2.2 μM Zn²⁺) did not stimulate phytochelatin production. Combination of Zn with Cu resulted in a similar phytochelatin production compared with a single Cu addition. The simultaneous exposure to Zn and Cd led to an antagonistic effect on phytochelatin production, which was probably caused by metal competition for cellular binding sites. Glutathione concentrations were affected only upon exposure to Cd (85% increase) or the combination of Cd with Zn (65% decrease), relative to the control experiment. Ratios of phytochelatins to glutathione indicated a pronounced metal stress in response to exposures to Cu or Cd combined with Zn. This study indicates that variabilities in phytochelatin and glutathione production in the field can be explained in part by metal competition for cellular binding sites.     

Photon requirement for growth of the diatom Phaeodactylum tricornutum Bohlin (Bacillariophyceae)

Abstract Photon requirements for growth (φ_g^?1) of the diatom Phaeodactylum tricornutum were determined under nutrient-sufficient conditions at two photon flux densities corresponding to light limited and near-saturating conditions for growth. The value of φ_g^?1 based on assimilated carbon was light-dependent and varied from 8.8 to 14.0 mol photon mol C^?1 with the minimum value at the lowest photon flux density. These results are lower than might be predicted for microalgal growth based on the Z scheme of photosynthesis. Conversion of these values for carbon fixation to estimates based on oxygen evolution is problematical due to uncertainty over the appropriate assimilatory quotient (Q_a= mol O₂ mol C^?1). Minimum values based on oxygen evolution rates ranged from 6.2 to 7.6 mol photon mol O₂^?1 using a Q_a of 1.41 mol O₂ mol C^?1 obtained by Myers (1980). These estimates are similar to our previous measurements for photosynthesis and indicate a high efficiency for light energy transforming reactions during growth. The values of (φ_g^?1 obtained in this work indicate a number of inadequacies in our understanding of the energetics of microalgal growth and are inconsistent with our present knowledge of photosynthetic energy coupling in plant cells.     

Content of photosynthetic pigments, growth, and cell size of microalga Phaeodactylum tricornutum in the copper-polluted environment

Effects of copper at concentrations of 0.13 and 0.25 mg/l on content of photosynthetic pigments, growth, size characteristics, and cell shape of Phaeodactylum tricornutum (Bacillariophyta) microalga were studied. The inhibitory effect of copper on growth parameters was shown to become stronger with increasing concentration of copper in the growth medium. In spite of considerable retardation of cell growth, the concentrations examined had no effect on the content of photosynthetic pigments though affected some morphometric parameters.     

Plastidic phosphoglycerate kinase from Phaeodactylum tricornutum: on the critical role of cysteine residues for the enzyme function

Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in photosynthetic organisms, catalyzing a key step in the Calvin cycle. We performed the molecular cloning of the gene encoding chloroplastidic PGKase-1 in the diatom Phaeodactylum tricornutum. The recombinant enzyme was expressed in Escherichia coli, purified and characterized. Afterward, it showed similar kinetic properties than the enzyme studied from other organisms, although the diatom enzyme displayed distinctive responses to sulfhydryl reagents. The activity of the enzyme was found to be dependent on the redox status in the environment, determined by different compounds, including some of physiological function. Treatment with oxidant agents, such as diamide, hydrogen peroxide, glutathione and sodium nitroprusside resulted in enzyme inhibition. Recovery of activity was possible by subsequent incubation with reducing reagents such as dithiothreitol and thioredoxins (from E. coli and P. tricornutum). We determined two midpoint potentials of different regulatory redox centers, both values indicating that PGKase-1 might be sensitive to changes in the intracellular redox environment. The role of all the six Cys residues found in the diatom enzyme was analyzed by molecular modeling and site-directed mutagenesis. Results suggest key regulatory properties for P. tricornutum PGKase-1, which could be relevant for the functioning of photosynthetic carbon metabolism in diatoms.     

Gram-scale purification of eicosapentaenoic acid (EPA, 20:5n-3) from wetPhaeodactylum tricornutum UTEX 640 biomass

Eicosapentaenoic acid (EPA, 20:5n-3) was obtained from the microalgaPhaeodactylum tricornutum following a three-step process: fatty acid extraction by direct saponification of wet biomass, polyunsaturated fatty acid (PUFA) concentration by formation of urea inclusion compounds and EPA isolation by preparative HPLC. Direct saponification of wet biomass was carried out with KOH-ethanol (96% v:v) (1 h, 60 °C), extracting 91% of the EPA. PUFAs were concentrated by the urea method with an urea/fatty acid ratio of 4:1 at a crystallization temperature of 28 °C using methanol as the urea solvent. An EPA concentration ratio of 1.5 (55.2/36.3) and recovery of 79% were obtained. This PUFA concentrate was used to obtain 95.8% pure EPA by preparative HPLC, using a reverse-phase column (C₁₈, 4.7 cm i.d. × 30 cm) and methanol-water (1% AcH) 80:20 w/w as the mobile phase. Ninety-seven per cent of EPA loaded was recovered and 70% EPA present in theP. tricornutum biomass was recovered in a highly pure form by means of this three-step downstream processing. In each of the HPLC preparative runs, 635 mg PUFA concentrate were loaded, obtaining 326 mg of a highly concentrated EPA fraction (2.46 g d^–1). Finally, a preliminary cost statement has been calculated.     

Effects of fertilizer-based culture media on the production of exocellular polysaccharides and cellular superoxide dismutase by <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> (Bohlin)

Microalgae cultures are receiving attention because of increasing biotechnological and biomedical production of active biomolecules. We evaluated various fertilizer-based culture media to scale up production of the marine microalga Phaeodactylum tricornutum for production of exocellular polysaccharides (EPS), soluble proteins, and cellular superoxide dismutase (SOD). The standard source of sodium nitrate was the same as that used in the synthetic f/2 culture medium and ammonium nitrate, urea, ammonium sulfate, and calcium nitrate as alternative sources of nitrogen. The maximum production of EPS was achieved in microalgae cells grown in the culture media containing 63 and 23% nitrogen from ammonium sulfate, and also in microalgae cells grown in the culture media containing 3% nitrogen from ammonium nitrate. The maximum production of cellular SOD was achieved in microalgae cells grown in the culture media containing 35 and 26% nitrogen from ammonium sulfate, and in the culture media containing 17% nitrogen from urea. The results suggest that it is possible to use a source of nitrogen, other than sodium nitrate, to scale up growth of P. tricornutum for production of EPS and SOD at reduced costs.     

Diffusion of Fe(II) from an iron propagation cage and its effect on tissue iron and pigments of macroalgae on the cage

Iron propagation cages were settled on sand and/or rock beds in coastal areas of Hokkaido. The cage was oxidized by dissolved oxygen and the released Fe(II) diffused into the seawater around the cage. Fe(II) concentrations in the range of 10–50 nM were detected within a 20-m distance around the cage. For comparison, in the Japan Sea, the total iron concentration is less than 2 nM.Laminaria japonica was grown in an indoor semi-continuous culture system. The critical Fe level for maintaining maximum growth, and the subsistence Fe level for survival were measured. The concentrations obtained were 14–21 and 8 g Fe g^–1 tissue, respectively. Iron found inL. japonica growing on rocks and/or rock beds in the Japan Sea was close to the subsistence level. However, the Fe level inL. japonica on the cage in the Japan Sea was considerably higher. The concentrations of chlorophyll-a and fucoxanthin collected from the cage were significantly higher for sporophytes, demonstrating that iron is a very important element for the growth of seaweeds.     

Immunocytochemical localization of photosystem I and the fucoxanthin-chlorophylla/c light-harvesting complex in the diatomPhaeodactylum tricornutum

Summary iserum against two polypeptides of the major fucoxanthin-chlorophylla/c light-harvesting complex of the diatomPhaeodactylum tricornutum and heterologous antiserum against purified photosystem I particles of maize were used to localize these two complexes on the thylakoid membranes ofP. tricornutum. As in many chromophyte algae, the thylakoids are loosely appressed and organized into extended bands of three, giving a ratio of 21 for appressed versus non-appressed membranes. Immunoelectron microscopy demonstrated that the fucoxanthin-chlorophylla/c light-harvesting complex, which is believed to be associated with photosystem II, was equally distributed on the appressed and non-appressed thylakoid membranes. Photosystem I was also found on both types of membranes, but was slightly more concentrated on the two outer non-appressed membranes of each band. Similarly, photosystem I activity, as measured by the photooxidation of 3,3-diaminobenzidine, was higher in the outer thylakoids than in the central thylakoid of each band. We conclude that the thylakoids of diatoms differ from those of green algae and higher plants in their macromolecular organization as well as in their morphological arrangement.Abbreviations BSA bovine serum albumin - DAB 3,3-diaminobenzidine - FCPC fucoxanthin-chlorophylla/c light-harvesting complex - LHC light-harvesting complex - PBS phosphate-buffered saline - PS photosystem     

Impacts of chlorination and heat shocks on growth, pigments and photosynthesis of Phaeodactylum tricornutum (Bacillariophyceae)

The main impacts of cooling water from thermal (nuclear) power plants on aquatic organisms were caused by chlorination and temperature increase. In this study, we investigated the impacts of residual chlorine and short-term heat shocks on growth, pigment contents and photosynthesis of Phaeodactylum tricornutum. Growth of P. tricornutum was completely inhibited; Chlorophyll a and carotenoids contents deceased about 63.3% and 61.4% in 24 h treated with 0.2 mg L^− 1 chlorine. The negative effects of chlorination increased with enhanced concentration and prolonged exposure time. Relative electrode transfer rate (rETR) of P. tricornutum was significantly suppressed when treated with 0.2 mg L^− 1 residual chlorine for 24 h. Furthermore, the effective quantum yield (F_v'/F_m') decreased first but then recovered with prolonged exposure when residual chlorine ranged between 0.1 and 0.2 mg L^− 1. The cells were less sensitive to heat shocks compared with chlorination: the rETR and F_v'/F_m' was suppressed only when the temperature exceeded 35 °C for 1 h. When P. tricornutum was exposed to chlorination combined with heat shocks, the rETR was further inhibited at 35 °C. It indicated that both chlorination and heat shocks had negative impacts on the primary producers living in discharging coastal waters; furthermore, there were synergistic effects of heat shocks on chlorination toxicity.     

Nannochloropsis (Eustigmatophyceae) as source of commercially valuable pigments

Pigment composition and its variation with culture agewere analyzed in six strains of Nannochloropsis(Eustigmatophyceae). The capacity for accumulationof the ketocarotenoids astaxanthin and canthaxanthinwas higher in N. salina and N. gaditanathan in the other strains studied here. Theinfluence of salinity (15 to 100 practical units) onpigment production was studied in N. gaditana,where a defined pattern of variation could not befound apart from a notable increase in zeaxanthin at100. In cultures grown in a photobioreactor and athigh cell densities of about 10⁹ cells mL^-1,pigment production reached: 350 mg L^-1 forchlorophyll a, 50 mg L^-1 for violaxanthin,5 mg L^-1 for canthaxanthin, 3 mg L^-1 forastaxanthin. The highest contents of canthaxanthin andastaxanthin obtained in experiments with N.gaditana were 19.4 and 14.6 ng pigment (10⁶cells)^-1, respectively, which accounts for 0.7%dry weight. By means of xanthophyll cycle inductionthrough exposure of cells to high irradiance and at40 ^°C, conversion of violaxanthin intozeaxanthin may attain up to 70% of the violaxanthincontent, which corresponds to 0.6% dry weight. Theresults indicate that interest in Nannochloropsis as a source of valuable pigments isnot related to its capacity for single pigmentaccumulation, but the availability of a range ofpigments such as chlorophyll a, zeaxanthin,canthaxanthin and astaxanthin, each with highproduction levels.     

Productivity analysis of outdoor chemostat culture in tubular air-lift photobioreactors

Net productivity and biomass night losses in outdoor chemostat cultures ofPhaeodactylum tricornutum were analyzed in two tubular airlift photobioreactors at different dilution rates, photobioreactor surface/volume ratios and incident solar irradiance. In addition, an approximate model for the estimation of light profile and average irradiance inside outdoor tubular photobioreactors was proposed. In both photobioreactors, biomass productivity increased with dilution rate and daily incident solar radiation except at the highest incident solar irradiances and dilution rates, when photoinhibition effect was observed in the middle of the day. Variation of estimated average irradiance vs mean incident irradiance showed two effects: first, the outdoor cultures are adapted to average irradiance, and second, simultaneous photolimitation and photoinhibition took place at all assayed culture conditions, the extent of this phenomena being a function of the (incident)¹ irradiance and light regime inside the culture. Productivity ranged between 0.50 and 2.04 g L^–1 d^–1 in the tubular photobioreactor with the lower surface/volume ratio (S/V = 77.5 m^–1) and between 1.08 and 2.76 g L^–1 d^–1 in the other (S/V = 122.0 m^–1). The optimum dilution rate was 0.040 h^–1 in both reactors. Night-time biomass losses were a function of the average irradiance inside the culture, being lower in TPB0.03 than TPB0.06, due to a better light regime in the first. In both photobioreactors, biomass night losses strongly decreased when the photoinhibition effect was pronounced. However, net biomass productivity also decreased due to lower biomass generation during the day. Thus, optimum culture conditions were obtained when photolimitation and photoinhibition were balanced.     

Optimization of eicosapentaenoic acid production byPhaeodactylum tricornutumin the flat panel airlift (FPA) reactor

Biomass and eicosapentaenoic acid (EPA) productivities were investigated in a flat panel airlift loop reactor ideally mixed by static mixers. Growth with ammonium, urea and nitrate as nitrogen source were performed at different aeration rates. Cultures grew on ammonium but the decay of pH strongly inhibited biomass increase. On urea biomass productivity reached 2.35 g L^–1d^–1at an aeration rate of 0.66 vvm (24 h light per day, 1000 mol photon m^–2s^–1). Aeration rates between 0.33 vvm and 0.66 vvm and maximal productivities on urea were linearly dependent. Productivity on nitrate never exceeded 1.37 g L^–1d^–1. In the range of maximum productivity photosynthesis efficiency of 10.6% was reached at low irradiance (250 mol photon m^–2s^–1). Photosynthesis efficiency decreased to 4.8% at 1000 mol photon m^–2s^–1. At these high irradiances the flat panel airlift reactor showed a 35% higher volume productivity than the bubble column. At continuous culture conditions the influence of CO₂concentration in the supply air was tested. Highest productivities were reached at 1.25% (v/v) CO₂where the continuous culture yielded 1.04 g L^–1d^–1(16 h light per day, 1000 mol photon m^–2s^–1). The average EPA content amounted to 5.0% of cell dry weight, that resulted in EPA productivities of 52 mg L^–1d^–1(continuous culture, 16 h light per day) or 118 mg L^–1d^–1(batch culture, 24 h light per day).     

Characterization of cadmium- and lead- phytochelatin complexes formed in a marine microalga in response to metal exposure

Phytochelatins (PC_n) are thiol-containing peptides with general structure (-Glu-Cys)_n-Gly enzymatically synthesized by plants and algae in response to metal exposure. They are involved in the cellular detoxification mechanism for their capability to form stable metal-phytochelatin complexes. The speciation of Cd and Pb complexes with phytochelatins has been studied in laboratory cultures of the marine diatom Phaeodactylum tricornutum. An approach based on size-exclusion chromatography (SEC) with off-line detection of phytochelatins, by reverse-phase HPLC, and metal ion, by atomic absorption spectrometry, has been used. The formation of Cd- and Pb-PC_n complexes with n-value from 3 to 6 was demonstrated. The metal-PC_n complexes formed with Cd appear to be different from those formed with Pb for the number of molecules of peptide involved in the complex and for the amount of the metal ion bound. The chromatographic behaviour of metal-PC_n complexes is consistent with Pb-PC_n complexes in which only a molecule of peptide binds the metal ion, and with Cd-PC_n complexes containing two or more molecules of peptide. The metal/peptide molar ratio in Cd-PC_n complexes was higher that in Pb-PC_n complexes. The formation of Cd- or Pb-PC₂ complexes was not demonstrated, probably for a dissociation during the cellular extract preparation. The effectiveness of phytochelatins in the detoxification of these two metal ions in this alga is discussed.     

Field observations in the Oosterschelde (The Netherlands) onCoscinodiscus concinnus andCoscinodiscus granii (Bacillariophyceae) infected by the marine fungusLagenisma coscinodisci (Oomycetes)

During 1985–1990Coscinodiscus concinnus andCoscinodiscus granii from the Oosterschelde were infected by the marine fungusLagenisma coscinodisci, although not every year with the same intensity. Infected cells were only observed during the period July–October at water temperatures between 13.2 and 20.2°C. In 1986 and 1987 the course of the infection withL. coscinodisci inC. concinnus andC. granii, was recorded at three stations. The highest infection percentages varied between 22.2 and 58.3% inC. concinnus and between 7.1 and 41.9% inC. granii. It is concluded that the water temperature may play an important role in the appearance ofL. coscinodisci inC. concinnus andC. granii and that silicate limitation is of minor importance. Finally it is discussed that the infection was not of great importance for the phytoplankton community in the Oosterschelde.     

Die Lage des Chromatophors in Beziehung zur Systematik vonCymbella-Arten (Bacillariophyceae)

InCymbella the position of the chromatophore corresponds to the taxonomic differentiation into sections. The pyrenoid always shows the same right hand — left hand inclination.

     

Examination of the type materials ofNavicula subtilissima Cleve (Bacillariophyceae)

Navicula subtilissima was described by Cleve as a new species from ‘a dry rivulet at the shore of Imandra, at the foot of Chibinä, Lapland’. However, the examination of the type slides and the unmounted original material using transmission and scanning electron microscopy revealed that there are at least three finely striated similar taxa distinguishable at the specific level. From these taxa, application of the nameN. subtilissima Cleve is established by selecting a lectotype; a second one is described as a new species,N. parasubtilissima, and the third is identified asN. subtilissima var.micropunctata Germain. The last one warrants a specific status by its fine structural differences in having continuous striae consisting of a single elongate areola occluded by a hymen with perforations arranging hexagonally. The occurrence of these three species is shown in Japanese collections.