COMPARATIVE PHYSIOLOGICAL STUDY OF MARINE DIATOMS AND DINOFLAGELLATES IN RELATION TO IRRADIANCE AND CELL SIZE. I. GROWTH UNDER CONTINUOUS LIGHT1,2

Cell division rates and chlorophyll a and protein contents for ten diatom and dinoflagellate species were measured. Species were chosen to include a wide range of cell size in terms of both cell volume and cell protein: from 0.004 ng protein/cell for a small Chaetoceros sp. to 2.2 ng protein/cell for Prorocentrum micans Ehrenberg. Experiments were conducted in batch or semi-continuous cultures at 21 C under continuous illumination from 8–256 μEin ^.m^-2'^.s^-1. Light saturation of cell division occurred at 32–80 μEin m^-1 s^-1 for all species, with no observable difference between the two phylogenetic groups. When the light-saturated cell division rates were plotted against cell size as protein/cell, the diatoms and dinoflagellates fell on two separate lines with the diatoms having higher rates. Chl a /protein ratios (μg/μg) decreased with increasing irradiance. The diatoms had higher chl a per unit protein. The relationship between cell division rate and the chl a/protein ratio is discussed.     

THE IMPORTANCE OF DIATOM CELL SIZE IN COMMUNITY ANALYSIS1

The large variation in size and shape in diatoms is shown by morphometric measurements of 515 benthic and pelagic diatom species from the Baltic Sea area. The largest mean cell dimension (mostly the apical axis) varied between 4.2 and 653 μm, cell surface area between 55 and 344,000 μm², and cell volume between 21 and 14.2 × 10⁶μm³. The shape‐related index, length to width ratio, was between 1.0 and 63.3 and the shape‐ and size‐related index, surface area to volume ratio, was between 0.02 and 3.13. Diatom community analysis by multivariate statistics is usually based on counts of a fixed number of diatom valves with species scores irrespective of cell size. This procedure underestimates the large species for two reasons. First, the importance of a species with higher cell volume is usually larger in a community. Second, larger species usually have lower abundances and their occurrence in the diatom counts is stochastic. This article shows that co‐occurring small and large diatom species can respond very differently to environmental constraints. Large epiphytic diatoms responded most to macroalgal host species and small epiphytic diatoms most to environmental conditions at the sampling site. Large epilithic diatoms responded strongly to salinity, whereas small epilithic diatoms did so less clearly. The conclusion is that different scale‐dependent responses are possible within one data set. The results from the test data also show that important ecological information from diatom data can be missed when the large species are neglected or underestimated.     

RELATIONSHIP BETWEEN PHOTOSYNTHESIS AND CELL SIZE OF MARINE DIATOMS12

Three photosynthetic parameters of 7 species of marine diatoms were studied using Na₂¹⁴CO₃ at 5–8 C using log phase axenic cultures. The cell volumes of the different species varied from 70 μm³ to 40 × 10⁵μm³. The present experiment is consistent with the interpretation that the initial slope α (mg C · [mg chl a]^?1· h^?1· w^?1· m²) of photosynthesis vs. light curves is controlled by self-shading of chlorophyll a in the cell. Pm, the rate of photosynthesis at light saturation (mg C · [mg cell, C]^?1· h^?1) and R, the intercept at zero light intensity (mg C · [mg cell C]^?1· H^?1) are both dependent on the ratio of surface area to volume of cell.     

GROWTH CHARACTERISTICS AND ANTIOXIDANT PROPERTIES OF THE BENTHIC DIATOM NAVICULA INCERTA (BACILLARIOPHYCEAE) FROM JEJU ISLAND,KOREA1

Benthic diatoms are a commonly used food source in shellfish aquaculture. Diatoms of the genus Navicula are the most abundant benthic diatoms occurring year‐round on the coast of Jeju Island, Korea. We isolated an axenic strain of N. incerta Grunow; estimated its growth characteristics under 27 different combinations of temperature, salinity, and nutrients; and determined its biochemical composition and antioxidant activities. The maximum specific growth rate (μ_max), defined as the increase in cell density per unit time, was 0.81–1.04 · d^?1, and the maximum cell density, 7.99 × 10⁵ cells · mL^?1, was reached at 0.88 · d^?1 μ_max, 20°C, 30 psu salinity, and F/2 nutrient concentration on day 12 of the culture period. The approximate cellular composition was as follows: 7.0 ± 0.04% protein, 1.7 ± 0.28% lipid, 12.8 ± 0.85% carbohydrate, 68.4 ± 0.09% ash, and 10.1 ± 0.44% moisture. The antioxidant properties of N. incerta were determined for various extracts. The rates of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free‐radical scavenging for Neutrase and methanol extracts were 81.6% and 62.8%, respectively. Flavourzyme extract had a superoxide‐scavenging rate of 57.7%. Kojizyme and Ultraflo extracts had nitric‐oxide‐scavenging rates of 42.2% and 40.6%, respectively, significantly higher than commercial antioxidants, such as α‐tocopherol and butylated hydroxytoluene (BHT). The metal‐chelating activities of the methanol, Neutrase, and Termamyl extracts were 68.5%, 45.2%, and 41.2%, respectively, four to six times higher than commercial antioxidants. The Termamyl extract showed the highest linoleic acid peroxidation inhibition, exceeding α‐tocopherol and on par with BHT.     

SIZE DEPENDENCE OF GROWTH RATE,RESPIRATORY ELECTRON TRANSPORT SYSTEM ACTIVITY,AND CHEMICAL COMPOSITION IN MARINE DIATOMS IN THE LABORATORY1

The dependence of growth, electron transport system activity and chemical composition on the size of diatoms was examined during the exponential phase of growth. The six different marine centric species compared ranged in volume from 7.7 μm³ to 62 × 10⁵μm³. A size dependence was observed for growth, ¹⁴C uptake, respiration and the productivity index (¹⁴C/chl a). Although the size dependence of all parameters was similar, the results indicate that on a carbon basis, growth efficiency decreases with increasing size. The C/N and C/chl a ratios were not size dependent. The importance of the surface area to cell volume ratio, and the importance of carbon per unit volume in determining the observed size dependence are discussed.     

METABOLIC AND ECOLOGICAL CONSTRAINTS IMPOSED BY SIMILAR RATES OF AMMONIUM AND NITRATE UPTAKE PER UNIT SURFACE AREA AT LOW SUBSTRATE CONCENTRATIONS IN MARINE PHYTOPLANKTON AND MACROALGAE1

Marine phytoplankton and macroalgae acquire important resources, such as inorganic nitrogen, from the surrounding seawater by uptake across their entire surface area. Rates of ammonium and nitrate uptake per unit surface area were remarkably similar for both marine phytoplankton and macroalgae at low external concentrations. At an external concentration of 1 μM, the mean rate of nitrogen uptake was 10±2 nmol·cm^?2·h^?1 (n=36). There was a strong negative relationship between log surface area:volume (SA:V) quotient and log nitrogen content per cm² of surface (slope=?0.77), but a positive relationship between log SA:V and log maximum specific growth rate (μ_max; slope=0.46). There was a strong negative relationship between log SA:V and log measured rate of ammonium assimilation per cm² of surface, but the slope (?0.49) was steeper than that required to sustain μ_max (?0.31). Calculated rates of ammonium assimilation required to sustain growth rates measured in natural populations were similar for both marine phytoplankton and macroalgae with an overall mean of 6.2±1.4 nmol·cm^?2·h^?1 (n=15). These values were similar to maximum rates of ammonium assimilation in phytoplankton with high SA:V, but the values for algae with low SA:V were substantially less than the maximum rate of ammonium assimilation. This suggests that the growth rates of both marine phytoplankton and macroalgae in nature are often constrained by rates of uptake and assimilation of nutrients per cm² surface area.     

Growth and grazing rates of Protoperidinium hirobis Abe, a thecate heterotrophic dinoflagellate

Growth and feeding rates of a laboratory-reared small thecateheterotrophic dinoflagellate, Protoperidinium hirobis Abè,grown on the diatom Leptocylindrus danicus, were measured inbatch cultures. Ingestion rates were determined directly bythe enumeration of empty diatom frustules produced by dinoflagellatefeeding. Both growth and feeding rates saturated at diatom concentrationsof {small tilde} 10⁴ cells ml^–1, and reached maximum valuesof 1.7 divisions day^–1 and 23 diatoms grazer^–1 day^–1,respectively. This rate of cell division is notably high comparedto photosynthetic dinoflagellates, which seldom grow fasterthan 1 division day^–1. A maximal clearance rate of 0.5µl h^–1 was measured. Mean cell size varied proportionallywith food abundance, with food-saturated cells having doublethe mean volume of food-depleted cells. Tuning of cell divisionand grazing rate patterns were also examined; while mitosisoccurred chiefly during the dark period, no diel variationsin feeding rate were detected. These rates represent the firstdirect growth and ingestion measurements to be made for a thecateheterotrophic dinoflagellate. They serve to underscore one functionthese dinoflagellates perform within the microzooplanktonicfood web: that of transforming large diatoms into particlesmore easily ingested by microzooplankters.     

INFLUENCE OF SALINITY AND SULFATE ON THE TOXICITY OF CHROMIUM(VI) TO THE ESTUARINE DIATOM THALASSIOSIRA PSEUDONANA1

The acute toxicity of Cr(VI) to the diatom Thalassiosira pseudonana (Hasle and Heimdal) clone 3H was determined in artificial media of 3.2 and 0.32 ppt salinity and with variations of sulfate concentration in the media independent of salinity. Inhibitory concentrations of Cr(VI) ranged from 6.6 μM for growth rate and 4.9 μM for cell yield at 3.2 ppt salinity and 2.8 μM sulfate to 0.04 μM for growth rate and 0.02 μM for cell yield at 0.32 ppt salinity and 0.019 mM sulfate. The inhibition by Cr(VI) was a function of the ratio of Cr(VI) to sulfate. Inhibition occurred when-this ratio exceeded about 500:1. It is suggested that the mechanism for the toxicity of Cr(VI) to diatoms and perhaps other aquatic organisms involves a site at which sulfate and chromate compete.     

IMPORTANCE OF PHYSICAL VARIABLES ON THE SEASONAL DYNAMICS OF EPILITHIC ALGAE IN A HIGHLY SHADED CANYON STREAM1

The seasonal abundance of epilithic algae was correlated with major physico-chemical parameters in a first-order, heavily shaded stream in northern Arizona. Diatoms made up over 85%, by numerical abundance, of the epilithon community Light energy, water temperature, and stream discharge were most highly correlated with seasonal abundance of epilithic diatom taxa when analyzed with stepwise multiple regression. None of the chemical variables measured in the study (NO₃-N, O-PO₄, SiO₂, including PH) was found to be significantly correlated with the seasonal community structure of epilithic diatoms. Total diatom cell densities showed a significant negative correlation to stream bed light energy. Likewise, total diatom cell densities along a transect in the stream bed showed a negative correlation to current velocity during those months when base flow was low and stable, and current velocity was ≤25 cm·sec-¹. Most diatom taxa had highest cell densities at temperatures < 16°C and at daily mean stream bed light levels < 400 μE·m^?2·s^?1. Highest cell densities of green algae occurred at temperatures between 6–16°C and at daily mean stream bed light levels of > 400 μE·m^?2·s^?1. Blue-green algae (cyanobacteria) grew best at the highest recorded water temperatures and daily mean stream bed light energy (16–20°C and 900–1200 μE·m^?2·s^?1). Abrupt increases in NO₃-N coincided with a brief pulse of Nostoc pruniforme colonies during June, and leaf drop from Alnus oblongifolia during October.     

GROWTH RATES AND ULTRASTRUCTURE OF SILICEOUS SETAE OF CHAETOCEROS GRACILIS (BACILLARIOPHYCEAE) 12

The growth of setae in post-division Chaeloceros gracilis Schütt was triphasic. Following cell separation there was an initial lag phase of about 30 min after which setae grew linearly at rates ranging from 0.20 to 0.38 μm-min^?1. The growth rate was independent of whether diatoms cultured in medium containing 200 μM Si were transferred for experimentation to media containing 10 or 200 μM Si. When developing setae had attained a length of approximately 2 μm, their growth rate slowed as they entered a clearly defined plateau phase. The amount of silicon per unit length of setae was found to be 0.02 pg Si μm^?1, and the deposition rate was estimated as 0.005 Pg Si min^?1. Transmission electron microscopy revealed an axial structure resembling a microlubule extending the length of each seta and membrane bound polyphosphate bodies postulated to be the energy source for growth and associated biomineralization processes.     

Unimodal size scaling of phytoplankton growth and the size dependence of nutrient uptake and use

Phytoplankton size structure is key for the ecology and biogeochemistry of pelagic ecosystems, but the relationship between cell size and maximum growth rate (μ_max) is not yet well understood. We used cultures of 22 species of marine phytoplankton from five phyla, ranging from 0.1 to 10⁶ μm³ in cell volume (V_cell), to determine experimentally the size dependence of growth, metabolic rate, elemental stoichiometry and nutrient uptake. We show that both μ_max and carbon‐specific photosynthesis peak at intermediate cell sizes. Maximum nitrogen uptake rate (V_maxN) scales isometrically with V_cell, whereas nitrogen minimum quota scales as V_cell^0.84. Large cells thus possess high ability to take up nitrogen, relative to their requirements, and large storage capacity, but their growth is limited by the conversion of nutrients into biomass. Small species show similar volume‐specific V_maxN compared to their larger counterparts, but have higher nitrogen requirements. We suggest that the unimodal size scaling of phytoplankton growth arises from taxon‐independent, size‐related constraints in nutrient uptake, requirement and assimilation.     

EFFECT OF ZINC AVAILABILITY ON GROWTH,MORPHOLOGY, AND NUTRIENT INCORPORATION IN A COASTAL AND AN OCEANIC DIATOM1

We investigated the effect of Zn availability on growth rate (μ), cell morphology, and elemental stoichiometry and incorporation rate in two marine diatoms. For the coastal diatom Skeletonema costatum (Grev.) Cleve, the half‐saturation constant (K_S) for growth was 4.1 pM Zn²⁺, and growth ceased at ≤ 2.6 pM Zn²⁺, whereas for the oceanic diatom Thalassiosira oceanica Hasle, K_S was 0.5 pM Zn²⁺, and μ remained at ～40%μ_max even at 0.3 pM Zn²⁺. Under Zn‐limiting (Zn‐L) conditions, S. costatum decreased cell size significantly, leading to an 80% increase in surface area to volume ratio (SA/V) at Zn²⁺ of 3.5 pM compared to Zn‐replete (Zn‐R) conditions (at Zn²⁺ of 13.2 pM), whereas T. oceanica’s morphology did not change appreciably. Cell quotas of C, N, P, Si, and chl a significantly decreased under Zn limitation in S. costatum (at Zn²⁺ of 3.5 pM), whereas Zn limitation in T. oceanica (at Zn²⁺ of 0.3 pM) had little effect on quotas. Elemental stoichiometry was ～85C:10N:9Si:1P and 81C:9N:5Si:1P for S. costatum, and 66C:5N:2Si:1P and 52C:6N:2Si:1P for T. oceanica, under Zn‐R and Zn‐L conditions, respectively. Incorporation rates of all elements were significantly reduced under Zn limitation for both diatoms, but particularly for Si in S. costatum, and for C in T. oceanica, despite its apparent tolerance of low Zn conditions. With [Zn²⁺] in some parts of the ocean being of the same order (～0.2 to 2 pM) as our low Zn conditions for T. oceanica, our results support the hypothesis that in situ growth and C acquisition may be limited by Zn in some oceanic species.     

COMPARATIVE PHYSIOLOGICAL STUDY OF MARINE DIATOMS AND DINOFLAGELLATES IN RELATION TO IRRADIANCE AND CELL SIZE. II. RELATIONSHIP BETWEEN PHOTOSYNTHESIS,GROWTH, AND CARBON/CHLOROPHYLL a RATIO1,2

Photosynthetic rates, growth rates, cell carbon, cell protein, and chlorophyll a content of two diatom and two dinoflagellate species were measured. The microalgae were chosen to have one small and one large species from each phylogenetic group; the two size categories differed from each other by 1.5 orders of magnitude in terms of cell carbon or cell protein. The cultures for the experiments were grown under continuous light at an irradiance high enough for the light-saturation of growth for all four species. The four species were found to have similar maximum photosynthetic rates per unit chlorophyll a. The diatom species showed lower carbon/chlorophyll a ratios and higher photosynthetic rates per unit carbon than the dinoflagellates. The higher growth rates of the diatoms were shown to be related to their higher photosynthetic rates per unit carbon. The ecological significance of the physiological difference between these two groups of microalgae is discussed.     

KINETICS OF SILICON-LIMITED GROWTH IN THE FRESHWATER DIATOM ASTERIONELLA FORMOSA1,2

Growth rates of two clones of the freshwater planktonic diatom Asterionella formosa Hass. were measured under conditions in which external silicon concentrations controlled growth. Clone AfOH2 from Lake Ohrid, Yugoslavia, had a higher maximum growth rate (μ_max= 1.11 doublings/day) and apparent half-saturation constant (K_si] + Si_o= 1.93 μM Si) than clone L262 from Lake Windermere, England. (μ_max= 0.61 doublings/day; K_si+ Si_o= 1.09 μM Si). K_lim, the silicon concentration at μ= 0.9 μ_max, is 13.8 μM Si for clone AfOH2 and 6.5 μM Si for clone L262. These values agree well with published field observations showing A. formosa populations decreasing below 0.5 mg/l SiO₂ (= 8.4 μM Si). Calculations of yield gave a range of 0.5–1.5 μM Si/10⁶ cells for clone AfOH2 and 0.6–1.9 μM Si/10⁶ cells for clone L262.     

EFFECTS OF LIGHT AND SUBSTRATE ON THE BENTHIC DIATOMS IN AN OLIGOTROPHIC LAKE: A COMPARISON BETWEEN NATURAL AND ARTIFICIAL SUBSTRATES1

Benthic diatoms form a particularly important community in oligotrophic lakes, but factors influencing their distribution are not well known. This study reports the depth distribution of living motile and total diatoms (living plus dead diatoms) on both natural (from sand to fine organic mud) and artificial substrates in an oligotrophic lake. On artificial substrates, motile diatom densities peaked in abundance (24–30 cells · mm^?2) between 0.6 and 1.9 m depth; on natural sediment surfaces, motile diatoms were generally more numerous and peaked in abundance (925 cells · mm^?2) at 1.3 m depth. Total diatom densities on artificial substrates were highest (1260 valves · mm^?2) at 0.6 m depth, with very low values below 3 m depth; on natural sediment surfaces, total diatom abundances were generally much higher (21600 valves · mm^?2) at 3 m depth and declined gradually with depth. Significant relationships were found between light and diatom densities on the artificial substrate. Ordination analysis indicated that substrate type significantly correlated with the variation of diatom composition on artificial and natural substrates. Our results suggest that in oligotrophic lakes, light influences benthic diatom abundance, whereas substrate type has more influence on benthic diatom composition.     

COMPARISONS OF NITRATE UPTAKE, STORAGE, AND REDUCTION IN MARINE DIATOMS AND FLAGELLATES

Diatoms, but not flagellates, have been shown to increase rates of nitrogen release after a shift from a low growth irradiance to a much higher experimental irradiance. We compared NO₃ ^? uptake kinetics, internal inorganic nitrogen storage, and the temperature dependence of the NO₃ ^? reduction enzymes, nitrate (NR) and nitrite reductase (NiR), in nitrogen‐replete cultures of 3 diatoms (Chaetoceros sp., Skeletonema costatum, Thalassiosira weissflogii) and 3 flagellates (Dunaliella tertiolecta, Pavlova lutheri, Prorocentrum minimum) to provide insight into the differences in nitrogen release patterns observed between these species. At NO₃ ^? concentrations <40 μmol‐N·L ^{? 1}, all the diatom species and the dinoflagellate P. minimum exhibited saturating kinetics, whereas the other flagellates, D. tertiolecta and P. lutheri, did not saturate, leading to very high estimated K _s values. Above ～60 μmol‐N·L ^{? 1}, NO₃ ^? uptake rates of all species tested continued to increase in a linear fashion. Rates of NO₃ ^? uptake at 40 μmol‐N·L ^{? 1}, normalized to cellular nitrogen, carbon, cell number, and surface area, were generally greater for diatoms than flagellates. Diatoms stored significant amounts of NO₃ ^? internally, whereas the flagellate species stored significant amounts of NH₄ ⁺ . Half‐saturation concentrations for NR and NiR were similar between all species, but diatoms had significantly lower temperature optima for NR and NiR than did the flagellates tested in most cases. Relative to calculated biosynthetic demands, diatoms were found to have greater NO₃ ^? uptake and NO₃ ^? reduction rates than flagellates. This enhanced capacity for NO₃ ^? uptake and reduction along with the lower optimum temperature for enzyme activity could explain differences in nitrogen release patterns between diatoms and flagellates after an increase in irradiance.     

Radioisotopic Method for Measuring Cell Division Rates of Individual Species of Diatoms from Natural Populations

Silicon is an essential element for diatom frustule synthesis and is usually taken up only by dividing cells. With ⁶⁸Ge, a radioactive analog of Si, the cell cycle marker event of frustule formation was identified for individual species of diatom. The frequency of cells within a population undergoing this division event was estimated, and the cell division rate was calculated. In laboratory cultures, these rates of cell division and those calculated from changes in cell numbers were similar. By dual labeling with ⁶⁸Ge(OH)₄ and NaH¹⁴CO₃, rates of cell division and photosynthesis were coincidently measured for diatoms both in laboratory cultures and when isolated from natural populations in estuarine, offshore, and polar environments. These techniques permit the coupling between photosynthesis and cell division to be examined in situ for individual species of diatom.     

EFFECT OF POLYCARBONATE CONTAINERS ON THE GROWTH OF TWO SPECIES OF MARINE DIATOMS1

Two species of marine diatoms [Skeletonema costatum (Greville) Cleve and Thalassiosira pseudonana (Hustedt) Hasle and Heimdal] were grown in glass and polyarbonate containers. S. costatum exhibited a signzJicantly lower exponential growth rate and maximal yield and a signajcantly longer lag phase when grown in polycarbonate. Exponential growth rate and maximal yield of T. pseudonana was significantly reduced (P < 0.05 in all cases). This study suggests that a difference in diatom growth between glass and polyarbonate containers might arise in certain cases. However, such a difference may not be detectable with all biomass measurement techniques or with low within-treatment replication.     

OPTIMAL GROWTH CONDITIONS AND ANTIOXIDATIVE ACTIVITIES OF CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE)1

We isolated the unialgal strain of Cylindotheca closterium (Ehrenb.) Reimann et J. C. Lewin and produced an axenic strain using an antibiotic cocktail of enriched f/2 artificial seawater medium. The optimal growth conditions were estimated under 27 different combinations of temperature, salinity, and nutrients, and mass culture was performed based on the best specific growth conditions. Its antioxidant activities were determined from the extracts of methanol, water, and enzymes (proteases and carbohydrases). The maximum specific growth rate (μ_max) varied from 0.63 to 0.97 · d^?1. The maximum cell density was 7.20 × 10⁴ cells · mL^?1, while the μ_max was 0.82 · d^?1 in culture conditions of 20°C, 30 psu (practical salinity unit), and “F” nutrient concentrations on day 10 of the culture period. The scavenging rates for 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free radical were 72.5% and 69.4% from Viscozyme and methanol extracts, respectively. The enzymatic extracts of C. closterium prepared by the hydrolyses of Amyloglucosidase (AMG) and Viscozyme showed 45.8% and 45.5% nitric‐oxide‐scavenging rates, slightly lower than the activity of alpha‐tocopherol (α‐tocopherol) but similar to butylated hydroxytoluene (BHT). The extract from methanol and water showed 44.8% and 44.4% scavenging rates, statistically similar with BHT. The metal‐chelating activities of the Kojizyme, Alcalase, methanol, Viscozyme, and Neutrase extracts were 67.1, 53.9, 53.2, 52.1, and 50.2 %, respectively, five to six times higher than the commercial antioxidants. The AMG, Viscozyme, and Neutrase extracts showed a remarkable linoleic acid peroxidation inhibition, which was higher than BHT and statistically similar with α‐tocopherol.