Biomass production in mass cultures of marine phytoplankton at varying temperatures

Natural populations of marine phytoplankton obtained from a large outdoor pond were grown on waste water-sea water mixtures in laboratory continuous cultures in the temperature range 5–33 °C. Virtually all of the influent inorganic nitrogen (14.0 mg l^?1) was assimilated at every temperature tested. There was, however, a distinct change in dominant species with temperature: below 19.8 °C Phaeodactylum tricornutum was dominant, at 27 °C Nilzschia sp. was the main species, and as the temperature increased above 27 °C a blue-green alga, Oscillatoria sp., became increasingly dominant. There is some indication that the excellent growth of P. tricornutum below 10 °C was related to a dramatic increase in the nutrient content per cell as the temperature decreased. Thus at low temperatures reduced division rates are compensated for by increased nutrient uptake rates. It follows that there is a transfer of phytoplankton protein from numerous small cells at intermediate temperatures to large cells that are reduced in numbers at lower temperatures but which represent the same total organic matter. The effect of this phenomenon on annual food chain efficiencies in both controlled and natural marine ecosystems is unknown.     

EFFECTS OF VARIATION IN TEMPERATURE. I. ON THE BIOCHEMICAL COMPOSITION OF EIGHT SPECIES OF MARINE PHYTOPLANKTON1

The influence of temperature on the biochemical composition of eight species of marine phytoplankton was investigated. Thalassiosira pseudonana Hasle and Heim-dal, Phaeodactylum tricornutum Bohlin and, Pavlova lutheri Droop (three of eight species studied) had minimum values of carbon and nitrogen quotas at intermediate temperatures resulting in a broad U-shaped response in quotas over the temperature range of 10 to 25°C. Protein per cell also had minimum values at intermediate temperatures for six species. For T. pseudonana, P. tricornutum, and P. lutheri, patterns of variation in carbon, nitrogen, and protein quotas as a function of temperature were similar. Over all species, lipid and carbohydrate per cell showed no consistent trends with temperature. Only chlorophyll a quotas and the carbon: chlorophyll a ratios (θ) showed consistent trends across all species. Chlorophyll a quotas were always lower at 10°C than at 25°C. Carbon: chlorophyll a ratios (θ) were always higher at 10°C than at 25°C. We suggest that although θ consistently increases at lower temperatures, the relationship between temperature and θ ranges from linear to exponential and is species specific. Accordingly, the interspecific variance in θ that results from species showing a range of possible responses to temperature increases as temperature declines and reaches a maximum at low temperatures. High photon flux densities appear to increase the potential interspecific variance in the carbon: chlorophyll a ratio and therefore exacerbate these trends.     

Nutrient-induced competition between two species of marine diatoms

Competition experiments betweenPhaeodactylum tricornutum andSkeletonema costatum showed that even at temperatures higher than 10°C (i.c. 14°C), the development ofSkeletonema can be favoured by adjusting nutrient levels and nutrient ratios. Low NSi ratios were found to favourSkeletonema. Additionally, high NP ratios further enhanced the ability ofSkeletonema to dominate the cuftures. Contrary to some statements in literature, it seems that high concentrations of silicates are more important for the dominance ofSkeletonema costatum in large-scale cultures than just low temperatures. This finding is important with regard to stimulating the blooming ofSkeletonema costatum in natural phytoplankton populations as food for bivalve molluscs.     

Studies on N-limited growth of diatoms in dialysis culture

N-limited growth of Skeletonema costatum (Grev.) Cleve in dialysis culture has been studied. The division rate of exponentially growing cells was independent of the nitrate concentration in the growth medium in the range from 886 down to 0.25 μM N-salt, while no growth beyond one division took place in cultures to which no nitrogen salt was added. The half saturation constant, K₃, for growth must, therefore, be in the range 0–0.13 μM, provided the growth-nutrient relationship is hyperbolic for S. costatum.Contrary to growth rate, cellular chlorophyll and protein were markedly reduced in media poor in nitrogen salts. A dialysis culture chamber was used to demonstrate that the measurement of half saturation constants for S. costatum was influenced by stirring, the stirred culture growing almost twice as fast as the unstirred control under identical conditions. The ability of diatoms to grow rapidly at low nitrogen levels was used to remove nutrients from sewage enriched media. Removal efficiencies corresponding to 80 and 90 % were obtained for nitrate and ammonia, respectively, using the diatom Phaeodactylum tricornutum Bohlin. It was found that both this diatom and S. costatum as well as Thalassiosira pseudonana Hust (Hasle) tolerated ammonia up to at least 450 μM with no deleterious effects on growth rate.     

Heavy metal tolerance of marine phytoplankton. II. Copper tolerance of three species in dialysis and batch cultures

The tolerance to copper ions of three diatoms, namely, Skeletonema costatum, (Grev.) Cleve, Thalassiosira pseudonana (Hust.) Hasle and Phaeodactylum tricornutum Bohlin grown in dialysis and batch cultures in the local fjord water has been established. Reduction of growth rates was observed by the addition of 10, 25 and 400 μg/1 of copper ions, respectively for the three species investigated. At the higher levels of copper addition (400 and 700 MS/1) cells of P. tricornutum in dialysis culture increased their copper content to more than 200 times over those of the controls, the ratio of copper to chlorophyll in the cells increasing 150 times.All three species showed marked increases in copper content when a copper salt was added to batch cultures of the algae. The two clones of Skeletonema costatum tested showed nearly identical sensitivity to copper ions, but they differed markedly in their zinc tolerance.     

13C AND 15N UPTAKE BY MARINE PHYTOPLANKTON. I. INFLUENCE OF NITROGEN SOURCE AND CONCENTRATION IN LABORATORY CULTURES OF DIATOMS1

Two species of marine diatoms, Skeletonema costatum (Grev.) Cleve and Phaeodactylum tricornutum Bohlin were grown in batch and continuous cultures on four different nitrogen compounds (nitrate, nitrite, ammonium, urea). Carbon and nitrogen uptake were measured simultaneously with the stable isotopes ¹³C and ¹⁵N. Nitrogen uptake generally increased with N concentration in the medium, but no clear difference existed between the N sources. Carbon fixation was decreased for up to 5 h following the addition of the N compound. Nitrite generally had the greatest inhibitory effect on C uptake. Carbon-to-nitrogen uptake ratios decreased with increasing dissolved N concentration, becoming lower than one in nutrient-limited cultures. In contrast, batch cultures exhibited C:N uptake ratios greater than one. These effects are essentially short-term and differ from long-term influences of the N source on the cellular chemical composition.     

Interactive effect of diet and temperature on the growth of juvenile clams

Small juveniles (0.3–3 mg live weight) of three clam species, Tapes semidecussata Reeve, T. decussata L., and Mercenaria mercenaria L., were fed eight single-species algal diets at a range of temperatures. Nutritional value of the algae tested was in the order: Isochrysis aff galbana (T. ISO) = Skeletonema costatum >Chaetoceros calcitrans = Chroomonas salina = Thalassiosira pseudonana >Tetraselmis suecica >Phaeodactylum tricornutum >Chlamydomonas coccoides, when the same weight of these species was given in the diet to each clam species. Reasons for differences in nutritional value are discussed with reference to the biochemical content of the algae and the relative growth efficiency of the animals. Respiration rate, food cell clearance rate, and growth (as increase in organic weight) of M. mercenaria and T. semidecussata increased with temperature from 10 to 25 °C. Growth rates decreased at > 25 °C. T. decussata showed only a slightly increased growth response at > 15 °C.     

Heavy metal tolerance of marine phytoplankton. I. The tolerance of three algal species to zinc in coastal sea water

Tolerance levels to zinc ions of three diatoms (Skeletonema costatum (Grev.) Cleve, Thalassiosira pseudonana (Hust.) Hasle and Phaeodactylum tricornutum (Bohlin) grown in dialysis culture in the local fjord water were studied. Declining relative growth rates were observed by addition of 50, 250 and 25,000μg/l of zinc ions, respectively, for the three algae. Reduced final cell concentrations were found at lower zinc levels. At least for one species a significant increase in zinc uptake by the cells took place at zinc levels which did not seem to influence the growth and development of the alga. Two clones of Skeletonema costatum studied showed significant intraspecific differences regarding the tolerance to zinc pollution. Dialysis bioassay was found suitable for monitoring heavy metal pollution of aquatic recipients.     

EFFECTS OF LIGHT INTENSITY AND TEMPERATURE INTERACTIONS ON GROWTH CHARACTERISTICS OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cell division rate, carbon fixation per cell, cell width and chloroplast length of Phaeodactylum tricornutum Bohlin were determined at 30 different combinations of light intensity and temperature. Division rate peaked at 23° C or less depending on light intensity. For each light intensity studied, carbon fixation increased directly with growth temperature from 14 to 25° C. The slope of this relationship was modified by light intensity. Cells grown at 23–25° C tended to be larger than those grown at lower temperatures, possibly due to increased carbon fixation per cell coupled with lower division rates. Chloroplasts were largest at a combination of temperatures above 21° C and low light intensities. This effect could cause cells to sink at a higher than normal rate due to reduced vacuole size and is presented as a possible mechanism affecting the distribution of P. tricornutum.     

Temperature dependence of nitrate reductase activity in marine phytoplankton: biochemical analysis and ecological implications

The temperature dependence of NADH:NR activity was examined in several marine phytoplankton species and vascular plants. These species inhabit divergent thermal environments, including the chromophytes Skeletonema costatum (12–15° C), Skeletonema tropicum (18–25° C), Thalassiosira antarctica (?2 to 4° C), and Phaeocystis antarctica (?2 to 4° C), the green alga Dunaliella tertiolecta (14–28° C), and the vascular plants Cucurbita maxima (20–35° C) and Zea mays (20–25° C). Despite the difference in growth habitats, similar temperature response curves were observed among the chromophytic phytoplankton, with temperatures optimal for NR activity being between 10–20° C. In contrast, the chlorophyll b‐containing alga and vascular plants exhibited optimal temperatures for NR activity above 30° C. Such dramatic differences in NR thermal characteristics from the two taxonomic groups reflect a divergence in NR structure that may be associated with the evolutionary diversification of chromophytes and chlorophytes. Further, it suggests a potential contribution of the thermal performance of NR to the geographic distributions, seasonal abundance patterns, and species composition of phytoplankton communities. NR partial activities, which assess the individual functions of Mo‐pterin and FAD domains, were evaluated on NR purified from S. costatum to determine the possible causes for high temperature (>20° C) inactivation of NR from chromophytes. It was found that the FAD domain and electron transport among redox centers were sensitive to elevated temperatures. S. costatum cells grown at 5, 15, and 25° C exhibited an identical optimal temperature (15° C) for NADH:NR activity, whereas the maximal NR activity and NR protein levels differed and were positively correlated with growth temperature and growth rate. These findings demonstrate that thermal acclimation of NO₃^? reduction capacity is largely at the level of NR protein expression. The consequences of these features on NO₃^? utilization are discussed.     

High predictability of direct competition between marine diatoms under different temperatures and nutrient states

The distribution of marine phytoplankton will shift alongside changes in marine environments, leading to altered species frequencies and community composition. An understanding of the response of mixed populations to abiotic changes is required to adequately predict how environmental change may affect the future composition of phytoplankton communities. This study investigated the growth and competitive ability of two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, along a temperature gradient (9–35°C) spanning the thermal niches of both species under both high‐nitrogen nutrient‐replete and low‐nitrogen nutrient‐limited conditions. Across this temperature gradient, the competitive outcome under both nutrient conditions at any assay temperature, and the critical temperature at which competitive advantage shifted from one species to the other, was well predicted by the temperature dependencies of the growth rates of the two species measured in monocultures. The temperature at which the competitive advantage switched from P. tricornutum to T. pseudonana increased from 18.8°C under replete conditions to 25.3°C under nutrient‐limited conditions. Thus, P. tricornutum was a better competitor over a wider temperature range in a low N environment. Being able to determine the competitive outcomes from physiological responses of single species to environmental changes has the potential to significantly improve the predictive power of phytoplankton spatial distribution and community composition models.     

Changes in fatty acid profiles of thermo-intolerant and thermo-tolerant marine diatoms during temperature stress

Fatty acid composition and degree of fatty acid saturation during temperature stress in thermo-intolerant (Phaeodactylum tricornutum) and thermo-tolerant (Chaetoceros muelleri) marine diatoms were investigated. A greater number of fatty acids were observed in C. muelleri than in P. tricornutum regardless of treatment. The major fatty acids detected were 14:0, 16:0, 16:1, 16:2, 16:3, 18:0, 18:1(n-9)c, 18:2(n-6) and 20:5(n-3) with additional fatty acids 18:1(n-9)t and 20:4(n-6) detected in C. muelleri. Short duration (2 h) temperature increase above optimal growth temperature had a greater effect on fatty acid composition in C. muelleri than in P. tricornutum and the degree of fatty acid saturation was affected more by temperature in C. muelleri than in P. tricornutum during both short and long duration (24 h) treatments. Total protein assay results suggest that P. tricornutum, but not C. muelleri, was undergoing stress under our growing conditions although lipids in both diatoms were affected by increased temperature. Immunodetection of proteins with anti-rubisco indicates that the rubisco large subunit was undergoing greater turnover in C. muelleri than in P. tricornutum. However, the integrity of rubisco as a suitable indicator of lipid status needs further study. This work supports the hypothesis that a particular temperature, and not treatment duration, has the greater effect on changes in fatty acid composition. Furthermore, changes in fatty acid composition and degree of fatty acid saturation occurred more quickly in the diatoms in response to increased temperature than previously observed in nutrient starvation studies. Since diatom lipids represent an important resource for growth and reproduction of marine animals, the rapid alteration of their lipid composition under temperatures normally encountered in marine environments warrants further study.     

Photosynthetic Rates, Gross Patterns of Carbon Dioxide Assimilation and Activities of Ribulose Diphosphate Carboxylase in Marine Algae Grown at Different Temperatures

Studies of the marine green flagellate Dunaliella tertiolecta have confirmed and extended previous observations of Steemann Nielsen and his colleagues. Algae, grown at 12°C, assimilated carbon dioxide under light-saturated conditions more rapidly than did those grown at 20°C; for both, the assimilation rate being higher at 20°C than at 12°C. Cells grown at the lower temperature contained higher concentrations of soluble protein, higher activities of ribulose diphosphate carboxylase and showed an enhanced relative rate of protein synthesis during the photosynthetic assimilation of carbon dioxide. This appears to represent true adaptation since it allowed the growth rate at 12°C to be almost the same as that at 20°C. Studies of the marine diatom Phaeodactylum tricornutum have not revealed the same picture of temperature adaptation. Cultures grown at 5°C had significantly higher rates of photosynthesis than did those grown at 10°C, but the same was not true when algae grown at 10°C were compared with those grown at 20°C. In this organism, growth at the lower temperatures reduced its ability to photosynthesize at 20°C. Cells grown at the lower temperatures contained more protein than did those grown at 20°C; this was particularly marked in cells growing at 5°C, a temperature which reduced the growth rate. The relative rate of protein synthesis was higher in Phaeodactylum grown at lower temperatures; but this difference was most marked when the measurements were made at 20°C.     

Biochemical compositions of two dominant bloom- forming species isolated from the Yangtze River Estuary in response to different nutrient conditions

Variations of cellular total lipid, total carbohydrate and total protein content of two dominant bloom-forming species (Skeletonema costatum and Prorocentrum donghaiense) isolated from the Yangtze River Estuary were examined under six different nutrient conditions in batch cultures. Daily samples were collected to estimate the cell growth, nutrient concentration and three biochemical compositions content during 7 days for S. costatum and the same sampling procedure was done every other day during 10 days for P. donghaiense. Results showed that for S. costatum, cellular total lipid content increased under phosphorus (P) limitation, but not for nitrogen (N) limitation; cellular carbohydrate were accumulated under both N and P limitation; cellular total protein content of low nutrient concentration treatments were significantly lower than that of high nutrient concentration treatments. For P. donghaiense, both cellular total lipid content and total carbohydrate content were greatly elevated as a result of N and P exhaustion, but cellular total protein content had no significant changes under nutrient limitation. In addition, the capability of accumulation of three biochemical constituents of P. donghaiense was much stronger than that of S. costatum. Pearson correlation showed that for both species, the biochemical composition of three constituents (lipid, carbohydrate and protein) had no significant relationship with extracellular N concentration, but had positive correlation with extracellular and intracellular P concentration. The capability of two species to accumulate cellular total lipid and carbohydrate under nutrient limitation may help them accommodate the fluctuating nutrient condition of the Yangtze River Estuary. The different responses of two species of cellular biochemical compositions content under different nutrient conditions may provide some evidence to explain the temporal characteristic of blooms caused by two species in the Yangtze River Estuary.     

Sterols of the phytoplankton—effects of illumination and growth stage

The sterol compositions of different species of cultured phytoplankton, (two diatoms—Phaeodactylum tricornutum and Skeletonema costatum, two green algae—Danaliella minuta and Tetraselmis tetrathele and a brown alga—Monochrysis lutheri) were compared with that of a diatom field population ( > 98 % Thalassionema nitzschioides) using GC-MS techniques. The effect of culture age in the cultured specimen; was examined by harvesting in both the exponential and stationary growth phases and was found to produce considerable differences in the sterol composition in some species. The influence of the intensity and different spectral illumination on a cultured specimen of a green alga (Danaliella minuta) was also examined and found to produce changes in the sterol composition.     

Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum

Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post-resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.     

Etude de la fertilité des eaux marines au moyen de tests biologiques effectués avec des cultures d'algues. II. Limitation nutritionnelle et viabilité de l'inoculum

Ten cultures of phytoplankters, including four strains of Skeletonema costatum from different origins, were used to improve some aspects of the bioassay technology. Special attention was paid to the preliminary nutrient limitation of the inocula. When the cells are maintained in nutrient starvation, their carbon, nitrogen, carbohydrates, proteins and above all chlorophyll a contents decrease. The minimum appears after a range of 2–7 days, according to species. The survival of these limited cells and their capacity to give rise to active growing cultures when sub-cultured are different with species, but efficiency in the inocula they provide usually can occur only until the minimum content in cellular components appears. Starved cells and enriched cultures of Chaetoceros lauderi and Skeletonema costatum were used to inoculate several samples of sea water, in order to test the effect of the starvation on the experimental results. It appears that the starved cells increase the sensitivity of the method, but they are more susceptible to substances limiting their growth. In the opinion of the authors the best way would be to use both starved and enriched cells as inocula, but, when this is impossible, cautiously starved cells should be used with unpolluted sea waters.     

Kinetic exploration of nitrate-accumulating microalgae for nutrient recovery

Within sustainable resource management, the recovery of nitrogen and phosphorus nutrients from waste streams is becoming increasingly important. Although the use of microalgae has been described extensively in environmental biotechnology, the potential of nitrate-accumulating microalgae for nutrient recovery has not been investigated yet. The ability of these marine microorganisms to concentrate environmental nitrate within their biomass is remarkable. The aim of this study was to investigate the application potential of nitrate-accumulating diatoms for nutrient recovery from marine wastewaters. The intracellular nitrate storage capacity was quantified for six marine diatom strains in synthetic wastewater. Amphora coffeaeformis and Phaeodactylum tricornutum stored the highest amount of nitrate with respectively 3.15 and 2.10 g N L^?1 of cell volume, which accounted for 17.3 and 4.6 %, respectively, of the total nitrogen content. The growth and nitrate and phosphate uptake of both diatoms were further analyzed and based on these features P. tricornutum showed the highest potential for nutrient recovery. A mathematical model was developed which included intracellular nitrate storage and the kinetic parameters were derived for P. tricornutum. Furthermore, a simulation study was performed to compare the performance of a proposed microalgal nutrient recovery unit with a conventional denitrification system for marine wastewater treatment. Overall, this study demonstrates the potential application of P. tricornutum for saline wastewater treatment with concurrent nitrogen and phosphorus recycling.     

Publisher's note

During the late spring and early summer 1976, laboratory cultured cells of five marine algae were enclosed in dialysis sacks and grown in situ, in Ligurian coastal waters.Skeletonema costatum (Greville) Cleve did not grow at all. Hemiselmis virescens Droop stayed alive, but did not grow significantly. Platymonas suecica (Butcher) Manton &; Parke grew on cell reserves. Only Phaeodactylum tricornutum Bohlin and Thalassiosira pseudonana Hasle &; Heimdal took up nutrients from sea water when all reserves were exhausted. When division ended, all cells were capable of growing when sub-cultured, but physiologically were only slightly active. During immersion in oligotrophic waters, cell contents adapted quickly from nutrient-rich to nutrient-limited growth conditions. During adjustment to equilibrium with natural conditions, the three species became impoverished mainly in phosphorus (67–88% of initial values), ATP (54–92%), and chlorophyll a (59–89%); losses of nitrogen were lower (24–53%).To obtain algal material similar to that in the wild, the data suggest that the cells have to be kept in situ only until they divide five times, which usually requires no more than a week. Counting the cell density each day is probably the most convenient method of control, and each culture should be counted, because the difference of growth between aliquot dialysis cultures can be important (up to 28%).Data obtained for “wild-like” cells of Platymonas suecica, Phaeodactylum tricornutum, Thalassiosira pseudonana, and a wild contaminant population of Cylindrotheca closterium Rabenhorst with biochemical analysis used to run the Droop's model and with enrichment bioassays demonstrate that, during the period studied, phosphorus was the limiting factor for the algal growth. Bioassays also demonstrate that there were some specific variations; some algae were permanently limited by phosphorus, while for a few others nitrogen and phosphorus could be equally limiting.     

Influence of temperature and cell size on the division rate and chemical content of the diatom Chaetoceros curvisetum Cleve

Cell size and temperature influenced the division rate and chemical content of the diatom Chaetoceros curvisetum Cleve when grown at 15, 20 and 25°C in nutrient replete media. Cell-size dependent trends of division rate in individual clones changed with temperature in a complex fashion. Considerable interclonal variability in division rate within a restricted range of cell sizes was also found. Cellular levels of carbon, nitrogen, protein, chlorophyll a, and silicon were linearly related to cell size. Cellular levels of carbon and chlorophyll per unit volume and silicon per unit surface area changed with temperature. No temperature effect on cellular levels of nitrogen and protein was found.