Marine Diatom Phaeodactylum Tricornutum Bohlin Research Articles

Hormesis-like growth and photosynthetic physiology of marine diatom Phaeodactylum tricornutum Bohlin exposed to polystyrene microplastics

Hormesis-like growth and photosynthetic physiology of marine diatom Phaeodactylum tricornutum Bohlin exposed to polystyrene microplastics

Pilot-scale production of antibacterial substances by the marine diatom Phaeodactylum tricornutum Bohlin

The production and extraction of antibacterial substances from Phaeodactylum tricornutum biomass were tested on a pilot scale in a photo-bioreactor greenhouse installation. Using column chromatography two antibacterial active fractions were obtained from the algal biomass: a pure fraction of eicosapentaenoic acid (EPA) and a potentially new antibacterial substance(s) (identified as fraction B) produced by P. tricornutum. Both fractions exhibited antibacterial activities against three human pathogens: Vibrio vulnificus, V. parahaemolyticus, and V. cholerae. Minimal inhibitory concentrations against Bacillus subtilis of active fractions and one antibacterial commercial product were determined revealing a higher antibacterial activity of EPA in comparison to the commercial product. Consequently, EPA as well as the raw algal biomass with both EPA and the fraction B compounds could be used as food additives and feeds in aquaculture, poultry, and livestock breeding. Two types of phytohormones, methyl jasmonate and salicylic acid, as well as one phytotoxin, coronatine, were tested for induction of the defense system of P. tricornutum. However, only a minor change in the fatty acids profile and antibacterial effects of the treated cultures indicated that P. tricornutum must employ another strategy to regulate its antibacterial defense system.

The Effect of Ni2+, Co2+, Zn2+, Cd2+ and Hg2+ on the Growth Rate of Marine Diatom Phaeodactylum tricornutum Bohlin: Microplate Growth Inhibition Test

The toxicity tests were carried out with the unicellular marine diatom Phaeodactylum tricornutum Bohlin 1897 strain CCAP 1052/1A in the f/2 medium at 20°C under continuous white light exposed to irradiance by PAR 120 μ mol m-2s-1 measured with flat sensor. The basic design of toxicity tests followed the International Standard ISO 10253 marine algal growth inhibition test (ISO 1995) and were modified according to Lukavský (1992) and Lukavský and Simmer (2001). To determine the toxicity of metals (Ni2+, Co2+, Zn2+, Cd2+ and Hg2+), on the growth rate of marine phytoplankton diatom P. tricornutum Bohlin, nickel sulphate (NiSO4 • 6H2O), cobalt chloride (CoCl2 • 6H2O), zinc sulphate (ZnSO4 • 7H2O), cadmium nitrate (Cd(NO3)2 • 4H2O), mercury chloride (HgCl2) and mercury sulfate (HgSO4) of analytical grade (99%, Kemika, Zagreb) and cobalt nitrate (Co(NO3)2 • 6H2O) of analytical grade (99%, Fluka) were used. By comparing these toxicity curves the differences in slopes could be observed. Zn, Cd, Co and Ni have a gradual “ slow” curve, while Hg has a “ sharp” one thus indicating high toxicity of Hg. The following order of toxicity was achieved: Hg>Cd>Ni>Co>Zn in the exponential phase of growth (72 h), and Hg>Ni>Co>Zn>Cd after 336 h of experiment duration (in the stationary phase of growth) with mercury as the most toxic metal. Although toxic, the effect of Hg could be attenuated under chronic conditions.P. tricornutum in the exponential phase of growth showed more tolerance to nickel than cadmium. In the stationary phase of growth a high tolerance to cadmium, zinc and cobalt was observed, while nickel showed an increase in toxicity. Besides providing screening toxicity profiles this method can be useful in determining the effects of pollutants on cell growth and viability of organisms. Also, establishing the maximum tolerance levels for organisms, this method could be used for evaluating the potential impact of pollution on aquatic system.

Regulation of the expression of intracellular beta-carbonic anhydrase in response to CO2 and light in the marine diatom Phaeodactylum tricornutum.

Cells of the marine diatom Phaeodactylum tricornutum Bohlin (UTEX 642) grown in 5% CO(2) were transferred to air-level CO(2) in the light or dark and allowed to acclimate to air. No accumulation of the transcript of the P. tricornutum beta-carbonic anhydrase 1 (ptca1) was detected in 5% CO(2)-grown cells, but ptca1 mRNA accumulated and reached a peak after 6 h acclimation to air but decreased over the next 18 h. A similar accumulation time course was observed in cells air-acclimated in the dark, except that levels of mRNA were <50% those in the light. These results suggest that air-level [CO(2)] is required to trigger the transcription of ptca1 and that light affects the extent of acclimation. During acclimation to air for 120 h in the light, levels of ptca1 mRNA exhibited a periodic oscillation with a cycle of about 24 h, which, however, was not reflected in protein accumulation levels. A 5'-upstream region from the transcription-start site toward -1,292 bp of ptca1 was cloned by inverse polymerase chain reaction, and 5'-truncations were carried out on this fragment. The truncated promoter regions were fused with the beta-glucuronidase gene (uidA) and introduced into P. tricornutum. The promoter fragments, truncated at positions -1,292, -824, -484, -225, and -70 bp, conferred on transformants clear CO(2)-responsive beta-glucuronidase expressions. In contrast, the CO(2)-responsive regulation was severely impaired or completely abolished by truncations, respectively, at position -50 or -30 bp. These results indicate that critical cis-elements required for CO(2)-responsive transcription of ptca1 may be located between -70 and -30 bp relative to the transcription start site.

Identification and characterization of a new carbonic anhydrase in the marine diatom Phaeodactylum tricornutum

A cDNA encoding a new isoenzyme of β-type carbonic anhydrase (CA; EC 4.2.1.1) in the marine diatom Phaeodactylum tricornutum Bohlin has been cloned. The cDNA contained an open reading frame of 819 bp, which encodes a polypeptide of 273 amino acids. This gene, which is designated as ptca2, was found to be highly homologous (83% at the nucleotide level) to the previously isolated intracellular β-CA gene from Phaeodactylum tricornutum (ptca1). Comparison of the deduced amino acid sequence of ptca2 with β-CAs from other sources demonstrated that PtCA2 possesses the completely conserved zinc coordination residues of β-CA. The N-terminus 19 amino acid sequence of PtCA2 was predicted to be an endoplasmic reticulum-targeting signal, suggesting localization of the protein in an organelle or in the periplasmic space. Quantitative analysis of mRNA accumulation of ptca2 using real-time polymerase chain reaction revealed a significant level of mRNA accumulation even under 5% CO2 and a 3.5-fold increase in accumulation upon acclimation of the diatom to air. This indicates that ptca2 belongs to a constitutive class of enzyme that responds only weakly to the ambient CO2 concentration. The sequences of both ptca1 and ptca2 were shown to be grouped into a phylogeny that is composed of mixture of sequences from the eucarya and procarya domains, including sequences from the red alga Porphyridium purpureum, the green alga Coccomyxa, the red mold Neurospora crassa, and the yeast Saccharomyces cerevisiae.Key words: carbonic anhydrase, marine diatom, inorganic carbon concentrating mechanism (CCM), Phaeodactylum tricornutum.

EFFECT OF OLIGOMYCIN ON DARK RESPIRATION IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE): IMPLICATIONS FOR DETERMINATION OF MAINTENANCE RESPIRATION

Oligomycin is an inhibitor of the mitochondrial ATP synthase. In nitrogen-replete cells of the marine diatom Phaeodactylum tricornutum Bohlin, the rate of dark respiration was high and markedly inhibited (62%–74%) in the presence of oligomycin. In contrast, the rate of dark respiration in nitrogen-deprived cells was about half that in nitrogen-replete cells but was only slightly inhibited (16%–30%) by oligomycin. Consistent with these effects on rates of dark respiration, oligomycin decreased the ATP level and the ATP:ADP ratio by about 40% in nitrogen-replete cells incubated in darkness but had a negligible effect on the ATP level and ATP:ADP ratio in nitrogen-deprived cells. In sodium and nitrogen-deprived cells, the rate of dark respiration was greater than that in nitrogen-replete cells, but there was little effect of oligomycin on the rate of dark respiration. In light-limited cells, the rate of dark respiration was similar to that in nitrogen-deprived cells, but the inhibition (57%) in the presence of oligomycin was greater. These results suggest that most of the O2 consumption by nitrogen-replete cells was linked to mitochondrial ATP synthesis and that the rate of mitochondrial ATP synthesis in nitrogen-deprived and sodium and nitrogen-deprived cells was low. The potential implications of these results for our understanding of maintenance respiration are discussed.

Steady-states of semicontinuous cultures of a marine diatom: Effect of saturating nutrient concentrations

The evolution of nitrogen incorporation, the C–N ratio and chlorophyll content were studied in semicontinuous, light–dark synchronised cultures of the marine diatom Phaeodactylum tricornutum Bohlin, maintained under conditions of limiting to highly saturating nutrient concentrations and at different dilutions. The behaviour of cultures diverged from the expected since a decrease in the nitrogen cellular quota and a stable chlorophyll a–C ratio was recorded under nutrient-sufficient conditions, indicating that current theories on continuous cultures cannot be applied directly to semicontinuous cultures when maintained under highly nutrient-saturated conditions.

Removal of cadmium ions by the marine diatom Phaeodactylum tricornutum Bohlin accumulation and long-term kinetics of uptake

The marine diatom Phaeodactylum tricornutum Bohlin was exposed to different cadmium concentrations (1–100 mg l −1) for 4 days. The amount of cadmium removed was recorded, with particular attention paid to long-term uptake kinetics, and to the cellular location of cadmium. Cadmium accumulation occurred at all concentrations assayed. The EC 50 of cadmium to P. tricornutum was 22·39 mg l −1 after 4 days of exposure. Cadmium uptake followed a saturation kinetic at cadmium concentrations ≥25 mg l −1. However, at lower cadmium concentrations, the uptake of this metal followed a linear trend for all days of culture. At cadmium concentrations in the medium lower than 25 mg l −1, P. tricornutum removed cadmium mainly within the cell. At higher cadmium concentrations, the amount of cadmium removed by adsorption to the cell surface was higher than intracellular cadmium, because of the toxic effects of cadmium on P. tricornutum cells. This toxicity reduced the cadmium accumulation within the cells.

Long-chain class III metallothioneins as a mechanism of cadmium tolerance in the marine diatom Phaeodactylum tricornutum Bohlin

Cadmium tolerance of the marine diatom Phaeodactylum tricornutum Bohlin was studied. P. tricornutum growth was significantly reduced by cadmium concentrations of 5 mg l −1 or higher. The EC 50 value was 22.39 mg l −1 of cadmium after four days of exposure. Therefore, P. tricornutum is more tolerant to cadmium than other microalgal cells. In response to exposure to cadmium, P. tricornutum synthesize class III metallothioneins which sequester the metal in a harmless form. However, cadmium tolerance of P. tricornutum cells is not only due to the ability of this microalga to synthesize class III metallothioneins (γ-Glu-Cys) n -Gly, but also because these metallothioneins are composed of long-chain polypeptides. The cadmium-metallothionein complexes of P. tricornutum cells contain predominantly polypeptides of n = 4−9. Long-chain metallothioneins are more efficient in binding heavy metals. Capillary electrophoresis was used to separate class III metallothioneins. This is a recent technique used to separate many molecules. With this technique, the length of the separated polypeptides can also be estimated.

Dissolved inorganic carbon utilization and the development of extracellular carbonic anhydrase by the marine diatom Phaeodactylum tricornutum.

The presence of extracellular carbonic anhydrase (CA) in relation to medium composition was investigated using cultures of the marine diatom Phaeodactylum tricornutum Bohlin. Large-volume cultures, with low initial cell inocula were grown on ASP-2 (no dissolved inorganic carbon (DIC), 550 μM NOa - ), f/2 (2-0 mM DIC, 880μM NO3 - ) and modified f/2 (2.0 mM DIC, 20 μM NO3 - media. Cells growing on ASP-2 showed extracellular CA in the early stages of growth, whereas extracellular CA was not detected until partial depletion of total DIC in the stationary phase for cultures on f/2 or modified f/2. Both HCO3 - and CO2 were important in carbon limitation, extracellular CA being present when the free-CO2 concentration fell below 5 μM, but the HCO3 - concentration needed to be below 1 mM. When carbon-replete cells were transferred to carbon-limited conditions, extracellular CA was recorded within minutes, the process being light-dependent and completely inhibited by 3,3,4-dichlorophenyl-l, 1-dimethylurea (DCMU). The addition of DIC to carbon-limited cells resulted in a rapid decrease in extracellular CA activity. The membrane-impermeable inhibitor of carbonic anhydrase, dextran-bound sulphonamide (DBS) was used to inhibit extracellular CA activity in relation to photosynthetic rate in carbon-replete and carbon-limited cells. At the lowest DIC concentration (O'lOniM), for cells with maximum external CA activity, DBS gave over 80% inhibition of the photosynthetic rate, demonstrating the key role of external CA in maintaining high photosynthetic rate under conditions of carbon limitation. It is proposed that the key factor in the regulation of extracellular CA activity is the total Hux of inorganic carbon (C.) into the cell. This determines the Ci , flux into the chloroplast and when this is inadequate to support the photosynthetic rate attained by a carbon-replete chloroplast at optimum photon flux density, extracellular CA is activated. This mechanism would explain the observed interaction of CO2 and HCO3 - in the regulation of extracellular CA activity.

CHANGES IN CELL COMPOSITION AND LIPID METABOLISM MEDIATED BY SODIUM AND NITROGEN AVAILABILITY IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

ABSTRACTThe effects of nitrogen starvation in the presence or absence of sodium in the culture medium were monitored in batch cultures of the marine diatom Phaeodactylum tricornutum Bohlin. During nitrogen starvation in the presence of sodium, cell nitrogen and chlorophyll a decreased, mainly as a consequence of continued cell division. These decreases were accompanied by decreases in the rates of photosynthesis and respiration. There was no change in either cell volume or carbohydrate, but both carbon and lipid increased. During nitrogen starvation in the absence of sodium, cell division ceased. Cell nitrogen and chlorophyll a remained constant, and respiration did not decrease, but the changes in the photosynthetic rate and the lipid content per cell were similar to cultures that were nitrogen‐starved in the presence of sodium. The carbon‐to‐nitrogen ratio increased in both cultures. Nitrogen, in the form of nitrate, and sodium were resupplied to cultures that had been preconditioned in nitrogen‐ and sodium‐deficient medium for 5 d. Control cultures to which neither nitrate or sodium were added remained in a static state with respect to cell number, volume, and carbohydrate but showed slight increases in lipid. Cells in cultures to which 10 mM nitrate alone was added showed a similar response to cultures where no additions were made. Cells in cultures to which 50 mM sodium alone was added divided for 2 d, with concomitant small decreases in all measured constituents. Cell division resumed in cultures to which both sodium and nitrate were added. The lipid content fell dramatically in these cells and was correlated to metabolic oxidation via measured increases in the activity of the glyoxylate cycle enzyme, isocitrate lyase. We conclude that lipids are stored as a function of decreased growth rate and are metabolized to a small extent when cell division resumes. However, much higher rates of metabolism occur if cell division resumes in the presence of a nitrogen source.

Eicosapentaenoic acid-rich biomass production by the microalga Phaeodactylum tricornutum in a continuous-flow reactor

The marine diatom Phaeodactylum tricornutum Bohlin is a potential source of the pharmaceutically valuable ω3 polyunsaturated fatty acid eicosapentaenoic acid (EPA). The results of indoor continuous growth of Phaeodactylum tricornutum Bohlin are reported. The relationships between dilution rate ( D), nitrate concentration and chemical composition were studied. Higher biomass and lipid productivities were obtained at low D values. EPA was found to be an intermediate metabolite and the best productivity (6 mg l −1 day −1 was achieved for D values ranging from 0·32 to 0·50 day −1. Under optimum conditions, 84 and 11%, respectively, of total recovered EPA were present in monogalactosyldiacylglycerol (MGDG) and in triacylglycerol (TG) moieties, respectively. Recorded EPA/AA and EPA/20·4 ω3 ratios for all tested dilution rates were among the highest values ever reported, showing EPA purification to be easier to perform from this starting material than from many others commonly in use.

In Situ Glutamine Synthetase Activity in a Marine Unicellular Alga (Development of a Sensitive Colorimetric Assay and the Effects of Nitrogen Status on Enzyme Activity).

A malachite green colorimetric assay for glutamine synthetase is described. Glutamine synthetase activity was determined in situ in the marine diatom Phaeodactylum tricornutum Bohlin using cells permeabilized by freeze/thawing. Higher activities were obtained with cells permeabilized in N-2-hydroxyethylpiperazine-N[prime]-2-ethanesulfonic acid compared with N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, tris(hydroxymethyl)aminomethane, or imidazole, and the optimum pH was 7.9. Activities were higher in cells permeabilized in the presence of reductant, particularly dithiothreitol. Glutamine synthetase activities were markedly decreased in the presence of methionine sulfoximine. In the presence of saturating concentrations of glutamate and ATP, the apparent Km for ammonia was 320 [mu]M, but this value decreased to 110 [mu]M with subsaturating concentrations of glutamate and ATP. The apparent Km values for glutamate and ATP, in the presence of saturating concentrations of ammonia, were 9.7 and 2.9 mM, respectively. Ammonia-grown cells had lower glutamine synthetase activities than did nitrate-grown cells. During nitrogen starvation of both ammonia- and nitrate-grown cells, glutamine synthetase activities increased rapidly during the first 8 h, reaching maximum values after 24 to 48 h. Moreover, the time course for the increases in glutamine synthetase activities and rate of methylamine uptake following the transfer of nitrate-grown cells to nitrogen-deficient medium were very similar. In nitrate-grown cells and cells deprived of combined nitrogen, glutamine synthetase activities and maximum rates of ammonia uptake gave comparable values when measured at the same temperature (20[deg]C).

Arrest of cell division but not protein synthesis in sodium-deficient cells of the marine diatom Phaeodactylum tricornutum Bohlin

Cell division in the marine diatom Phaeodactylum tricornutum was prevented when cultures were maintained in the absence of sodium, regardless of the nitrogen status of the cells or medium. Addition of 10 mM ammonium and 50 mM sodium to cultures preconditioned in nitrogen and sodium-deficient medium for 5 d led to a recovery in cell division and chlorophyll a, and net protein synthesis. Sodium added in the absence of ammonium led to a recovery in cell division, but not net protein synthesis. Ammonium added in the absence of sodium was partially assimilated (as NH3) and resulted in a small amount of protein synthesis, but without cell division. This effect was enhanced if the cells had lower protein quotas prior to ammonium addition, with total consumption of the added 1 mM ammonium and appreciable net protein synthesis. Respiration was enhanced by 1 or 10 mM ammonium or 10 mM methylammonium addition to nitrogen-deficient cultures maintained in the presence or absence of sodium. In contrast to respiration, photosynthesis was inhibited by these additions in sodium-replete cultures, but was enhanced in sodium-deficient cultures.

INTRACELLULAR AND EXTRACELLULAR AMINO ACID POOLS OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) GROWN ON UNENRICHED SEAWATER IN HIGH‐CELL‐DENSITY DIALYSIS CULTURE1

ABSTRACTThe consumption of inorganic macronutrients (NO3−+ NO2−, NH4+, and PO4−3) and the composition of intra‐ and extracellular dissolved free amino acid pools (IDFAA and EDFAA, respectively) were determined in continuous‐reservoir batch dialysis cultures of the marine diatom Phaeodactylum tricornutum Bohlin maintained on unenriched natural seawater as a growth medium. Nutrient diffusion (Nd), which equals the nutrient uptake of the culture, increased with the cell density and the age of the culture. A concentration of 6.77 × 107 cells · mL−1 was obtained in stationary phase, which coincided with the NO3−+ NO2− diffusion limit (Ndmax) of the dialysis apparatus. The Ndmax for NH4+ occurred much earlier, at the end of exponential growth, whereas Ndmax for PO4−3 was not attained during the growth cycle of the culture, even in early stationary phase. A significant depletion (77%) of the IDFAA pool during exponential phase was followed by a reestablishment–to approximately 60% of the initial level–of internal pools during linear and stationary growth phases. This recovery occurred during the illuminated portion of the photoperiod (12:12 h LD) and involved principally the amino acids GLN, GLU, β‐GLU, and ASN. The recovery of GLN and ASN levels was particularly significant, because the intracellular concentrations of these amino acids were higher at the end of the growth cycle than before. The EDFAA pool was generally dominated by the amino acids SER and GLY+THR; however, during active growth, ORN and LYS often constituted an important fraction. The EDFAA concentration increased until linear growth phase was reached, during which a higher concentration of total free amino acids was attained in darkness than under illumination. The EDFAA component diminished afterward, and in stationary phase this fraction returned to concentrations equivalent to those observed at the beginning of the growth cycle. The variations in EDFAA concentrations were expressed by a pronounced decrease in the cellular excretion of amino acids with increasing cell density. These cellular responses of Phaeodactylum tricornutum in dense culture, specifically the regulation of amino acid excretion and intracellular pool size, may affect the N‐conversion coefficient (YN). Consequently, by prolonging the linear phase of growth and reducing the concentration of autoinhibitory metabolites by diffusion, a markedly enhanced final cell density can be achieved in cultures grown on natural unenriched seawater.

Biosynthetically active glutamine synthetase in the marine diatom Phaeodactylum tricornutum: optimization of the forward-reaction assay

The activity of glutamine synthetase (GS) was measured in the marine diatom Phaeodactylum tricornutum Bohlin (Strain SME) by a biosynthetic assay, based on γ-glutamyl hydroxamate synthesis, and referred to as the forwardreaction assay. The effects of pH, temperature and different homogenizing buffer preparations on enzyme activity, linearity of reactions, and substrate-saturation kinetics were investigated. The resultant data provide the basis for establishing optimum experimental conditions for a standard assay. Affinities of P. tricornutum GS for glutamate, ATP and Mg2+ were similar to those recorded elsewhere for a variety of other phytoplankton species using “true” biosynthetic assays based on release of inorganic phosphate, whereas the affinity for hydroxylamine was two orders of magnitude lower than that for ammonium, with an apparent Km value in the millimolar range. This, together with negative results obtained during earlier attempts to detect GS activity in P. tricornutum using the “true” biosynthetic assay, indicates that the GS of this alga has a lower affinity for ammonium than that of other phytoplankton species. Dual substrate kinetics demonstrated that apparent Km and Vm values for glutamate were directly proportional to the concentration of ATP, thus giving indirect evidence of a correlation between GS activity and the adenylate energy charge. Comparisons between synthetase activities obtained with the optimized forward-reaction assay and transferase activities reported from other studies on various phytoplankton species revealed discrepancies which, to a great extent, probably arise from differences in the growth conditions of the organisms.

Effects of 9?10 dihydroanthracene and its biodegradation products on the marine diatom Phaeodactylum tricornutum

Growth, photosynthetic capacity and chlorophyll a content of the marine diatom Phaeodactylum tricornutum Bohlin were observed after exposure to the aromatic hydrocarbon 9–10 dihydroanthracene and its biodegradation products. Growth was inhibited after exposure to the aromatic hydrocarbon, whereas no inhibition occurred in the presence of the biodegradation products alone. The degradation products were found to enhance the chlorophyll a cellular content. Synergistic effects between dihydroanthracene and its biodegradation products increased the toxicity of this aromatic hydrocarbon.

The simultaneous assimilation of ammonium and l-arginine by the marine diatom Phaeodactylum tricornutum Bohlin

Nitrogen-replete cells of Phaeodactylum tricomutum Bohlin assimilated ammonium and the amino acid l-arginine simultaneously. Arginine was taken up at rates expected to supply at least 30% of the cells' requirement for nitrogen; arginme-carbon mainly entered protein but, when uptake was in darkness, ≈40% was respired. Cells grown in a 12:12 h light:dark cycle with ammonium as the sole nitrogen source took up ammonium throughout the growth cycle, whereas cells grown with the addition of arginine took up little ammonium during the dark phase. The uptake of ammonium over the course of the cycle was reduced by 30% when arginine was present. Cells grown with arginine as the sole nitrogen source took up the amino acid at the rate required for growth. In contrast, cells grown on ammonium, while growing at the same rate as those on arginine, assimilated nitrogen at twice the rate. Cells grown with both sources of nitrogen present, took up arginine at the same rate as before, but more of the arginine-carbon was respired (60% as compared with 40% when ammonium was absent). The uptake of ammonium was reduced by 30%, but the total nitrogen assimilation again exceeded immediate requirements. A high uptake rate of arginine was indicative of cells assimilating ammonium only; a low uptake rate of ammonium during the dark phase of growth was indicative of cells assimilating arginine. It is not known whether the findings with P. tricomutum are applicable to other marine phytoplankton. If they are, arginine may be of greater significance as a natural source of nitrogen for phytoplankton than is generally thought.

Light-limited growth of two strains of the marine diatom Phaeodactylum tricornutum Bohlin: Chemical composition, carbon partitioning and the diel periodicity of physiological processes

Two isolates of the marine pennate diatom Phaeodactylum tricornutum Bohlin were grown in semi-continuous, nutrient-sufficient culture at varying irradiances on a 12-h light, 12-h dark illumination cycle. The reponse of the isolates to varying degrees of light limitation differed with respect to all of the compositional parameters measured, including growth rates, elemental composition, chlorophyll content, and the partitioning of cellular carbon into four biochemical classes: proteins, lipids, polysaccharides, and low-molecular weight intermediates. The isolates also differed with respect to the relative contributions of light-period and dark-period uptake to the total uptake of ammonium and phosphate ions, although in all cases uptake took place at a reduced rate in the dark. They did not differ with respect to the diel periodicity of cell division, chlorophyll synthesis, and biochemical synthesis. Slightly more cell division took place during the dark period than during the light period. The specific rate of chlorophyll synthesis in the light period, when expressed as a function of irradiance, saturated rapidly; the rate was nearly constant for all irradiances > 100 βE · m −2 · s −1. Chlorophyll synthesis in the dark was positively correlated with irradiance over the entire range of irradiances, except where photoinhibition was involved. Protein was synthesized in both the light and dark periods, but at a reduced rate in the dark. Polysaccharides were synthesized during the light period and consumed during the dark period. Lipids and low molecular weight intermediates were synthesized during the light period, but showed little net change during the dark period.

Photosynthetic carboxylating enzymes in Phaeodactylum tricornutum: Assay methods and properties

Rapid freezing (in liquid nitrogen) of the marine diatom Phaeodactylum tricornutum Bohlin followed by thawing permits a convenient and sensitive measurement of the activities of carboxylating enzymes without the need to prepare a cell-free extract. Using this method, the properties of RuDP and PEP carboxylases have been compared with those assayed in cell-free extracts. The most significant difference was in the Michaelis' constants (K m 's); the values being lower in the freeze/thaw assay. The absolute rate of carbon-dioxide fixation by the enzymes was less than the rate of photosynthesis by the intact alga. Significantly, the activity of PEP carboxylase was comparable (in some experiments, greater) to that of RuDP carboxylase. The significance of this and the possibility of an enzymatic approach to measurements of marine primary productivity are discussed.