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.     

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.     

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.     

Natural chelators in sea water: Detoxification of Zn2+ by brown algal polyphenols

We have examined growth responses of several species of marine phytoplankton, cultured with and without heavy metal stress, to supplements of polymeric polyphenols from the brown algae Ascophyllum nodosum (L.) Le Jol. and Fucus vesiculosus L. In the absence of additional heavy metals. supplements of up to 4000 μg. 1 of polyphenols had a small effect on initial growth rates for three of these microalgae and had no effect on maximum cell densities for four species. One very common, heavy metal-sensitive diatom, Skeletonema costatum (Grev.) Cl., showed significantly increasing maximum cell density in the cultures, with increasing addition of polyphenols to the medium. The toxicity of Zn²⁺ (0.5–2.0 mg·1 ¹) to cultures of the diatom Phaeodactylum tricornutum Bohlin was relieved by supplements (100–200 μg·1 ¹) of the brown algal polyphenols. Exudation of these polyphenols from brown seaweeds may contribute to the natural chelating capacity of inshore sea water.     

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.     

Temperature-influenced variations in speciation and chemical composition of marine phytoplankton in outdoor mass cultures

Between September, 1976 and July, 1977 Phaeodactylum tricornutum Bohlin was replaced as the dominant species by Skeletonema costatum (Grev) Cleve as temperatures fell below 10°C in the fall in an outdoor pond supplied with a mixture of waste water and sea water. Phaeodactylum tricornutum returned in the spring as the major species when temperatures rose above 10°C. In an adjacent pond in which only nitrogen and phosphorus were added in excess, however, P. tricornutum dominated throughout the entire study period even through the temperature varied between 0 and 25°C. We suspected that the difference inspecies dominance in the two ponds occurred because Skeletonema costatum requires silicon, which was present in sufficient quantities only in the waste-water-enriched pond. whereas Phaeodatylum tricornutum does not have a specififc requirement for this nutrient. The cellular chemical composition of P. tricornutum varied in a U-shaped fashion with changing temperature: minimum values for the cellular carbon, nitrogen, and chlorophyll contents were displayed at 15–20°C and maximum values at 3 and 15°C. Both the cellular carbon: nitrogen and carbon: chlorophyll ratios by weight were invariant with changing temperatures at ≈6: 1 and 50: 1 respectively, indicating nutrient saturation. Only under conditionsof nutrient saturation, which can be established in various ways, can the influence of temperature on phytoplankton physiology be separated from nutrient-related factors.     

Heavy metal tolerance of marine phytoplankton. III. Combined effects of copper and zinc ions on cultures of four common species

The combined effects of copper and zinc ions on the growth of three marine diatoms and one dinoflagellate in culture have been studied. The two metals were found to act synergistically to all algae except Phaeodactylum tricornutum Bohlin. With this species an antagonistic effect was observed. Addition of zinc ions reduced the inhibition of growth caused by the more toxic copper ions. Zinc toxicity to this alga increased at low concentration of magnesium, indicating a common route for divalent metal ions in general.     

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.     

COPPER TOXICITY TO SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1,2

Copper toxicity to Skeletonema costatum (Grev.) Cleve has been studied in batch cultures of chemically defined culture media. The alga is relatively insensitive to cupric ion activity, demonstrating no effect on growth up to (Cu²⁺) = 10^?8.5 M. Cultures inoculated from stationary phase stocks exhibit a prolongation of the lag phase with increasing copper concentrations near and above the point of precipitation of the copper. The toxicity of copper is a function of the silicic acid concentration in the medium. This effect is observed in a range of Si(OH)₄ concentrations (10^?5 M to 10^?4 M) above known values for the saturation of silicon uptake kinetics, thus suggesting an influence of copper on silicate metabolism.     

BRACKISH WATER AND FRESHWATER SPECIES OF THE DIATOM GENUS SKELETONEMA. II. SKELETONEMA POTAMOS COMB. NOV.1

Electron microscope investigations of the siliceous frustule show that the diatom described by Hustedt as Stephanodiscus subsalsus (A. Cleve) Hust. is not Skeletonema subsalsum (A. Cleve) Bethge (Melosira subsalsa A. Cleve) but is Microsiphona potamos Weber. This species is so similar to Skeletonema costatum (Grev.) Cleve and Skeletonema subsalsum that the combination Skeletonema potamos (Weber) Hasle is suggested. Present records classify Skeletonema potamos as a freshwater species of lakes and rivers. In Sandusky Bay, Lake Erie (U.S.A.) and in River Wümme, a tributary of the River Weser (Germany) it grows with Skeletonema subsalsum. In nature, and when grown in cultures at a salinity of 0%, the processes are extremely short; when grown at salinities of 2% or more, the processes are much longer.     

Alkaline and acid phosphatases of the marine diatoms Chaetoceros affinis var. willei (Gran) Hustedt and Skeletonema costatum (Grev.) Cleve

Alkaline phosphatase activity in cultures of the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt was higher than in Skeletonema costatum (Grev.) Cleve. The enzyme activity was localized in coarse cell particles. Acid phosphatase activity was found in the cytoplasmic fraction. Induction of alkaline phosphatase depended on the $\text{N}\text{P}$ ratio in the culture medium. A $\text{N}\text{P}$ ratio > 40 in dilution/batch culture and > 30 in large scale batch culture, respectively, induced alkaline phosphatase.Cell phosphorus showed a critical value below which alkaline phosphatase was induced. Alkaline phosphatase in natural phytoplankton from the Trondheimsfjord is unlikely to occur except possibly in special situations.     

The effect of pH in intensive microalgal cultures. II. Species competition

The results of a series of competition experiments between the chlorophyte Dunaliellatertiolecta (Dun) Butcher and the diatom Phaeodactylum tricornutum (TFX-1) Bohlin demonstrate conclusively that Phaeodactylum tricornutum dominates in intensive marine cultures when the pH rises above ≈ 10. This dominance results because of the diatom's unique ability among marine species to tolerate alkaline conditions. When the pH is regulated both freshwater and marine algae, once firmly established in culture at their respective pH optima, can resist invasion from competing species. Hence. pH control may be a method for maintaining species other than P. tricornutum in mass culture. When Dunaliella tertiolecta, however, is grown under even slight pH stress it becomes susceptible to invasion by Phaeodactylum tricornutum. This susceptibility to takeover by P. tricornutum increases with increasing pH. In contrast, the freshwater chlorophyte Chlorella vulgaris Beij., which also is sensitive to increasing pH, is capable of remaining dominant at any pH within its tolerance range when invaded by the pH-insensitive chlorophyte, Scenedesmus obliquus (Turp.) Kutz. Although allelopathic interactions may be responsible for the success of Chlorella vulgaris under seemingly stressful conditions, the success of Phaeodactylum tricornutum at increasingly higher pH seems to be related primarily to the alga's pH tolerance characteristics and not to any chemical interactions with competing species.     

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.     

Does allelopathy affect co-culturing <Emphasis Type="Italic">Haslea ostrearia</Emphasis> with other microalgae relevant to aquaculture?

Haslea ostrearia is a marine diatom known to produce marennine, a water-soluble blue-green pigment responsible for the greening of oysters in ponds along the French Atlantic coast. This phenomenon occurs seasonally when H. ostrearia blooms in oyster ponds, and it increases the economic value of cultured oysters. From an ecological perspective, H. ostrearia blooms are accompanied by a decrease in the abundance of other microalgae, suggesting that this diatom produces allelochemicals. Recent studies showed that purified marennine has other biological activities, for instance antioxidant, antibacterial, and antiviral activities, which could be used in aquaculture to promote this pigment as a natural antipathogen agent. One important issue regarding the possible use of H. ostrearia in aquaculture as a mixed algal diet, however, is the importance of marennine allelopathy. In this study, we investigated the allelopathic effect of H. ostrearia on the growth of five microalgal species relevant to aquaculture: Chaetoceros calcitrans, Skeletonema costatum, Phaeodactylum tricornutum, Tetraselmis suecica, and Tisochrysis lutea. Allelopathic tests were realized by co-culturing these microalgae with H. ostrearia in batch and in semi-continuous mode, based on initial biovolume ratios. Our findings showed that inhibition of the growth of microalgae due to the presence of H. ostrearia and marennine was species dependent. Skeletonema costatum, C. calcitrans, and T. lutea were significantly more sensitive, whereas T. suecica and P. tricornutum appeared to be more resistant. Growth irradiance significantly influenced the allelopathic effect against the sensitive species S. costatum, and the H. ostrearia production of marennine increases with irradiance. Data presented in this study partly support the hypothesis that marennine released into the culture medium possibly acts as an allelochemical compound, thus explaining the dominance of H. ostrearia and the loss of sensitive algae in oyster ponds, but also that some species are insensitive, which allows co-culturing and use in a mixed algal diet in aquaculture.     

Using laser scattering to identify diatoms and conduct aggregation experiments

Laser in situ scattering and transmissometry (LISST) instruments are used to measure particle size distributions (PSDs) and volume concentrations in water. For populations of regularly shaped non-spherical particles, such as phytoplankton, the PSD produces a ‘scattering signature’ that corresponds to the shape of the particles. The objectives of this research were to describe the scattering signatures of six diatom species and to determine whether LISST instruments can be used as a tool to measure the aggregation of diatoms into larger particles. The scattering signatures of Chaetoceros muelleri var. subsalsum, Coscinodiscus wailesii, Thalassiosira weissflogii, Phaeodactylum tricornutum, Skeletonema costatum and S. marinoi were measured. The scattering signatures of individual species were consistent over time in batch culture and there were clear differences between species in terms of peak location, peak width, and relative peak height in the PSD. LISST was used to non-destructively follow the formation of diatom aggregates in the laboratory. Both rolling and warming cultures of S. costatum caused the cell chains to form aggregates, resulting in a change in the PSD, with a shift in peak position towards larger size bins. These experiments showed that the scattering signatures of unaggregated diatom species are conservative and that LISST instruments are useful tools to investigate the factors affecting diatom aggregation and disaggregation, with potential applications both in the laboratory and field.     

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.     

The adenylate energy charge in marine phytoplantkon: The effect of temperature on the physiological state of Skeletonema costatum (Grev.) Cleve

The adenylate energy charge $\text{([ATP] +}\text{1}\text{2}\text{[ADP])}\text{[0ATP+ADP+AMP]}$ was measured in axenic batch cultures of Skeletonema costatum (Grev.) Cleve at 2°, 10°, 15°, 20°, 24° and 30°C. The results suggest that this eurythermal diatom is physiologically capable of adapting to the 28 °C range of temperature with little apparent difference in the potential energy available to the cell. In N-limited continuous cultures at 15 °C, the energy charge values were lower than those observed in batch culture by 0.2, implying nutrient stress may result in decreased intracellular chemical energy. The utilization of the adenylate energy charge as an indicator of physiological state is suggested.     

Effect of growth conditions on accumulation of internal nitrate,ammonium, amino acids,and protein in three marine diatoms

The capacity of marine phytoplankton to change their cellular content of nitrate, ammonium, amino acids, and protein in response to different growth conditions was systematically investigated. Cellular concentrations of these compounds were measured in N-starved, N-deficient, and N-sufficient Skeletonema costatum (Grev.) Cleve and in N-deficient Chaetoceros debilis Cleve and Thalassiosira gravida Cleve, both before and after the addition of a pulse of nitrogen.N-sufficient Skeletonema costatum contains high concentrations of protein, large persistent pools of amino acids, and, if it is growing on nitrate, sizeable amounts of nitrate. As it becomes N-starved, the total cellular nitrogen decreases, the internal nitrate and amino acids become entirely depleted, and the protein content is drastically reduced. After nitrogen additions to N-deficient and N-starved cultures, transient pools of unassimilated nitrogen form which can account for a large fraction of newly taken up nitrogen. The size and kind of pool which accumulates is determined by the preconditioning of the cells, the nitrogen compound which is added, and the species identity. The pools which form in S. costatum indicate that nitrate reduction is the slowest step in nitrogen assimilation, the synthesis of protein from amino acids is the next slowest, and the incorporation of ammonium into amino acid is the fastest. However, the rate limiting steps may vary between diatom species.For the first time, measurements of the variation in cellular nitrogen compounds over a wide range of environmental conditions reveal the ability of some phytoplankton to buffer the effects of a changing, and sometimes growth-limiting, nitrogen supply. They accomplish this by utilizing stored internal nitrogen for growth when the external supply is low and by quickly storing unassimilated nitrogen when the external supply is suddenly increased beyond their ability to immediately assimilate it. The accumulation of large pools of unassimilated nitrogen compounds can explain the often observed difference between nitrogen uptake rates and growth rates.     

DIVERSITY IN THE GENUS SKELETONEMA (BACILLARIOPHYCEAE): III. PHYLOGENETIC POSITION AND MORPHOLOGICAL VARIABILITY OF SKELETONEMA COSTATUM AND SKELETONEMA GREVILLEI,WITH THE DESCRIPTION OF SKELETONEMA ARDENS SP. NOV.1

Skeletonema costatum (Grev.) Cleve emend. Zingone et Sarno and S. grevillei Sarno et Zingone were known only from the type material collected from Hong Kong waters more than a century ago. Both species have now been collected as live material, and their morphology and phylogenetic position are investigated in this study. Eight Skeletonema strains isolated from Florida, USA; Uruguay; and Brazil are attributed to S. costatum, while one strain from Oman is ascribed to S. grevillei based on morphological similarity to the type material of these species. In addition, a new Skeletonema species, S. ardens Sarno et Zingone, is described for a strain from Singapore and two from northern Australian waters. Skeletonema ardens has terminal fultoportula processes ending in a tapered, undulate protrusion and long intercalary fultoportulae with 1:1 junctions. The rimoportula of terminal valves is located at the margin of the valve face. No major morphological variations were observed within S. grevillei and S. ardens along a salinity gradient, whereas in S. costatum, the processes shortened and the valves came into close contact at low salinities, as already described for S. subsalsum (Cleve) Bethge. Consistent with their morphology, Skeletonema costatum and Skeletonema subsalsum also had similar rDNA sequences. Skeletonema grevillei and S. ardens were distinct in the large subunit (LSU) phylogeny. Skeletonema ardens exhibited consistent intraspecific genetic differences in both the LSU and small subunit (SSU) rDNA.     

Allelopathic effects of the dinophyte Prorocentrum minimum on the growth of the bacillariophyte Skeletonema costatum

We investigated growth interactions between the dinophyte Prorocentrum minimum and the bacillariophyte Skeletonema costatum using bi-algal cultures under axenic conditions. When low cell densities of P. minimum and high cell densities of S. costatum were inoculated into the same medium, growth of P. minimum was suppressed. Other inoculum combinations resulted in reduced S. costatum maximum cell densities. A mathematical model was used to simulate growth and interactions of P. minimum and S. costatum in bi-algal cultures. The model indicated that P. minimum always outcompeted S. costatum over time. Enriched filtrate from low-density P. minimum cultures significantly stimulated S. costatum growth, but enriched filtrate from high-density P. minimum cultures notably inhibited the growth of S. costatum. Growth of P. minimum was not affected by enriched filtrate from cultures of P. minimum at any density. Filtrates of P. minimum cultures were fractionated by ultrafiltration (molecular weight cutoff >3000 Da), and retentate that included polysaccharide(s) significantly inhibited the growth of S. costatum.