| Occurrence of Microcystis blooms becomes more and more serious, many blooms even extend to deep winter. Overwintering cells as "seeds" are crucial for next bloom forming. After winter, what effects of two cold shock patterns "temperature plummeting-into the winter(no warming up)" and "temperature plummeting-warming up-droppimg down", even the shock strength (temperature X time) on overwintering, have not been reported when a winter comes.As a result, the paper firstly focused on two patterns of "single cold shock" and "repeated cold shock (5 times, after each cold shock, samples were cultured in the algae culture chamber)", then discovered the growth condition of colonial Microcystis XW01 after cold shock by setting different shock strengths (0.5 h 2 h 4 h).The results showed:only the 2 h group exhibited stronger cold tolerance than the control group under the single cold shock condition and a further comparison in maximum growth rate suggested it was 8.5% higher than the control. Besides, despite 2 h group could grow better than others under the repeated cold shock condition, it remained worse than the control and its maximum growth rate was 16.7% than the control.As we know, Microcystis will initiate the non specificity anti cold stress mechanism in cold stress environment. Therefore, we studied the change of oxidative system,osmotic adjustment solutes,membrane fluidity and cellular polysaccharide content in Microcystis. The results showed:(1)Cold stress can induce theantioxidative enzyme systems of cells to eliminate damage caused by free radicals. As a result, SOD and POD reached the maximum after 2 hours cold shock(2.39 and 1.61 times than the control, respectively), while CAT activities peaked at 4 h and were 43.9% higher than the control. Among the repeated groups, the highest activities of SOD and POD also appeared at 2 h group(2.16 and 2.78 times than the control, respectively). In addition, CAT activities were greatest at 4 h group(l.9 times than the control)(2)Our research showed that contents of glutathione, ascorbic acid,phycocyanin and carotenoids would be increase to resist cold stress. Under the single cold shock pattern, the contents of them increased with chilling time and reached a maximum at 4 h groups. Similarly, the levels of ascorbic acid and phycocyanin were observed to increase with increasing time under repeated cold shock treatment(5.11 and 3.56 times than the control, respectively).However, the highest levels of glutathione were measured at 2 h group (2.3 times of control values) and longer chilling time inhibited its accumulation. We observed a trend of increase in the amount of carotenoids that they peaked at 4 h group with treatments of single and repeated cold shock(20.7% and 29.7% higher than the control).(3)Proline and glycine betaine play important roles in cytoprotection and their accumulation have been correlated with tolerance to cold stress. Under the single cold shock pattern, their contents increased relative to the control with increasing time(18.35 and 2.63 times than the control,2 h group). However, longer time reduced their accumulation, levels of proline and glycine betaine were observed to decrease at 4 h group.The same situation also appeared in the repeated cold shock. Their contents were greatest at 2 h group(10.5 and 4.25 times of the control).(4)Malondiadehyde(MDA)content and cell membrane permeability reflect the damage degree of membrane system, respectively. With the single cold shock treatment(0.5 h 2 h 4 h), MDA levels were 20.6%,17.9% and 20.9% higher than the control, but the cell membrane permeability did not change significantly. Under the repeated cold shock pattern, they reached the maximum at 4 h group(30.1% and 216.7% higher than the control).(5)Cold stress greatly increased polysaccharide production in Microcystis. After exposure to longer time(2 h 4 h)of single cold shock, their contents were 19.4%and 20.1% higher than the control, respectively.Furthermore, contents of polysaccharide were obviously increased with increasing chilling time under the repeated pattern(14.7% 20.8% and 21.5% higher than the control).Moreover, the concentrations of chlorophyll, soluble protein and N, P metabolic enzyme(nitrate reductase and alkaline phosphatase) activities can be affected in cold-stress environments. The results showed:single cold shock treatment did not affect soluble protein production,whereas the repeated group could change a lot(0.5、2 and 4 h:8.1%、16.2% and 40.5% lower than the control).Cold stress significantly affected the levels of photosynthetic pigments in Microcystis. Chla levels were lowest at 4 h group both in single and repeated group(22.7% 33.4% lower than the control). Cold stress also inhibit the activity of nitrate reductaseand it decreased by 16.2%, 35.3% and 60.2% respectively with single cold shock treatment(0.5、2 and 4 h). Meanwhile,0.5、2 and 4 hgroup were decreased by 9.9%,38.7% and 58.1% under the repeated pattern. Cold stress can enhance the activity of alkaline phosphatase. It would reach the maximum both in 2 h group no matter in single or repeated cold shock.(17.9% 80.9% higher than the control)In general, two cold shock patterns would improve the antioxidant enzyme system activity of Microcystis cells and the contents of osmotic adjustment substances and polysaccharide. At the same time, two kinds of cold shock pattern exacerbated the cell membrane damage and inhibit the synthesis of chlorophyll and protein.Furthermore, we focused on exploring the effects of two cold shock patterns on the recovery of the growth of Microcystis after overwintering. The results showed:Compared with other groups, shorter time (0.5 h) cold shock could better maintain the activity of cells after overwintering whether in single or repeated cold shock.Compared with the algae without cold treatment, shorter duration of cold shock could maintain the activity of the cells during the overwintering and the recovery was faster. At the same time, during the overwintering, we found that in two different temperature settings (0 C and 4-9 C), the former group of Microcystis could not grow normally,but the latter group could still grow.Through the study of this paper, it is found that different cold shock patterns have different effects on the physiological metabolism.Appropriate cold shock intensity was more favorable for the recovery of the cells in the winter. |
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