320 J. Phycol. 38, 320–331 (2003) INFLUENCE OF PHOSPHORUS LIMITATION ON TOXICITY AND PHOTOSYNTHESIS OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) MONITORED BY IN-LINE DETECTION OF VARIABLE CHLOROPHYLL FLUORESCENCE 1 Sebastian Lippemeier 2 Research and Technology Centre Westcoast of Kiel University, Hafentörn 1, 25761 Büsum, Germany Dion M. F. Frampton, Susan I. Blackburn CSIRO Marine Research, GPO Box 1538 Hobart, Tasmania 7001, Australia Stephanie C. Geier and Andrew P. Negri Australian Institute of Marine Science, PMB 3 Townsville MC, 4810, Australia The effects of phosphorus (P) limitation on growth, toxicity, and variable chl fluorescence of Alexan- drium minutum were examined in batch culture ex- periments. Cell division was greatly impaired in P-limited cultures, but P spiking of these cultures after 9 days stimulated high levels of cell division equiva- lent 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 in- crease 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 val- ues 2-fold higher than the P-replete cultures. Toxin profiles of the A. minutum strain used in these exper- iments were dominated by the N 1 -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 N 1 -hydroxy toxins increased signifi- cantly in control cultures as they advanced through logarithmic growth. In-line measurements of the vari- able chl fluorescence of light-adapted cells indicated consistent photochemical efficiency under P-replete conditions. P limitation induced a drop in fluores- cence-based photochemical efficiency that was re- versible by P spiking. There was an inverse linear re- lationship between in-line fluorescence and cell toxin quota (r = -0.88). Monitoring fluorescence in-line may be valuable in managing efficient biotechnologi- cal production of toxins. Key index words: Alexandrium minutum; biotechnol- ogy; dinoflagellate; Dinophyceae; fluorescence; limi- tation; PAM; paralytic shellfish toxins; phosphorus; saxitoxin Abbreviations: GTX, gonyautoxins; PAM, Pulse-Ampli- tude-Modulation fluorometer; PST, paralytic shell- fish toxin; PAR, photosynthetically active radiation Alexandrium minutum Halim is a paralytic shellfish toxin (PST)-producing bloom-forming dinoflagellate. An apparent increase in the global occurrence of harmful algal blooms, including those of A. minutum, has been at least partly attributed to increased nutri- ents from anthropogenic sources such as land clear- ing, farming, and aquaculture (Hallegraeff 1993). Be- cause dinoflagellate blooms often occur in stratified waters, artificial nutrient loading can lead to limita- tions in nitrogen or phosphorus at the end of such blooms depending on the available nutrient concen- trations at the beginning of stratification. Changes in nutrient supply can affect not only the abundance of cells but also the cellular toxicity of a species (Boyer et al. 1987, Anderson et al. 1990, Siu et al. 1997). In gen- eral, nitrogen (N) limitation has been reported to cause a decrease in cell division and toxin synthesis (Anderson et al. 1990, Flynn et al. 1994, Maestrini et al. 2000). This decrease in toxin production could be over- come by N spiking of N-deprived A. minutum (Flynn et al. 1994). In contrast to the effects of N supply, the influence of phosphorus (P) supply on toxin produc- tion is not as well defined. Although P limitation re- duces cell division, some investigations have shown that the cellular toxin content increases under these con- ditions (Boyer et al. 1987, Anderson et al. 1990, Mae- strini et al. 2000). In a recent study John and Flynn (2000) showed that P limitation in Alexandrium fundy- ense only influences cellular toxicity if N is colimiting. Even though there is still a debate about the reasons for toxin synthesis in Alexandrium, there are indica- tions that the toxins might create a decreased grazing pressure (Guisande et al. 2002). Nutrient availability also has the potential to influ- ence photosynthetic efficiency. By measuring variable chl fluorescence, the photosynthetic efficiency of cells can be assessed. Many studies have revealed that the supply of N, P, silicate, and iron have strong influ- ences on phytoplankton fluorescence in the field and 1 Received 15 February 2002. Accepted 5 December 2002. 2 Author for correspondence: e-mail lippem@ftz-west.uni-kiel.de.