In-situ Detecting Of Stress Indexes And Lipids In Microalgae

With the continuous growth of population and the increasing demand for energy and high-quality food,microalgae have attracted more and more attention because of their high lipid ac-cumulation and rich metabolic products.Different from common crops,most of the important metabolites such as lipids,fatty acids and carotenoids are accumulated by microalgae under stress.Inorganic salt stress is one of the most important methods to obtain these useful organic compounds.The culture environment optimation and high-yielding algae strains cultivation are key ways to prompt the production of microalgal products.A deep understanding of the metabolic mechanism of microalgae under inorganic salt stress,and the monitoring of stress indicators and key meat-bolites dynamics in culture process will help us to dynamically regulate the growth environment of microalgae,ensure the efficient production of microalgae,and accelerate the development and application of high-yield microalgae strains.In order to realize the efficient accumulation of mi-croalgal lipids and promote the comprehensive development and utilization of microalgal lipids,this study investigated the metabolic mechanism and detection methods of main components of microalgae under inorganic salt stress combined with isotope tracer,electrochemical analysis tech-nology,Hyperspectral imaging technology and Raman spectroscopy.The synthesis pathway of main fatty acids of microalgae under nitrogen inorganic salt stress was analyzed with the help of isotope probes.The main organic component changes of microalgae under inorganic salt stress were explored,then a novel metabolite tracing method was proposed through Raman spectroscopy combined with isotopes.Electrochemical analysis was used to detect the extracellular hydrogen peroxide content of microalgae at macro and micro scales.Then,the main component changes in microalgae was further analysised through Raman spectroscopy and feature extraction method.Moreover,an in situ and non-destructive detection mehtod of total lipid concentration of microal-gae was realized through hyperspectral imaging technique,and a visual analysis method for lipid accumulation and distribution monotoring of microalgae was proposed.The variation of fatty acid content in microalgae under nitrogen inorganic salt deficiency stress with stress time and stress degree was further analyzed.Combined with visible/near-infrared hyperspectral imaging,the pre-diction of fatty acid content and the visualization of main fatty acid distribution were realized.The main conclusions if this study are as follows:（1）The¹³C isotope was used to analyze the synthesis pathways of fatty acids in T.obliquus cells under nitrogen deficiency stress.Then,a visual tracer method for main components in mi-croalgae under nitrogen deficiency stress was proposed combined with Raman spectroscopy.The metabolic flux and distribution of carbon atoms in microalgae under nitrogen inorganic salt defi-ciency were analyzed by high resolution mass spectrometry.Then,the potential synthesis pathway of fatty acids in microalgae under nitrogen inorganic salt deficiency was discussed.The results show the accumulation of palmitic acid in microalgae under nitrogen deficiency stress was mainly related to de novo synthesis pathway while linolenic acid was mainly accumulated by non-de novo synthesis pathway.The changes of main components in T.obliquus under nitrogen deficiency stress were studied by combining chemical analysis and Fourier transform infrared spectroscopy（FTIR）methods,and results show the content of total lipid,total starch was increased during the stress process.The hydrogen peroxide content increased after nitrogen deficiency stress,suggesting that oxidative stress was occured.Fourier infrared spectroscopy and Raman spectroscopy were used to analyze the characteristic peak shift of some carbon compounds,such as lipids and carotenoids,which showed that the¹³C isotope was synthesized to these metabolites.This study also proposed a carotenoids visualization method by using the red-shift phenomenon of Raman peak induced by isotopes.Compared with traditional Raman imaging which only relies on the intensity of charac-teristic peaks,this method is more robust and can also provide information about the distribution of these organic molecules in the cell.（2）Hydrogen peroxide,an important indicator of microalgae oxidative stress,was detected by electrochemical method,and real-time tracking of hydrogen peroxide released by cells at macro and single cell scale was realized.The standard curve of hydrogen peroxide concentration-current response established by using standard hydrogen peroxide has a good linearity,R²up to 0.9624.The effects of different levels of cadmium ion on the oxidative stress level of microalgae were an-alyzed by the proposed detection method,and it was found that results display a volcanic curve of the detected hydrogen peroxide concentration extracellularly released from E.gracilis against the increase Cd（II）concentration.This study also investigated the effects of different stress levels on cells by combining cell phenotype and Raman spectroscopy.A quantitative analysis model of stress concentration based on Raman spectrum was established by using the characteristic wavenum-ber selected by successive projection algorithm（SPA）with a determination coefficient of R²up to 0.8383.Then,the main component changes of microalgae under different stress levels were analyzed and visualized based on the characteristic wavenumbers selected by SPA.Finally,the ox-idative stress mechanism of microalgae under different concentrations of Cadmium ion stress was analyzed through proteomics.（3）An in-situ and visual detection method of total lipid content in microalgae was proposed based on hyperspectral imaging technology.Transmission hyperspectral imaging method was used to remove the influence of water molecules on near infrared spectrum.Then,a set of total lipid prediction models based on full spectrum,competitive adaptive reweighting algorithm（CARS）and continuous projection algorithm（SPA）were established through the mean spectra that extracted from regions of interest of samples.The results show Savitzky-Golay smoothed CARS-MLR model has the best prediction performance,and the correlation coefficient of prediction set can reach R_p=0.9419.Based on the optimal model,the total lipid distribution of microalgae was further visualized.This method is of great significance to the determination of the optimum harvest time of microalgae and the cultivation of high-yield algal strains.（4）The comprehensive development and utilization of microalgae lipids is the inevitable trend of microalgae resources development.The content and composition of fatty acids determine the application potential of microalgae lipids.In this study,the changes of fatty acid content in T.obliquus under nitrogen inorganic salt deficiency stress were analyzed by gas chromatography-mass spectrometry（GC-MS）and visible/NIR hyperspectral imaging.Then,the rapid and nonde-structive detection of fatty acid contents in microalgae was achieved.By comparing the changes of microalgal fatty acid content with the days of stress and different stress levels,it was found that different stress levels had a certain impact on the composition of microalgal fatty acid,and the accumulation direction of microalgal fatty acid could be effectively controlled by regulating the nitrogen deficiency stress level and harvest time.Under nitrogen free culture environment,the content of tetrachylinoleic acid（C18:1）was significantly accumulated at the end of stress culture period.However,mild nitrogen deficiency induced the microalgae to obtain more polyunsaturated fatty acids（C16:4）.Then,prediction models of fatty acid content in T.obliquus were established based on hyperspectral imaging technology.It was found that the visible/NIR hyperspectral pre-diction models of myristic acid（C14:0）,palmitic acid（C16:0）,oleic acid（C18:1）and linoleic acid（C18:2）all achieved very good prediction results with R²more than 0.9.The neural network model of myristic acid（C14:0）achieved the best performance,while the CARS-MLR model of palmitic acid（C16:0）,oleic acid（C18:1）and linoleic acid（C18:2）was the best.The models of remaining fatty acids,such as lauric acid（C12:0）,only gets a passably good result,possibly due to the fact that these fatty acids were less characteristic in the visible/NIR part of the spectrum.Moreover,the visualization of two major fatty acids,palmitic acid（C16:0）and oleic acid（C18:1）,showed a good performance compared with GC-MS result.This study can provide a theoretical basis and methodological support for the real-time monitoring of fatty acid productions of microalgae in the actual culture process,and provide technical support for real-time decision making in the culture process of microalgae.