The demonstrated modified spectrophotometric method makes use of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and its specific absorbance properties. The absorbance decreases when the radical is reduced by antioxidants. In contrast to other investigations, the absorbance was measured at a wavelength of 550 nm. This wavelength enabled the measurements of the stable free DPPH radical without interference from microalgal pigments. This approach was applied to methanolic microalgae extracts for two different DPPH concentrations. The changes in absorbance measured vs. the concentration of the methanolic extract resulted in curves with a linear decrease ending in a saturation region. Linear regression analysis of the linear part of DPPH reduction versus extract concentration enabled the determination of the microalgae's methanolic extracts antioxidative potentials which was independent to the employed DPPH concentrations. The resulting slopes showed significant differences (6 - 34 μmol DPPH g−1 extract concentration) between the single different species of microalgae (Anabaena sp., Isochrysis galbana, Phaeodactylum tricornutum, Porphyridium purpureum, Synechocystis sp. PCC6803) in their ability to reduce the DPPH radical. The independency of the signal on the DPPH concentration is a valuable advantage over the determination of the EC50 value.

PAM (pulse-amplitude-modulated) fluorescence measurements of motile microphytobenthic algae were carried out in June 1996 at Sylt, Germany. Compansons between "C-based and fluorescence-based production rates were made. A very high correlation between 14C-and fluorescence-based production rates was found for maximal production rates (P, , values). I4C-based maximal production rates var~ed during the study penod between 0.65 and 1.7 mg C mg chl a-' h-', comparable to variations of P,,, measured with the fluorescence-based method. For other photosynthetic parameters [a (maximum light utilization coefficient). Ek (light saturation index), E,,, (light intensity at which P, , , , , is reached)], differences between the 2 methods were much larger. Highest carbon quantum yields ( @, , , ) (m01 C m01 quanta-' absorbed) were obtained at low irradiances. Considering the whole range of investigated carbon quantum yields, we found that initially these values decreased at low to moderate irradiances without a concomitant decline of the actual photochemical efficiency (F,' -F)/F,,' (Fand F,'. m~nimal and maximai iiuoresce~lct' signals in :he !igh!) Therefcre, e high !ine~rit)r between the actual photochemical efficiency and the carbon quantum yield could only be observed up to values of 0.018 m01 C m01 quanta-' This is different to higher plants, for which linearity can be observed up to carbon quantum yields of 0.042 m01 C m01 quanta-' It was shown that, for the calculation of the overall production rates based on the fluorescence method, it is necessary to carefully measure the mean specific absorption coefficient (a') of the algae. Unless this is achieved. PAM measurements cannot be used to calculate absolute production rates.

The effects of phosphorus (P) limitation on growth, toxicity, and variable chl fluorescence of Alexandrium minutum were examined in batch culture experiments. Cell division was greatly impaired in P‐limited cultures, but P spiking of these cultures after 9 days stimulated high levels of cell division equivalent to P‐replete cultures. The cellular concentration of paralytic shellfish toxins was consistent over the growth cycle of control cultures from lag phase into logarithmic growth phase, with toxins repeatedly lost to daughter cells during division. The low level of cell division in P‐limited cultures resulted in a 10‐fold increase of cellular toxin compared with controls, but this dropped upon P spiking due to increased rates of cell division. The history of phosphorus supply had an important effect on toxin concentration, with the P‐limited and the P‐spiked cultures showing values 2‐fold higher than the P‐replete cultures. Toxin profiles of the A. minutum strain used in these experiments were dominated by the N1‐hydroxy toxins, gonyautoxins (GTX) GTX1 and GTX4, which were approximately 40 times more abundant than their analogues, GTX2 and GTX3, in P‐limited cultures. The dominance of the N1‐hydroxy toxins increased significantly in control cultures as they advanced through logarithmic growth. In‐line measurements of the variable chl fluorescence of light‐adapted cells indicated consistent photochemical efficiency under P‐replete conditions. P limitation induced a drop in fluorescence‐based photochemical efficiency that was reversible by P spiking. There was an inverse linear relationship between in‐line fluorescence and cell toxin quota (r = −0.88). Monitoring fluorescence in‐line may be valuable in managing efficient biotechnological production of toxins.

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Turbidostat cultures of Dunaliella salina (Chlorophyceae) and Thalassiosira weissflogii (Bacillariophyceae) were grown at fluctuating concentrations of nitrate, phosphate and silicate. In-line measurements of PAM fluorescence were used to monitor the effects of fluctuating nutrient supply on the photochemical efficiency of photosystem II reaction centres of light-adapted cells (∆F\F h m ). Besides the maximal photochemical efficiency of photosystem II reaction centres of dark-adapted cells (F v \F m ), chlorophyll a, particulate organic carbon, nitrogen and phosphorus, and the cell number were measured frequently during the experiments. Following nutrient-replete growth, the cells were supplied with medium from which either nitrate, phosphate or silicate was omitted. When significant effects of nutrient starvation were indicated by the fluorescence parameters, a pulse of the deficient nutrient was added to the cultures. Our experimental set-up for in-line fluorescence measurements provided sensitive and reproducible detection of the various fluorescence signals, revealing strong influences of nutrient supply on the photochemical efficiency of photosystem II. In general the fluorescence values changed substantially within 1-30 min after re-addition of the deficient nutrient. Addition of phosphate and silicate induced an immediate characteristic decrease in fluorescence, whereas nitrate addition was characterized by a strong, delayed increase in fluorescence. Complete recovery to pre-starvation fluorescence values took about 48 h in all experiments. The physiological background of nutrient uptake is used to explain the observed tight couplings between fluorescence responses and nutrient re-addition. Our study clearly demonstrates that in-line fluorescence measurements provide a new tool for the investigation of phytoplankton reactions to fluctuating nutrients and offer the possibility to detect nutrient starvation in the field.