|Other Abstract||Phytoplankton evolved in the Archaean oceans more than 2.8 billion years ago and are of crucial importance in regulating aquatic food webs, biogeochemical cycles and the Earth’s climate. For a better understanding of phytoplankton dynamics, it is important to know the processes that affect both the increase and decline of a population. In addition, release of intracellular content, such as Microcystin, odours compound, during the cell death, contaminated the drinking water, posing a threat to human and livestock health. However, loss via cell death has been poorly understood. This paper focused on the cell death process of harmful bloom algae Microcystis (Cyanobacteria), characterized the physiological change when Microcystis suffered from different artificial or natural stresses to death, and developed viability assessment methods for Microcystis pure culture and filed colonies. The main results are as followings:
1. The decaying process of M.aeruginosa FACHB 905 under nitrogen starvation, phosphorus starvation, dark or low temperature (10 ℃) was investigated. The decrease of biomass and the concomitant change of photosynthetic activity, esterase activity and antioxidant system, including superoxide dismutase (SOD) and catalase (CAT) activities were studied. The results indicated that the growth was significantly inhibited under dark and low temperature. Compared with the physical stress, nutrition stress such as nitrogen and phosphorus starvation did not limit the growth effectively in the early stage. After being kept in the phosphorus free medium for 20 days, Chlorophyll a reached peak, about the twice of the primary concentration, and then decreased at a rate of 0.1 mg.L-1.d-1, the highest decaying rate among the four stress factors, which indicated its significance of available phosphorus for Microcystis long survival. Esterase activity increased significantly under low temperature, also increased slightly under nitrogen and phosphorus stresses. Only under dark stress, the decrease of esterase activity correlated with the decrease of photosystem activity. The increase of SOD and CAT activity presented the similar behaviour as the decrease of Fv:Fm and ETRmax. SOD activity increased in the early stress stage, while CAT activity did not increase significantly until the content of Chlorophyll a decreased.
2. Several assay methods were screened for viability assessment in cyanobacteria using M.aeruginosa FACHB 905. Compared with FDA, Evan’s Blue and autofluorescence, the MTT assay, which was based on the ability of viable cells to reduce MTT to formazan, was found to be reliable and was selected for further study. MTT concentration, incubation time and temperature were optimized for M. aeruginosa. Improvements to the sensitivity and reproducibility of the MTT assay included performing it in the dark to reduce the effects of formazan light sensitivity when extracted in DMSO. Another improvement involved collecting viability data by cell counting rather than colourimetrically, which was concluded from the fact that oxidoreductase activity, responsible for MTT reduction, would elevate or decrease under stress conditions. The MTT assay was also found to be applicable to other Cyanobacteria and Diatoms, including field samples, but not for algae belonging to Chlorophyta, Euglenophyta, Pyrrophyta or Chrysophyta.
3. The differences of floating and sediment Microcystis colonies from water surface were compared. The floating colony presented healthier color green, owned higher photosynthetic activity and MTT reducing ability. The ratio of the sediment in the mixed sample correlated significantly with the viability tested by the MTT, Evan’s Blue staining. The results suggested that the sediment of Microcystis should be related to the physiological status. The ratio of the sediment could be employed to estimate the viability of field Microcystis without disaggregating the colony.
4. Decline and sedimentation of Microcystis in Dianchi Lake was investigated in the winter of 2007. Photosynthetic activity decreased significantly in winter. Microcystis sank in two types, as structured or blank colony. Based on the observation, Microcystis died at least in two ways: cell broke down and left intracellular content or shrunk and died. The death process was completed in the capsule, which was speculated from the fact that the capsule remains kept the colony shape and the percentage of the single cell in the water body was less than 3%.
5. Cell death of M.aeruginosa FACHB 905 upon Ca2+ exposure was studied. Upon 0.1 mol.L-1 Ca2+ exposure, cell membrane permeability changed quickly before the decrease of photosynthetic activity and MTT reducing ability, however, intracellular microcystin release was not significant. The death of M.aeruginosa FACHB 905 should belong to programmed cell death since the markers of programmed cell death-DNA fragmentation and the Caspase-3 like activity were detected. In addition, the death could be blocked by the protein synthesis inhibitor chloromycin, also inhibited under low temperature (4 ℃) condition with light. The specific Caspase-3 inhibitor Z-VAD-FMK did not block the cell death, on the contrary, it accelerated the cell death even without excess Ca2+ exposure.
6. Cell death of M.aeruginosa FACHB 905 upon 0.25 mg.L-1 Cu2+ exposure was studied. After 30 min in BG11 medium containing 0.25 mg.L-1 Cu2+, all of the cells were unable to reproduce. The indicator of viable cell including photosynthetic activity, MTT reducing ability lost till 8 h later. The total content of Chlorophyll a decreased to 42% in 48 h, but the cell number did not changed significantly since the remains kept the cell shape. Microcystin was released dramatically in the early 4 hours, but the change of membrane permeability tested by the Evan’s Blue staining was not as significant as expected, and only 20% of the cells presented ruptured membrane. The results suggested that the death of M.aeruginosa upon 0.25 mg.L-1 Cu2+ exposure would be a perfect model to study the essence of prokaryotic life and to develop more accurate viability assessment methods.|