Studies On Luxury Nitrogen Uptake And Dynamics Of Intracellular Nitrogen Pool In Graesiella Emersonii WBG-1

With the development of economy,the amount of wastewater discharge in China is increasing rapidly.The emission of wastewater rich in nitrogen and phosphorus causes a series of problems such as water eutrophication,which poses a threat to ecological environment,and in turn restricts the development of industrial and agricultural production.Microalgae has unique advantages over other microorganisms in wastewater denitrification,and its feasibility and potential have been confirmed in many studies.However,more knowledges about the dynamic process of nitrogen assimilation,accumulation and re-distribution in microalgae cells should be investigated before further application.Graesiella emersonii WBG-1 is a microalga with many outstanding characteristics,such as fast growth,wide p H adaptability,rich in TAGs,easy sedimentation,etc.It shows great research value and application potential in outdoor large-scale cultivation,development of high value-added products,sewage treatment and so on.In this study,growth and nitrate absorption of G.emersonii WBG-1 were analyzed under both batch and continuous culture modes.Responses of five kinds of intracellular nitrogenous compounds of G.emersonii WBG-1 to different extracellular nitrogen concentrations were investigated.Combined with differential proteomics analysis,the biochemical substances which functioned as nitrogen pool in G.emersonii WBG-1 and their dynamics in cells were determined.The main results are as follows:(1)In batch culture mode,G.emersonii WBG-1 grew fast and accumulated biomass rapidly.After nitrogen depletion,the biomass dry weight was still increasing,which indicated that there may be a nitrogen storage mechanism in the cells.In chemostat culture mode,biomass dry weight,Chl a and photosynthetic activity(F_V/F_M)increased with increasing nitrate concentration in the feeding medium.On the other hand,by cutting down 95%of the nitrate supply from 400 mg L^-1to 20 mg L^-1,steady growth-state of Graesiella emersonii WBG-1 could be reached and sustained at the same growth rate of 0.96 d^-1.This suggested that the over-assimilated nitrogen is not a necessity for basic life cycles of Graesiella.(2)Under the batch culture mode,G.emersonii WBG-1 showed typical characteristic of luxury nitrogen uptake.When extracellular nitrate was added to the culture medium,the nitrate could be depleted by G.emersonii WBG-1 in a short time.While under the chemostat culture mode,the input of nitrogen was controlled as needed.Absorption of extracellular nitrate by G.emersonii WBG-1 was limited by the rate of nitrogen supply.In a certain range,the rate of nitrogen uptake was equivalent to that of nitrogen supply,until nitrogen absorption reached saturation.(3)Among the determined intracellular nitrogenous compounds(proteins,free amino acids,pigments,nucleic acid,intracellular inorganic nitrogen),protein nitrogen accounted for the highest proportion of the biomass TN,which was about 90%.By increasing extracellular nitrogen concentration,the protein content changed greatly,from the lowest 22.75%DW to the highest 49.05%DW.It was reasonable to assume that some proteins played the role of"intracellular nitrogen pool"under environmental nitrogen fluctuation,and participated in the process of luxury absorption,storage and reutilization of nitrogen.When exogenous nitrogen was sufficient,algal cells absorbed a large amount of nitrogen,transformed them into proteins and stored in cells.When exogenous nitrogen was deficient,non-essential proteins were transformed into other essential functional proteins to maintain the growth of G.emersonii WBG-1.(4)Isobaric tags for relative and absolute quantification(i TRAQ)technique was used to analyze the proteomics of G.emersonii WBG-1 under high and low nitrogen steady states.A total of 811 proteins with molecular weight ranging from 10 kDa to120 kDa were identified.And 165 differentially expressed proteins(DEPs)between high nitrogen steady state(ST20)and low nitrogen steady state(ST3)were screened,of which 80 proteins were significantly up-regulated and 85 proteins were significantly down-regulated.Compared with low nitrogen steady state(ST3),photosynthesis related proteins and ribosome related proteins were significantly up-regulated in cells of high nitrogen steady state(ST20),while carbon metabolism and proteasome related proteins were significantly down-regulated.More than 54%of the DEPs were subcellularly located in chloroplast and cytoplasm,and the proportion of chloroplast protein was the highest,accounting for 1/3 of the total DEPs.In addition,among the differentially expressed 8 proteins that related to photosynthesis,the FC value of photosystem?Psb27 protein(A8I5A0)was as high as 2.17,followed by the photosystem?CP47 reaction center protein(A0A218N8S0)and Oxygen-evolving enhancer protein 1 of photosystem?(A8J0E4),the FC value of them were both higher than 1.5.It was concluded that G.emersonii WBG-1 adapted to the fluctuation of extracellular nitrogen through regulation of photosynthetic activity,and the protein complex related to photosystem not only played the role of carbon assimilation,but also functioned as a intracellular nitrogen pool.