Stress Of Haematococcus Pluvialis And Raman Spectroscopy Study

Haematococcus pluvialis is a unicellular freshwater algae that accumulates astaxanthin in cells under extreme stress conditions(bright light,nitrogen deficiency,high salt concentration,etc.).Astaxanthin is one of the most known substances with the strongest antioxidant properties.It has a wide commercial value in the food,medicine and health care fields.Moreover,astaxanthin itself has a strong Raman signal,which makes Haematococcus pluvialis a good model organism for Raman imaging.Raman spectroscopy is widely used in the field of biological analysis and imaging because it can provide the fingerprint of the sample to be tested and the advantages of no fear of water interference.In this paper,the culture conditions of Haematococcus pluvialis and the conditions of Raman spectroscopy were optimized.Based on this,the algae were cultured under salt stress and nitrogen stress,Raman imaging was performed and multivariate analysis techniques were used to characterize the distribution of intracellular pigments in different periods of salt stress and nitrogen stress.The details are as follows:1.Optimization of Culture Condition of Haematococcus pluvialis and Characterization of Raman Spectra at Different Stages of Algae.First of all,we optimized the culture conditions of algae.We studied the effects of different p H value,nitrate concentration and salt concentration on the growth of Haematococcus pluvialis,and prepared for the subsequent experiments.In addition,the examination conditions of Haematococcus pluvialis in inverted confocal Raman spectroscopy were optimized,and the influences of the excitation wavelength,laser intensity,and integration time on the measurement results were optimized.In order to reduce the influence of glass slides on Raman spectra,quartz groove slides were designed and used to characterize Raman spectra of algae at different stages.2.Effect of Salt Stress on the Distribution of Pigments in Haematococcus pluvialis3.Stress concentrations of Haematococcus pluvialis were cultured with different concentrations of saline solution,and Raman spectra and intracellular pigment distributions during the stress of Haematococcus pluvialis were studied.Based on the conditions optimized in Chapter 2,Raman spectra of algae at various times of salt stress were obtained using inverted confocal Raman spectroscopy,and multivariate analysis techniques were used to separate astaxanthin,chlorophyll and ?-carotene from the total spectrum,and imaging techniques are used to visualize the distribution of different pigments in the cells.The results showed that the content of astaxanthin in the salt stress process was significantly higher than that in the control group,while the content of ?-carotene was slightly decreased,and the content of chlorophyll was also reduced to some extent.4.Effects of Nitrogen Stress on the Distribution of Pigments in Haematococcus pluvialis Using different concentrations of nitrogen for stress culture of Haematococcus pluvialis,the distribution of pigments in Haematococcus pluvialis under nitrogen deficiency conditions was studied.The Raman spectra of algae cells under nitrogen stress at different times were collected by an inverted confocal Raman spectrometer,and the common pigments were separated from the acquired total spectrum using a multi-component analysis technique.Finally,the distribution of various pigments within the cells are presented in images.From the change of pigment content,the results are basically similar to salt stress.To some extent,this result also proves that there is a certain similarity in the mechanism between salt stress and nitrogen stress.