Production of glycine betaine and dimethylsulfoniopropionate in marine phytoplankton. I. Batch cultures

The quantitative significance of the nitrogenous compound glycine betaine (GBT) and its sulfur analog dimethylsulfoniopropionate (DMSP) to intracellular pools in marine phytoplankton is not well known. In a series of experiments conducted in August 1993, we measured these compounds, as well as total organic sulfur, carbon, and nitrogen, over the growth cycle in six isolates of marine phytoplankton, Amphidinium carterae Hulburt, Chrysochromulina sp. Lackey, Emiliania huxleyi Hay et Mohler, Prorocentrum minimum (Pavillard) Schiller, Skeletonema costatum (Greville) Cleve, and Tetraselmis sp. At the same time, we measured cellular concentrations of protein, amino acids, chlorophyll, and inorganic nutrients. All six species produced DMSP, while three produced GBT at lesser levels. In the Chrysochromulina sp. isolate, levels of GBT were greater than DMSP during the exponential phase of growth, but declined sharply as the culture approached stationary phase. This change appeared to coincide with the onset of nitrogen limitation. Other nitrogenous osmolytes were produced in five of the six species but in much smaller quantities. DMSP contributed significantly to cellular sulfur throughout the growth cycle although, in some algae, the proportion of dissolved DMSP increased substantially during stationary growth. When present, GBT formed a sizeable fraction of the cellular nitrogen only during exponential growth. A significant percentage (ca. 50%) of the organic nitrogen could not be accounted for even when cellular pools of protein, amino acids, inorganic nitrogen, and nitrogenous osmolytes were combined. Based on these experiments, there does not appear to be a reciprocal relationship between DMSP and GBT production, although GBT production does appear to be correlated with nitrogen availability. Received: 5 January 1998 / Accepted: 29 June 1999     

Production of glycine betaine and dimethylsulfoniopropionate in marine phytoplankton. II. N-limited chemostat cultures

The influence of salinity on the time elapsed between two successive molts and the size reached after each molt were studied at 30, 21, 12 and 3‰S in juveniles of two co-occurring grapsid species, Cyrtograpsus angulatus and C. altimanus, cultured under identical conditions of temperature, photoperiod and food. Juvenile growth patterns were compared between these species (which differ in size-at-maturity and maximum size). C. angulatus grew faster than C. altimanus, reflecting a higher increment per molt and a shorter intermolt period. A significant difference existed between the number of instars preceding the size of maturity in both species: >11 in C. angulatus, 6 in C. altimanus. There was evidence of a differential effect of low salinity on growth. By the end of the experiment, individuals of both species were smaller at the lowest salinity (3‰) tested; the largest crabs developed at 21‰ (C. angulatus) and 30‰ (C. altimanus). The size difference between the “optimal” and the less suitable salinities in the sixth crab instar was 12.4% in C. angulatus and 35% in C. altimanus. During early juvenile development (Crab Instars 1 to 4), there were slight differences in intermolt period among salinities in C. angulatus, but large differences in C. altimanus. The longest intermolt period of C. altimanus was at 3‰S and the shortest at 30‰S. In the following instars (5 to 10 in C. angulatus and 5 to 6 in C. altimanus), the longest intermolt period occurred at 21‰S, the shortest at 3‰S, in both species. Interspecific differences in response to low salinities may explain why C. angulatus occurs throughout a whole temperate coastal lagoon, whereas C. altimanus is restricted to its mouth. Received: 12 July 1998 / Accepted: 24 February 1999     

Release of photoassimilated carbon as dissolved organic matter by marine phytoplankton

The release of photoassimilated carbon as dissolved organic matter was studied in situ in oligotrophic and eutrophic marine waters and in axenic laboratory cultures. Percentage extracellular release (PER), integrated for the trophogenic zone, ranged from 6 to 12% in eutrophic waters and from 17 to 27% in oligotrophic seas. Most of the algal cultures released low amounts of dissolved organic matter (5%) during exponential growth. The highest levels of PER were observed in surface and deep samples, possibly as a result of elevated photorespiration and senescent cells. The generally lower values for extracellular release reported in this work as compared to other studies may be partly due to improved experimental techniques which minimized previously encountered artifacts.     

Cycling of selenite and selenate in marine phytoplankton

The marine phytoplankton Dunaliella tertiolecta, Cachonina niei, Thalassiosira nordenskioldii, Phaeodactylum tricornutum, and Chaetoceros sp. were incubated with a range of molar concentrations of sodium-selenite (Na₂-SeIVO₃) and sodium-selenate (Na₂-SeVIO₄) to examine further their role in metabolic cycling of selenium in ocean waters. At low selenium concentrations, approaching those found naturally in seawater (10^-10 to 10^-9 M), all species distinguished between selenite and selenate, and actively concentrated selenite from the incubating medium while only marginally accumulating selenate. At much higher concentrations (10^-8 to 10^-6 M), selenate was also taken up. At the highest concentration tested, i.e., 10^-5 M with C. niei, after an immediate rapid uptake in the first 24 h, the intracellular selenite and selenate levels dropped to about 35 to 50% of the initial peak values. These observations suggest an uptake mechanism in these algae which, at normal ambient concentrations of selenium (10^-9 M), preferentially selects selenite and excludes selenate. At much higher concentrations (10^-8 M), the mechanism becomes overloaded and both selenium species enter the cells. Intracellularly, selenite became associated primarily with protein and amino acid fractions, in approximately equal proportions, while only ca. 4% of total intracellular selenium was found in the lipid fraction. Trace amounts of selenate that entered the cells, mainly during the first minutes of exposure, also entered the protein and amino acid components, but over time were increasingly associated with the protein fraction only. At the end of a 10-d incubation of algal cells in selenite-spiked medium, less than 25% of total Se in the medium could in fact be identified analytically as selenite. This suggests the presence of a non-selenite metabolite, possibly released back into the medium from the algae.     

Coagulation efficiency and aggregate formation in marine phytoplankton

Flocculation of phytoplankters into large, rapidly sinking aggregates has been implicated as a mechanism of vertical transport of phytoplankton to the sea floor which could have global significance. The formation rate of phytoplankton aggregates depends on the rate at which single cells collide, which is mainly physically controlled, and on the probability of adhesion upon collision (=coagulation efficiency, stickiness), which depends on physico-chemical and biological properties of the cells. We describe here an experimental method to quantify the stickiness of phytoplankton cells and demonstrate that three species of diatoms grown in the laboratory (Phaeodactylum tricornutum, Thalassiosira pseudonana, Skeletonema costatum) are indeed significantly sticky and form aggregates upon collision. The dependency of stickiness on nutrient limitation and growth was studied in the two latter species by investigating variation in stickiness as batch cultures aged. In nutrient repleteT. pseudonana cells stickiness is very low (< 5 × 10^?3), but increases by more than two orders of magnitude as cell growth ceases and the cells become nutrient limited. Stickiness ofS. costatum cells is much less variable, and even nutrient replete cells are significantly sticky. Stickiness is highest (> 10^?1) forS. costatum cells in the transition between the exponential and the stationary growth phase. The implications for phytoplankton aggregate formation and subsequent sedimentation in the sea of these two different types of stickiness patterns are discussed.     

Diel periodicity of photosynthesis in marine phytoplankton

Short-term changes in photosynthesis were documented for 17 of 24 marine phytoplankton species, representing a range of taxonomic groups. Periodicity in phytoplankton photosynthesis on light-dark cycles (diel periodicity) was widespread but not universal for the species studied. The centric diatoms Lauderia borealis, Ditylum brightwellii, Stephanopyxis turris, Coscinodiscus rex, Chaetoceros gracile, and Biddulphia mobiliensis had strong diel periodicity in photosynthetic capacity (P _max). Amplitudes of the daily variations ranged from 2.9 to >50, with maxima in the morning or near midday, and with minima during the dark period, and these variations were not dependent on changes in cell pigmentation. There was some evidence for sustained photosynthetic periodicity in constant conditions in several diatoms, and an endogenous rhythm may have been present. The photosynthesis-irradiance (P-I) relationship was time-dependent for representative marine diatoms, with both the initial slope () and the asymptote (P _max) of P-I curves exhibiting significant synchronous diel oscillations. Moreover, detailed studies of the amplitude and timing of photosynthetic periodicity for the diatoms L. borealis and D. brightwellii demonstrated large temporal variations in photosynthesis with morning maxima. These P-I oscillations are discussed with reference to models of primary production which use the relationship between photosynthesis and light as a component of predictive equations for phytoplankton growth in the sea.     

Light stimulation of phosphate uptake in marine phytoplankton

A phosphate uptake system responding positively to light would offer the algae a competitive advantage over bacteria, particularly when phosphorus is limiting. Effects of light on phosphate uptake in phytoplankton were investigated in view of conflicting literature reports, to test the hypothesis that both the P status and the light history of the population controlled the response. Maximum stimulation, to about 2.7x dark uptake, was observed at a Strait of Georgia, British Columbia, Canada station where phytoplankton was phosphorus sufficient but light limited. Stimulation was similar, about 1.8 x the dark rate, in the Northern Sargasso Sea, where phytoplankton was probably limited by phosphate but not by light. Minimal stimulation was observed in the Sheepscot Estuary, Maine, USA, where the population appeared to be neither phosphorus nor light limited.     

Iron-mediated effects on nitrate reductase in marine phytoplankton

The potential activity of nitrate reductase was determined in uni-algal cultures in the laboratory and in natural marine phytoplankton assemblages. In the laboratory bioassays, distinct differences in nitrate reductase activity were observed in iron replete versus depleted cultures for Emiliania huxleyi, Isochrysis galbana and Tetraselmis sp. Cells from iron-depleted cultures had 15 to 50 percent lower enzyme activity than those from iron-replete cultures. Upon addition of iron, nitrate reductase activity was enhanced in depleted cells up to levels comparable to those of the replete cells. Bioassays in the northern North Sea conducted in 1993, under low iron conditions, demonstrated similar results. Upon addition of 2.5 nM iron, a distinct enhancement, to a maximum of three times, of nitrate reductase activity was observed within 32 h after addition. Therefore, iron can stimulate nitrate reductase activity. In spite of the clean techniques used, some nitrate reductase activity was always observed. Iron deficiency was shown to impair nitrate reductase activity, but it is unlikely that nitrate reduction would cease completely.     

Circadian variations in photosynthetic assimilation and estimation of daily phytoplankton production

In August 1984, hourly measurements of photosynthetic characteristics were carried out during 96 h, at 5 and 10 m, on a natural population of phytoplankton in the St Lawrence Estuary. Synchronous circadian variations of similar amplitude (max./min.: 2 to 3) were observed at the two depths in both the photosynthetic capacity (P _m ^B ) and the photosynthetic efficiency (^B). Maximum values occurred at around noontime and minima during the night. Estimates of daily specific productivity were computed with and without the observed circadian variability. Large differences (15 to 70%) were evidenced between estimates.Contribution to the programs of GIROQ (Groupe interuniversitaire de recherches océanographiques du Québec) and of the Maurice Lamontagne Institute (Department of Fisheries and Oceans)     

Photosynthesis and chlorophyll a fluorescence rhythms of marine phytoplankton

Circadian rhythms in photosynthesis were defined in field populations of phytoplankton. Measurements of carbon-dioxide fixation rates demonstrated that a diurnal periodicity of photosynthesis in samples incubated under natural light-dark (LD) cycles also were observed to continue in similar samples which had been photoadapted to constant dim light (LL) for 48 h. These changes in photosynthetic rates preceded sunset and sunrise, had daily amplitudes that ranged from 1.5 to 2.0, appeared to be independent of light-intensity, and displayed maxima about midday, while rates of dark fixation of carbon dioxide and the photosynthetic pigment content per cell were constant over the circadian cycle. Similar rhythmicity also was detected in room-temperature (22°C) chlorophyll a fluorescence yield, in both the obsence and presence of the photosynthesis inhibitor DCMU [3-(3,4-dichlorophenyl)-1, 1-dimethylurea]. However, the magnitude and timing of the fluorescence rhythm maxima seem to depend on wavelengths monitored and, in part, on the measuring technique used. Also, the circadian changes in the fluorescence intensity were abolished at low temperature (-60°C), and the shape of the emission spectra of chlorophyll fluorescence of cells in LD and LL did not change over time. The significance of the fluorescence rhythms with regard to chlorophyll a determinations and photosynthetic rates is discussed. It was concluded that there was sufficient similarity between circadian rhythms of photosynthesis in natural phytoplankton populations and in laboratory cultures of dinoflagellates to suggest that the mechanism of regulation may be the same for both of them.     

Nitrate uptake in marine phytoplankton: Energy sources and the interaction with carbon fixation

Field studies of whole natural phytoplankton communities from Knight Inlet, B. C., Canada and laboratory cultures of the diatom Skeletonema costatum indicate inorganic carbon fixation may be temporarily suppressed following 10 to 15% enrichment with NO ₃ ^- or NH ₄ ⁺ . (This effect is suggested to be due to competition between inorganic carbon and nitrogen for adenosine triphosphate (ATP), and is reduced when chlorophyll a is increased intracellularly after 6 to 8 h.) Results imply that the source of ATP for nitrate uptake is primarily from Photosystem I (cyclic photophosphorylation) in the presence of light. It would appear that a transient nutrient-adaptive response occurs upon addition of extracellular nitrogen.     

Short-term variability of photosynthesis in natural marine phytoplankton populations

Harmonic regression analysis has been used to determine the short-term variability in the photosynthetic rate (mgC/mg chlorophyll a/h) of phytoplankton in three inlets of Japan. In natural water without large zooplankton present, the photosynthetic rate [log P=log (100xmgC/mg chlorophyll a/h)] can be expressed as (B+A cos T). Factor B represents the average photosynthetic rate, of which the maximum is usually designated as P _max, and Factor A corresponds to the slope of the regression line. The phase of the periodicity, represented by T, is adjusted to give the highest correlation: usually T is expressed as [360/24 x (local time + 4)] in degrees. The correlation between Factors A and B is very high (r=0.95, P<0.001), indicating that Factor A may depend upon Factor B (potential activity of chlorophyll a). Both Factors A and B decrease with decreasing irradiance, but the slope of each regression between Factor A and irradiance varies with season. Continuous darkness reduces the phase of the periodicity to one cycle a day when phytoplankton has multiple cycles of photosynthetic rate per day. Adequate nutrient supply from zooplankton regeneration may cause an increase in Factor B; however, excess density of zooplankton decreases Factor A.     

Correlation between oxygen utilization and electron transport activity in marine phytoplankton

Measurement of a respiratory control ratio is deemed very useful in determination of energy balance in respiration. We have defined and computed a control ratio as the ratio of the rate of oxygen consumption in vivo to the calculated maximum rate of oxygen consumption in vitro as measured by enzymatic electron transport rates. In order for a control ratio to serve a useful purpose in calculating in situ oxygen consumption in a water parcel, the ratio must be fairly constant. Our results indicated that this is precisely so in healthy cultures of the 9 species of phytoplankton studied. The data presented in this communication further point towards the potential for making reasonable estimates of phytoplankton oxygen consumption in an ocean water parcel by directly utilizing in vitro electron transport activity measurements.     

DMSP-lyase activity in five marine phytoplankton species: its potential importance in DMS production

Activity of DMSP-lyase, which cleaves dissolved DMSP (henceforth DMSP_d-lyase), was examined in five axenically cultured phytoplankton species, including both DMSP-producing and non-DMSP-producing species. High DMSP_d-lyase activity was found in two DMSP producers, Heterocapsa triquetra strain NIES-7 and Scrippsiella trochoidea strain NIES-369 (Dinophyceae). The DMS production rates at 100 nM DMSP_d were 0.5 fmol cell⁻¹ min⁻¹ for H. triquetra and 0.3 fmol cell⁻¹ min⁻¹ for S. trochoidea. In a non-DMSP producer, Heterosigma akashiwo strain NIES-6 (Raphidophyceae), the DMSP_d-lyase activity was not found. Two DMSP-producing Prymnesiophyceae species, Isochrysis galbana strain CCMP-1323 and Gephyrocapsa oceanica strain NIES-353, did not show any obvious activity either, in contrary to other authors' findings on Phaeocystis sp., another DMSP-producing Prymnesiophyceae species. The comparison of the DMSP_d-lyase activity of the two Dinophyceae species with bacterial DMSP consumption and DMS production activity in Tokyo Bay showed that the DMSP_d-lyase activity of H. triquetra and S. trochoidea could be an important mechanism for DMS production during their blooms. Received: 9 April 1999 / Accepted: 10 December 1999     

Questions on the mechanism of temperature adaptation in marine phytoplankton

The rate of light-saturated photosynthesis in 3 marine algae [Phaeodactylum tricornutum Bohlin, Nitzschia closterium (Ehrenberg) Smith and Dunaliella tertiolecta Butcher] varies during growth in batch culture. The photosynthetic rare declines most rapidly during growth at the higher temperatures. Because of these changes in photosynthesis rate, the previously reported enhanced photosynthetic abilities caused by growth at lower temperatures (generally interpreted as evidence for higher enzyme levels) can only be observed when measurements are made late in the exponential phase or after the onset of the stationary phase of growth. When allowance is made for the earlier peak of photosynthetic ability in cultures growing at higher temperatures, there is no evidence for adaptation to lower temperatures being caused by increased levels of the enzymes required for carbon-dioxide fixation. When the changes due to growth in batch culture are taken into account, certain effects of temperature can be recognized. the dry weight: chlorophyll ratio of all 3 algae increases with decreasing growth temperatures. For P. tricornutum and N. closterium, growth at lower temperatures reduces the cellular content of chlorophyll a, but has little effect on the chlorophyll content of D. tertiolecta. The dry weight: cell-number ratio of D. tertiolecta and P. tricornutum increases with lower growth temperatures, but growth temperature has little effect on the cell mass of N. closterium. Growth of the 3 algae at lower temperatures does not increase their ability to photosynthesize at these lower temperatures. Rather, it reduces their ability to assimilate carbon dioxide at the higher temperatures.     

Influence of high temperature and residual chlorine on marine phytoplankton

Marine phytoplankton forms are frequently exposed to sudden biological changes such as rapid rise in water temperature and chlorine content of their environment, resulting from the use of sea water for cooling purposes by electric generators. The direct influence of these effluents, i.e. inhibitory effects of high temperature and residual chlorine on growth and photosynthesis of Chlamydomonas sp. and Skeletonema costatum, were investigated experimentally. Chlamydomonas sp. and S. costatum exposed to high temperatures were affected in their growth from 43° and 35°C, respectively, by immersion of the respective cultures in a warm bath for 10 min. Treatment at high temperatures of 40 °C and 30° 35°C for 10 min, influenced their photosynthetic activities, which were completely inhibited immediately after 10 min exposure at 42° and 37 °C, respectively. S. costatum was killed by chlorine at a concentration of 1.5 2.3 ppm when exposed for exactly 5 or 10 min, while Chlamydomonas sp. was not irreversibly damaged even at 20 ppm chlorine or more with the same exposure period. These results lead to the conclusion that the high temperature of, and residual chlorine in, effuents from a power plant discharging into the open sea, should not cause great damage to marine phytoplankton in that area.     

Interspecific differences in the photosynthetic carbon metabolism of marine phytoplankton

Six species of marine phytoplankton of different sizes and taxonomic categories were grown in microcosms under identical experimental conditions; the species cultured were: Pavlova lutheri (Prymnesiophyceae), Dunaliella tertiolecta (Chlorophyceae), Phaeodactylum tricornutum (Baciollariophyceae), Eutreptiella sp. (Euglenophyceae), Alexandrium tamarense (Dinophyceae), and Phaeocystis pouchetii (Prymnesiophyceae). The photosynthetic carbon metabolism of these phytoplankton was studied throughout the exponential and lag phases of growth after nutrient depletion. The relative incorporation of carbon into protein was positively correlated with phytoplankton growth, while carbon assimilation into low molecular weight metabolites (LMWM) and storage products, i.e., lipid and polysaccharides, generally increased under nutrient-limiting conditions. Clear taxonspecific differences were observed in the proportions of carbon incorporated into cell constituents. A significant linear relationship was consistently found between the relative synthesis of protein to LMWM, and both the production normalised to chlorophyll (P:B) and the phytoplankton growth rate. However, ANCOVA revealed significant, interspecific differences in these relationships.     

Effects of diurnal variations in phytoplankton photosynthesis obtained from natural fluorescence

Effects of diurnal variation in phytoplankton photosynthesis on estimating daily primary production (DPP) were examined using field data from Sagami Bay, Japan. DPP at 5 m depth was calculated from the continuous data of chlorophyll a (Chl a) and light intensity monitored by a natural fluorescence sensor with and without considering time-dependent changes in the photosynthesis–irradiance (P–E) relationship. Chl a could be estimated from natural fluorescence examining the variations in the quantum yield of fluorescence (φ _f) and Chl a-specific light absorption coefficient (a*_ph), and relating them to Chl a. The P–E relationship was determined by water sampling three times daily. A distinct diurnal pattern was observed for the maximum photosynthetic rate (P*_max), being maximal at noon, while periodicity of the maximum light utilization coefficient (α*) was less obvious. The actual DPP was calculated by interpolating the P–E parameters from those obtained at dawn, noon, and dusk. For comparison, DPP was calculated by fixing the P–E parameters as the constants measured at dawn, noon or dusk for a day. The difference from the actual DPP was small when the P–E parameters measured at dawn (3% on average) and noon (5%) were used as the constants for a day. The difference was largest when the values at dusk were used (−43%). The medium values of P*_max at dawn, its low values at dusk, and the fact that a major part of the DPP was produced around noon were responsible for these results. The present study demonstrates that measurement of the P–E parameters at dawn or noon can give a good estimation of DPP from natural fluorescence.     

Tidal variations in the photosynthesis of estuarine phytoplankton isolated in a tank

In 1981 two large (1 200 1) seawater samples from the St. Lawrence Estuary were kept under constant temperature and light conditions for periods of 50 and 68 h, respectively. In both tank experiments, semidiurnal variations in NH₄ were observed that could be related to cyclical NH₄ uptake by the phytoplankton. Semidiurnal cycles in photosynthetic efficiency (^B) and intracellular chlorophyll a in the tank, phased on tides at sea, were also evidenced in both experiments. These results support the hypothesis that variations in phytoplankton photosynthetic activity, which are possibly endogenous, can be phased on semidiurnal variations in vertical tidal mixing (variations of the mean light conditions in the mixed layer). In addition, observed variations in intracellular chlorophyll a suggest the possibility of endogenous cycles of phytoplankton light and shade adaptation.Contribution to the program of GIROQ (Groupe interuniversitaire de recherches océanographiques du Québec)     

Inorganic nitrogen metabolism in marine bacteria: The intracellular free amino acid pools of a marine pseudomonad

The marine pseudomonad bacterium PL₁ contains an intracellular pool of free amino acids which consist mainly of glutamate with small amounts of glutamine and aspartate when grown in a nutrient medium containing 0.2 M NaCl. When the NaCl concentration of the growth medium is increased to 0.8 M, proline becomes a major component of the intracellular pool together with glutamate—at this molarity and under suitable nutrient conditions these amino acids comprise 20% of total bacterial amino acid nitrogen. When grown in a nutrient growth medium containing a constant level of NaCl, the intracellular pool size can vary by a factor of 4 depending on the concentration of carbon and nitrogen in the medium. Experiments show that the amino acid pool can act as a nitrogen reserve but has little function as a carbon reserve. At high NaCl concentrations there is a marked dependence for growth on the presence of sufficient potassium in the medium. However, no correlation between K⁺ and glutamate concentration in either nitrogen or K⁺-limited cultures has been found. None of the enzymes associated with glutamate biosynthesis was influenced by NaCl levels between 0.2 and 0.5 M. Neither Na⁺ or K⁺ stimulated the activity of these enzymes when tested in vitro.