Physiological And Biochemical Responses Of Microcystis Aeruginosa To Nitrogen And Phosphorus Deficiency

With the rapid development of economy,human activities are accelerating CO2 emissions and water eutrophication,which caused frequent outbreaks of water blooms.The quantities of CO2 were absorbed during the process of an algal bloom event.When nutrients in the water were exhausted,the blooms gradually faded.Therefore,how to use water bloom to reduce CO2 emissions and water eutrophication are attracting the most concern.Although the cause and mechanism of water bloom outbreaks were well studied,the fading process of the blooms still poorly understood.Here,we monitored physiological and biochemical changes for Microcystis aeruginosa to understand the mechanisms of the carbon flow and growth inhibition in response to nitrogen(N)and phosphorus(P)deficiency.The results of carbon metabolism and regulation showed that:(1)Algal cells in nutrient-rich culture grew well,and the cell density reached 13.7×l07 cells/mL.Compared with the control group,the N-deficiency group grew slower and entered stationary phase earlier,and the division cycle was blocked at S phase,the cell density was 5.4×107 cells/mL,at the stationary phase.Compared with the control group,algal cells in the P-deficiency group also grew slower and entered stationary phase earlier,and the cell division was blocked at G1 phase,the cell density in P-deficient culture was 4.1×107 cells/mL.(2)Photosynthesis of algal cells cultured in N-or P-deficient media were significantly inhibited.Compared with the value of control,the rETR and Fv/Fm of algal cells cultured in N-deficient medium,decreased by 33%and 11%,respectively.The values of rETR and Fv/Fm of algal cells cultured in P-deficient medium decreased by 30%and 16%,respectively,compared with the value of control.After 24 days,the carbohydrate content of the algal cells cultured in N-and P-starved treatments was 4 and 2.4 times that of control,respectively.(3)The Nile Red fluorescence intensities of algal cells in N and P deficiency groups were 8.7 and 1.7 times higher than that of the control,indicating N and P starvation induced lipid accumulation in M aeruginosa.Further analysis of fatty acid composition showed that the proportion of C16 saturated fatty acids in control,N deficiency,and P deficiency group were 62%,61%,and 57%,and C18 unsaturated fatty acids were 30%,28%,and 28%,respectively.(4)Consistent with the lipid accumulation,the expression of key enzyme genes involved in the lipid synthesis pathway,showed significant upregulation in algal cells grown under N and P deficiency.Among them,the expression of accB and accD genes were up-regulated by 3.8-and 3.3-fold in N deficiency group,and were up-regulated by 1.2-and 0.6-fold in P-deficient group.The expression of those genes involved in TCA cycle,glycolysis,and lipid metabolism pathways were up-regulated in N and P deficient group,while pyruvate kinase gene in N-deficient culture was down-regulated by 1.6 times.These results showed that the expression of these genes involved in various metabolic pathways,including lipid synthesis and carbon metabolism,were up-regulated in N and P deficiency cultures,which led to an increase in glycerophosphate and fatty acid synthesis precursors,and finally caused lipid accumulation.The results of the growth inhibition mechanism of algal cells cultured in N-or P-deficient media displayed as follows:(1)In response to N-deficiency,the cell cycle was blocked,which led cellular DNA content to increase,to 1.5 times of that of the control.The N-starved cells had lower content of protein than the control,which decreased by 66%after 24 days of culture.The chlorophyll a and carotenoid contents were significantly decreased to 29%and 53%of those of the control,respectively.The ROS level in algal cells cultured in N-deficient group was decreased significantly to 2%of algal cells after 19 days.While the SOD,GSH activities in N-starved cells were increased by 70%and 30%compared with those of control.In response to P-deficiency,the cellular DNA content was decreased by 20%compared with that of control.Cellular protein content in the P-deficient group was only 64%of that of the control group,as well as chlorophyll a and carotenoids significantly decreased,which were 32%and 56%those of the control.The ROS level in algal cells in P-deficient group was increased significantly to 80%of algal cells.Compared with control,the activities of SOD,POD,and CAT in algal cells cultured in P-deficient group were increased by 0.6,8,and 6 times,but the activities of GSH was decreased to 66%that of control.(2)Transcriptome data indicated that expression of antioxidant enzyme of algal cells grown under N-deficient conditions was significantly up-regulated compared with that of control.The expression of 8 enzyme genes,including antioxidant enzymes,cyclic proteases,and anti-toxin proteases,were significantly up-regulated in algal cells grown under P-deficient condition.The toxin-antitoxin family is the largest family of the defense system in the genome of Microcystis aeruginosa.In the N-deficient culture,7 toxin mazF genes for regulating growth inhibition,showed significant upregulation,and 7 tox:in higB genes were significantly up-regulated in P-deficient treatment group.In conclusions,the cell division was prevented and protein synthesis was significantly inhibited in N starvation.The contents of chlorophyll a and carotenoid per cell were decreased obviously in N-and P-starved cells.In P starvation,DNA replication was blocked,and protein synthesis was inhibited.The ROS level showed significant increases,and the activities of antioxidant system of algal cells were increased in P deficiency group.These physiological changes led to the cell growth inhibition of N-deficient and P-deficient groups.MazF and HigB probably played key roles in the growth inhibition of algal cells grown under N-deficient and P-deficient conditions,respectively.