Microcystis and Aphanizomenon are among the most dominant bloom-forming species of cyanobacteria in eutrophic waterbodies. The mechanism of their formation and disappearance in freshwater bodies has been always the focus of research due to their harmful effect on aquatic ecosystems and their potential hazard to human health. Phosphorus (P) is always considered the principal nutrient that influences phytoplankton growth in most freshwater bodies, and the oversupply of this nutrient from human sources underlies the eutrophication of water columns. In this thesis, the physiological and biochemical response to P and their competition for P were explored by means of batch culture and continuous culture. The difference in responses of Microcysti and Aphanizomenon to P was discovered. And competitive behaviors of two strains in P-limited continuous cultures condition and field condition were discussed. The main results are as followings:
(1) The effects of three phosphoric chemicals in different forms (pyrophosphate sodium, polyphosphate sodium and β-glycerophosphate disodium) on the growth and alkaline phosphatase activity (APA) of Microcystis aeruginosa, and total soluble phosphorus in medium were tested at orthophosphate-repleted and orthophosphate-limited conditions. The results showed that the addition of the three different phosphoric chemicals had no significant effects on the growth rate and APA activity under orthophosphate repleted condition, indicating that orthophosphate (K2HPO4) was the major phosphorus source by M. aeruginosa for its growth. Under orthophosphate-limited condition, however, the addition of the three different phosphoric chemicals promoted the growth rate of M. aeruginosa significantly. The phosphorus in different forms could be utilized effectively by M. aeruginosa but by different utilization ways. β-glycerophosphate disodium was uptaken fast than that of pyrophosphate sodium and polyphosphate sodium by M. aeruginosa. It was demonstrated that APA was increased upon the addition of β-glycerophosphate disodium, but not for pyrophosphate sodium and polyphosphate sodium.
(2) The physiological and biochemical response to P in unicellular and colonial Microcystis were compared. The two phenotype strains exhibit physiological differences mainly in terms of their response to low P concentrations. Unicellular strains were damaged when exposed to P-free medium for a long time; whereas colonial strains were endure P-free culture longer. The growth rate, the oxygen evolution rate, Fv/Fm, and ETRmax of unicellular Microcystis strains were significantly inhibited and APA, content of extracellular microcystin were significantly increased at a P concentration of 0.2 mg/L; however, that of the large colonial Microcystis strains were not significant changed. There was also exist difference in P uptake and storage between two phenotype strains. The results of phosphate uptake experiments conducted using P-starved cells indicated that the unicellular strains exhibited higher Km and Vmax values than those exhibited by the colonial strains, which indicates that the latter had a higher affinity for low levels of P. The unicellular strains consumed more P than the colonial strains. The reason for the difference in growth patterns and physiological traits under P-limited conditions may be attributed to the difference in the extracellular structure between the two phenotype strains. The better performance of the colonial strains at low Pi concentrations might be due to the role of mucilage in nutrient sequestration and processing.
(3) The effects of P on the growth of Aphanizomenon flos-aquae and uptake of P kinetics were studied using BG11 as basal medium in the laboratory. The results indicated that either lack or excessiveness of P concentration would inhibit growth of A. flos-aquae. The optimal P concentrations for growth of A. flos-aquae would be 20-100 μM; and the growth were significantly inhibited when P concentration less than 10 μM. The relationship between P uptake rate of A. flos-aquae and the external phosphate concentrations can fit by Michaelis-Menten model. The half saturation constant for P uptake and maximal uptake rate was 21.031±6.266 μM and 1.5093±0.263×10－5 µM P • cell-1 • h-1, respectively.
(4) The effects of dilution rates and agitation rates on the growth and physiological character of M. aeruginosa and A. flos-aquae were studied using unialgal chemostat cultures with various supply rates of culture medium where P limited algal growth. In unialgal continuous cultures, either lack or excessiveness of dilution rates was disadvantage for the growth of M. aeruginosa. The adaptable dilution rates for its growth would be 0.0036-0.0128 h-1. The growth rates of M. aeruginosa reached maximum under stable condition and decreased with the increasing of agitation rates. Comparatively, the growth rates of A. flos-aquae were correlated with dilution rate and increased with the increasing of dilution rate. There were no significant differences between growth rates of A. flos-aquae and agitation rates. The relationship between the P uptake rates and specific growth rate of two strains and extracellular P concentration in the culture vessel can fitted to Michaelis-Menten model and Monod model, respectively. The M. aeruginosa exhibited lower Km and Ks values than those exhibited by the A. flos-aquae, which implied that the former had a more competitive advantage for Pi.
(5) In order to figure out the effects of different environmental factors on competition between the cyanobacteria M. aeruginosa and A. flos-aquae. The competitive interaction between two strains was studied using mixed chemostat cultures under various dilution rates and various temperatures. In P limited mixed culture at 25 ºC, M. aeruginosa was dominate at dilution rates less than 0.00736 h-1; M. aeruginosa and A. flos-aquae can coexist in mixed culture at a higher dilution rate of 0.01472 h-1. Temperature can affect their competitive interactions. M. aeruginosa dominated at higher temperature (> 19 ºC), whereas A. flos-aquae out-competed at lower temperature (< 19 ºC). The APA was greater in the A. flos-aquae than in the M. aeruginosa. Addition of organic P can accelerate the growth of A. flos-aquae, which made it out-competed. These results revealed that the succession of M. aeruginosa and A. flos-aquae was the outcome of synergistic actions of nutrients and temperature. The result from chemostat cultures explains in part the occurrence of seasonal succession of Microcystis and Aphanizomenon in Dianchi Lake.