| Due to the limited reserves of fossil fuels and the environmental damage,biodiesel,as a renewable?environmentally friendly energy source,has gradually become a research focus.Microalgae has been a potential source of biodiesel due to its high oil production and land utilization.Because human and production activities produce numerous wastewater with a certain salinity,and salinity had been reported to promote lipid accumulation.Therefore cultivating microalgae with saline water can provide an integrated strategy to generate bioenergy and save limited water resources.Microalgae Botryococcus sp.NJD-1 can grow under certain salinity condition and has the potential for biodiesel sources under previous study.Therefore,this study investigated the growth characteristics?lipid accumulation and reactive oxygen species of the oleaginous microalga Botryococcus sp.NJD-1 under different saiinities.Metabolomics and proteomics were used to explore the mechanism of salt stress inducing reactive oxygen species production and promoting lipid accumulation.The main results were as follows:First,this study explored the effects of growth?lipid accumulation and reactive oxygen species levels under salt stress.The results showed that as the salt concentration increased,cell growth was inhibited,cell density decreased but single cel!mass increased.At the NaCl concentration of 10 g L-1,the maximum lipid content and lipid productivity can be obtained,which were 54.5%and 110.5 mg L-1d-1,respectively.As the salt concentration increased,the neutral lipid content increased,while the glycolipids and phospholipids decreased.Through fatty acid analysis,the main fatty acids were palmitic acid and oleic acid.As the salt concentration increased,the content of palmitic acid and the stearic acid increased but linolenic acid and docosadienoic acid decreased significantly.The level of reactive oxygen species and the number of free radical ions increased as the salt concentration increased.Meanwhile,the levels of intracellular enzyme antioxidants and non-enzyme antioxidants increased,indicating that the cells stimulate defense system in response to salt stress.Secondly,gas chromatography-mass spectrometry(GC-MS)was used to investigate the effects of intermediate metabolites under salt stress.GC-MS-based metabolomics analysis showed that citrate was significantly increased in the TCA cycle,while the levels of other metabolites were decreased,indicating that more citrate was used to synthesize acetyl-CoA and enterd the fatty acid synthesis pathway.In the malate/pyruvate cycle,the malic enzyme activity was consistent with the trend of lipid content,indicating that more NADPH provided more reducing power for lipid synthesis.Finally,laber-free proteomics was used to assess the effect of salt stress on intracellular protease activity.Results showed that under salt stress conditions,the potential inside and outside the cell is balanced by consuming ATP and related ion transporters.Mitochondria contributed more to ATP supply than chloroplasts.For the NADPH supply,the malate/pyruvate pathway contributed more than the pentose phosphate pathway while the levels of long-chain acetyl-CoA synthetase and glycerol3-phosphate rise,more triacylglycerols synthesized.Finally,we proposed a hypothesis about the relationship between salt stress,active oxygen and lipid accumulation:when Na+ions pass through the transporter to the cell,a higher potential difference generated,which was transferred to the organelle through the electron transport chain,thereby generating reactive oxygen species(ROS)around the organelle membrane.The toxic effect of ROS was mainly through the reduction of free radical ions,which required numerous supply of ATP and reducing agents to reduce ROS toxicity.So the TCA cycle and the malate/pyruvate cycle were stimulated to produce more ATP and NADPH,while numerous NADPH and acetyl-CoA would enter the fatty acid synthesis pathway and promoted more lipid accumulation.Long chain acetyl-CoA synthetase and diacylglycerol acyltransferase provided the necessary precursor and acyl capabilities for triacylglycerol synthesis.Therefore,this study provides new insights into the effects of salt stress on lipid synthesis,thus providing theoretical basis for the development of advanced biofuel technologies. |
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