Pierre Marsot

Distribution et effets du chlorure de tributylétain et de ses produits de dégradation sur la croissance de l'algue marine Pavlova lutheri en culture continue

Organotin compounds have been used as biocides, wood preservatives, plastic stabilizers and antifouling agents for many years (Maguire, 1991). The acute toxicity as well as the sublethal effects of tributyltin (TBT) derivatives have been tested for numerous freshwater and marine species (Alzieu, 1989; Kelly et al., 1990). The biodegradation of TBT in dibutyltin (DBT) and monobutyltin (MBT) is well established for some phytoplankton species (Lee et al., 1989) but little is known about the physiological adaptation of microalgae to a long term sublethal contamination by TBT. The objective of this study was to evaluate the ability of marine alga Pavlova lutheri, cultured in “chemostat mode” under axenic conditions, to accomodate increasing cocentrations of TBTCl in the culture medium and to determine its capacity to degrade TBT in DBT and MBT. Chemostat culture of P. lutheri were exposed to contaminated nutrient supplies containing 18.5, 74, and 185 nmol l−1 of TBT chloride (expressed as TBT+) respectively. For comparison purposes, a batch culture of P. lutheri was exposed to a concentration of 13 nmol l−1 of TBTCl for a 48 h-period. Organotin species (BuSn) were monitored by GC/ITD in three different fractions: dissolved in the culture medium, adsorbed on the external cellular walls, and dissolved in the cellular fluid. Within a 24 h-period, TBT was observed in the cellular fluid indicating a rapid uptake of the contaminant. The ability of P. lutheri to degrade TBT was confirmed and found more effective in a continuous culture than in a batch culture under similar contamination conditions. MBT was observed in both cellular fractions of all cultures (Tables 2 and 3) but P. lutheri seems particularly efficient to form MBT at low TBT concentration. The amount of BuSn adsorbed onto the cell was directly related to TBT concentration in the nutrient supply but intracellular BuSn decreased from 150 nmol g−1 (dry weight) to less than 60 nmol g−1 (dry weight) when the TBT concentration increased from 74 to 185 nmol l−1 (Fig. 2). The cell density and the growth rate of a continous culture of P. lutheri (Figs 3 and 4) were not affected by a contamination level of 18.5 nmol l−1 of TBT. In a continuous culture receiving 74 nmol l−1 of TBT P. lutheri suffered a toxic impact in the first few days of the experiment, loosing 40% of its cell density, However, the culture recovered its initial growth rate in the next 10 days in spite of an uninterrupted contamination. Finally, a severe toxic shock was observed for a culture receiving 185 nmol l−1 of TBT leading to a major dysfunction of the culture in only three days. The toxic effects observed in culture productivity (Fig. 5) for treatments at 74 and 185 nmol l−1 are also illustrated by a significant decrease of chlorophyll α concentration in culture [Fig. 6(A)] but not in the individual cells [Fig. 6(B)] which was an indication of the preservation of life functions of cells in all experiments. These results evidence the ability of P. lutheri, cultured in chemostat mode, to resist and accommodate to a continuous input of a high level of TBT to the culture medium degrading TBT into much less toxic species MBT and DBT. As a chemostat culture is comparable in many aspects to an open natural environment (Rhee, 1980; Wangersky and Maass. 1991). The behavior of P. lutheri observed in this study might be extended to the natural coastal environment where this alga is present. If such an assumption is correct at least regarding the general processes of bioaccumulation and degradation, this study clearly indicates that P. lutheri might play an important role in the food uptake of BuSn by filter feeders and other herbivorous species due to its ability to bioaccumulate large quantities of BuSn [Fig 2(A) and 2(B)] without being irreversibly affected. Fortunately, the natural environment as simulated by our continuous cultures seems to strongly accelerate the degradation rate of TBT into DBT and MBT, two much less toxic tin species. Results of previous studies on the toxicity effects of TBT on micro-algae, all conducted in batch cultures, should be interpreted with some caution since the ability of P. lutheri and possibly other marine algae to survive and grow in TBT contaminated media seems to have been grossly underestimated.

Growth kinetics and nitrogen-nutrition of the marine diatom Phaeodactylum tricornutum in continuous dialysis culture

The growth kinetics and nitrogen (N)-nutrition of the marine pennate diatom Phaeodactylum tricornutum Bohlin were determined in continuous dialysis culture at different cell densities. Inflow nutrient medium was supplied as natural unenriched estuarine seawater to a dialysis culture system with a high ratio of membrane surface area/culture volume (Am/Vc). Under the experimental conditions, the supply of inorganic macronutrients (NO3− + NO4− and PO4−3) by diffusion (Nd) was markedly greater than that provided by the dilution (FfCN) of the culture (Nd ≫ FfCN), thereby establishing an inverse relationship between the cell density and the dilution rate (D). This continuous dialysis system allows for the maintenance of prolonged growth (> two weeks) at various cell densities (1.4 to 27.2 × 109 cells 1−1) within a range of dilution rates between 0.30 to 1.08 d−1. In high cell density cultures, where the extracellular medium was characterized as nutrient deficient, a lower growth rate (μe) was exhibited than in cultures with lower cell densities. The growth rate (μe) remained equivalent to the dilution rate (D) throughout the culture cycle, indicating that equilibrated growth was achieved. High cell density cultures yielded higher productivity (P), relative to that of cultures grown at lower cell densities, in terms of cell-N and −C produced per unit time. However, cell quotas of both N and C declined with increasing cell concentrations. Denser cultures were characterized by an enhanced N-conversion efficiency (YN) and a higher cellular N/C atomic ratio. The nutritional response of this diatom in dense cultures reveals an efficient use of N-nutrients, presumably as a result of cellular nutrient adaptation to oligotrophic conditions.

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

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

Biocatalysis of chlorophyllase from Phaeodactylum tricornutum in organic solvent media

Abstract The biocatalysis of partially purified chlorophyllase from Phaeodactylum tricornutum in an organic solvent medium was investigated. The optimum amounts of water and organic solvent required for the chlorophyllase-catalyzed hydrolytic activity were measured in a wide range of solvents, including acetone, ethanol, propanol, butanol, pentanol, hexanol, toluene, pentane, hexane, octane, and heptane as a function of their hydrophobicities. The logarithm of partition coefficient (log P ) was used to define the quantity of solvent polarity. The amount of aqueous buffer required for the enzymic activity was lower in non-polar solvents (log P >1.8) than polar ones (log P V max values for the hydrolytic activity of chlorophyllase in water-miscible solvents (log P P > 0.8). The hydrolytic activity was decreased by approximately 12 times by increasing the carbon chain of alcohol from two to five. The results also indicated that phytol has a non-competitive inhibitory effect on the chlorophyllase activity in reaction media containing acetone, and activatory effect in that containing hexane.

Continuous microalgal culture using swine manure dialysate as a nutrient source

Abstract Nutrients diffusing from swine manure through hollow-fibre dialysis improve the dry matter yield of the marine diatom Phaeodactylum tricornutum grown under natural light by 40% (72·6 versus 51·6 mg liter −1 day −1 ) compared to natural estuarine water. The increase is achieved despite incomplete nutrient removal and is ascribed to the higher concentrations of ammonia and orthophosphate supplied by swine manure dialysate. Growth promoting factors aside, the exclusion of predators and particles causing turbidity as well as the limited dialysis of heavy metals eliminate or greatly attenuate the growth retarding effects of such undersirable constituents of swine manure.

Un nouveau procede de culture continue a dialyse pour le phytoplancton

SummaryEquipment and methods for continuous dialysis culture are described which make feasible the continous aseptic production of dense cultures of phytoplankton (unicellular marine algae). New features include the use of independently-replaceable fibre dialysis units and renewal of the nutrient medium (sea water) by controlled hydraulic diffusion. Cell densities of up to 4 × 107 are attained.

Rendements et caractéristiques biologiques de cultures à dialyse continues de Phaeodactylum tricornutum opérant à divers taux de dilution

Abstract Continous dialysis cultures of the marine diatom P. tricornutum , conducted under saturating light and using natural estuarine water as nutrient substrate, were grown at dilution rates varying between 0.30 and 0.82 d −1 . Results indicate that productivity varies iversely with dilution rate. Less rapid dilution, while maintaining denser cultures, affects the two principal factors determining productivity: nutrient mass transfer and nutrient conversion efficiency of the algae. Dense cultures increase nutrient diffusion across the dialysis membrane, hence the substrate utilization efficiency of the technique. Such cultures are characterized both by a higher nitrogen conversion coefficient ( Y n) and a lower maintenance coefficient ( Y e) for this nutrient. On the other hand, slower dilution of dialysis cultures decreases nutrient availability per cell. Biochemical composition is in turn altered as dry weight, carbon and nitrogen, on a unit cell basis, are impoverished at lower dilution rates. However, chlorophyll a concentrations are not similarly affected by varying rates of dilution.