Regulatory Mechanism Of Lipid Biosynthesis And Intracellular Signal Transduction In Chlorella Pyrenoidosa By Salt Osmotic Stress

In recent years,with the increasingly frequent and severe natural disasters caused by global warming,“carbon peak”and“carbon neutrality”have become the common goal of all mankind,and the emerging and sustainable bio-energy has received extensive attention.Microalgae is considered as the third generation of renewable energy that can produce biodiesel from CO₂ by photosynthesis,which has these advantages of fast propagation,high lipid production and low growth environment requirements（it can be cultivated with wastewater at low cost）.Salt-induced osmotic stress is an effective way to increase the lipid content in algae cells.In order to explore the regulation mechanism of lipid biosynthesis in microalgae under salt osmotic stress,Chlorella Pyrenoidosa（C.Pyrenoidosa）was chosen as an object in this work,and the interactive relationships among salt osmotic stress and cyclic adenosine phosphate（cAMP）signaling pathway and key genes of lipid synthesis were investigated.Furthermore,the effects of plant hormones such as abscisic acid on the synthesis of intracellular lipid in C.Pyrenoidosa under osmotic stress were studied,and the regulatory mechanism was revealed.In addition,the method of alleviating salt-induced osmotic stress to Chlorella Pyrenoidosa by ABA was applied to the treatment of high-salinity mustard tuber wastewater（MTWW）.In this work,effect of salt-induced osmotic stress on C.Pyrenoidosa growth and lipid synthesis characteristics were studied.The results show that salt-induced osmotic stress promoted the expression of key genes for lipid synthesis,lipid content and cAMP level in C.Pyrenoidosa.The content of the total lipids was up to 38.31%at 453 m Osm/kg,which was 63%higher than that of the control group（23.45%）.After the addition of cAMP inhibitor（atropine）under salt osmotic stress（453 m Osm/kg）,the cAMP signaling pathway was inhibited,and the intracellular lipid content（29.65%）and the transcription level of genes related to lipid synthesis were significantly decreased.These results indicates that cAMP signaling pathway was involved in the biosynthesis of lipid in microalgae cells under salt osmotic stress.In order to further improve the lipid yield of C.Pyrenoidosa under salt osmotic stress,the effects of plant hormones on the growth and lipid synthesis of C.Pyrenoidosa under salt-induced osmotic stress（453 m Osm/kg）were investigated by adding salicylic acid（SA）,jasmonic acid（JA）,and abscisic acid（ABA）.The results show that under the condition of salt-induced osmotic stress+250μmol/L ABA,the highest lipid content（42.30%）in C.Pyrenoidosa was obtained,which was 10.42%higher than that of the control group.At this condition,the levels of intracellular jasmonic acid（JA）,antioxidant and biomass were also significantly increased.In addition,when the intracellular jasmonic acid level was promoted or inhibited,the intracellular lipid content and total antioxidant level were increased or decreased.Furthermore,the transcriptome analysis shows that the expression levels of genes related to lipid synthesis（g10025,g8895,g6473,g2851）were increased and those related to lipid metabolism（g5675,g1137,g7621,g3439,g9586）were down-regulated under ABA+salt osmotic stress condition.Meanwhile,the expression levels of genes related to JA synthesis（g4994,g577,g6367,g3370）increased.In addition,these genes associated with the biosynthesis of chlorophyll,starch,sucrose,triacylglycerol and antioxidant were up-regulated.The result suggests that JA signaling pathway was involved in intracellular lipid biosynthesis of C.Pyrenoidosa under ABA+salt osmotic stress condition.Based on the mechanism of alleviating salt-induced osmotic stress by ABA,the MTWW was bio-treated using C.Pyrenoidosa.Firstly,the MTWW was pretreated by photo-Fenton oxidation,then,the pre-treated wastewater was further degraded by C.Pyrenoidosa with adding ABA（300μmol/L）.It is found that the removal rates of COD,TN,TP,NH₄⁺-N and salinity reached 99.31%,96.23%,98.29%,98.11%and 78.82%,respectively.The effluent met the Level 3 of Comprehensive Sewage Discharge Standard（GB18466-2005）,and the biomass productivity and lipid productivity of C.Pyrenoidosa cultured with mustard wastewater reached 152.65 mg/L/d and 56.59 mg/L/d,respectively.The oxidation stability and low temperature fluidity of the extracted lipids were also improved.The results shows that the high-salinity mustard tuber wastewater could be effectively treated by C.Pyrenoidosa under ABA added condition,and the growth and lipid biosynthesis of C.Pyrenoidosa were promoted.In conclusion,cAMP signaling mediated lipid synthesis of C.Pyrenoidosa under salt osmotic stress was revealed for the first time.And the regulatory mechanism of JA signaling pathway mediated the growth and lipid biosynthesis of C.Pyrenoidosa under ABA+salt osmotic stress was clarified.This regulatory mechanism has been applied in the treatment of high-salt mustard tuber wastewater by C.Pyrenoidosa.