| Astaxanthin is a safe and efficient colorant and antioxidant,which has great potential in aquaculture,cosmetics,nutraceutical and medicine.Haematococcus pluvialis,a unicellular Chlorophyceae,is widely known as the principal source of natural astaxanthin,is mass-cultivated in industrial-scale production.Continuous improvement of enhancing biomass and astaxanthin content of H.pluvialis is the key to develop astaxanthin production,and the researches on these two aspects have both theoretical and practical value.It has been confirmed that exogenous addition of sodium acetate significantly enhanced biomass accumulation and astaxanthin content of H.pluvialis,however,there is little information about physiological changes and the effects on the photoinhibition level and photoprotection capacity,by adding exogenous sodium acetate at the non-motile stage.Moreover,photorespiration has been proved to playing an important role in astaxanthin accumulation process,however,the metabolic responses allowing photorespiration to affect astaxanthin accumulation still need to be clarified.Furthermore,based on industrial-scale production,inoculation proportion also has an important impact on cell growth and subsequent astaxanthin accumulation.The alga H.pluvialis?strain H6?was used in this study,combined with two-stage culturing strategy.This study was focused on physiological characteristics and metabolic changes of the second stage,which is the astaxanthin accumulation stage.Firstly,the photosynthesis-irradiance?P-I?curve was used,to analyze the photoacclimation response of algal cells in the tubular photobioreactors,under the outdoor,changeable and high light conditions.Secondly,the effects of exogenous sodium acetate on the physiological changes during astaxanthin accumulation process,and on the photoprotection capacity at the non-motile stage were investigated.Finally,metabolomics analysis in absence or presence of sodium acetate during astaxanthin accumulation period were performed,using gas chromatography-mass spectrometry?GC-MS?,and the composition of astaxanthin geometrical isomers and the total lipid content were determined,in order to further explore the mechanism of exogenous sodium acetate affecting the biosynthesis and metabolism in H.pluvialis.The above researches would provide basic data and theoretical support for the promotion of adding sodium acetate at the astaxanthin accumulation stage in industrial-scale production.Moreover,to further investigate the interrelation between photorespiration and astaxanthin accumulation,metabolomics analysis of H.pluvialis during astaxanthin accumulationperiodwereperformed,inabsenceorpresenceof carboxymethoxylamine?CM?,an inhibitor of the photorespiratory pathway.Furthermore,it is necessary to study the effects of different inoculation proportion on cell growth during the green motile stage,and astaxanthin accumulation during the subsequent red non-motile stage,combined with changes of biomass and physiological level.The main results were as follows:1.Based on morphological and pigment changes,the process can be divided into the cell transformation stage?from green,motile cells to green,astaxanthin-deficient,non-motile cells?and the astaxanthin accumulation stage?forming red,astaxanthin-enriched,non-motile cells?.Dry weight and the diameter of the cellular protoplasm increased significantly in the former stage,while astaxanthin content of algal per cell significantly increased and the multilayered cell wall was formed in the latter stage.The net photosynthetic rates,maximum net photosynthetic rate,and saturation irradiance during the cell transformation stage were higher than those during the astaxanthin accumulation stage,indicating the former stage possessed better photosynthetic activities.We speculate that photosynthetic strategy of different stages were as follows:?i?during the cell transformation stage,the flux of photosynthetic substance and energy mainly flowed into biomass?especially the rounded and enlarged protoplast?;?ii?during the subsequent astaxanthin accumulation stage,photosynthesis performance reduced,and the substrates and energy required for astaxanthin accumulation and cell wall thickening were mainly derived from the transformation of other substances,suggesting adding exogenous carbon source would enhance astaxanthin accumulation at this stage.This study would provide physiological background for optimizing astaxanthin production using H.pluvialis in industry.2.The astaxanthin contents in H.pluvialis cells with 10 mM sodium acetate increased more than 1.26-fold as compared with that in cells without sodium acetate after 6 days of incubation,indicating that exogenous sodium acetate accelerated astaxanthin accumulation at the non-motile stage significantly.Addition of sodium acetate inhibited photosynthetic rates,indicating that exogenous sodium acetate suppressed photosynthetic activity at the non-motile stage.However,additional sodium acetate increased respiratory rates.It can be speculated that the enhanced respiration plays an important role in the acceleration of astaxanthin accumulation in the presence of sodium acetate.Moreover,the level of photoinhibition in H.pluvialis decreased after adding sodium acetate at non-motile stage,which is indicated by the fact that the decreased value of Fv/Fm determined at midday,compared with that determined at predawn,declined on day 4 and day 6.NPQ increased significantly with additional sodium acetate on day 4 and day 6,indicating that additional sodium acetate induced a mechanism to protect H.pluvialis cells against photoinhibition.Taken together,exogenous sodium acetate enhances astaxanthin accumulation and the photoprotection capacity of H.pluvialis at the non-motile stage.3.Adding exogenous sodium acetate?Ac?at the astaxanthin accumulation stage significantly enhanced the astaxanthin content,including both free astaxanthin monomer and astaxanthin ester,and had a certain influence on its geometrical isomers.A total of 78 metabolites were identified by GC-MS,including 27 amino acids,16organic acids,12 fatty acids,7 polyols,3 phosphoric acids,3 sugars,2 amines,and 8other compounds.After adding Ac,pyruvic acid significantly accumulated,as well as citric acid,succinic acid,fumaric acid and malic acid which involved in the tricarboxylic acid cycle?TCA cycle?were increased,moreover,the contents of oxalic acid and glyceric acid which involved in the glyoxylae cycle were higher than those in CK.At the same time,fructose and glucose content decreased,sucrose increased significantly.In addition,the detected free fatty acids in Ac was significantly lower than those in CK at the red phase,while the total lipid content and astaxanthin esters content in Ac were significantly higher than those in CK at both brown and red phases,and the content of malonic acid,the precursor of fatty acid biosynthesis,was also significantly higher than that in CK.The above results indicated that exogenous Ac promoted the biosynthesis of fatty acids and further be used to synthesize lipid or astaxanthin ester.It is speculated that acetate can be utilized by TCA cycle and glyoxylae cycle to generate the carbon skeletons and NAD?P?H for astaxanthin synthesis directly;it can also directly participate in fatty acid biosynthesis in the form of acetyl coenzyme A to increase total lipid content,and the synthesized fatty acids combined with free astaxanthin monoesters to form astaxanthin ester,to further enhance astaxanthin content indirectly.4.The inhibition of photorespiration by CM did not affect the biomass,had a slight effect on chlorophyll at the end of the incubation,but significantly suppressed astaxanthin accumulation.Algal samples were collected on days 0,6 and 12,which were named as the green phase with green,non-motile cells,the brown phase with astaxanthin-synthesizing,non-motilecells,andtheredphasewith astaxanthin-enriched,red,non-motile cells,respectively.Multivariate statistical analyses?principal component analysis,partial least squares-discriminant analysis?,and hierarchical cluster analysis revealed the clustering of the metabolites.Glycine and glycolic acid had accumulated substantially at both the brown and red phases,indicating that photorespiration was inhibited by CM.In presence of CM,the TCA cycle was restricted at the brown phase due to decreased intermediates,specifically,decreased levels of fructose and glucose.However,inhibiting photorespiration enhanced the levels of many intracellular cytoprotective metabolites,such as amino acids,polyamines,polyols and sucrose.A hypothetical metabolic regulation model of the photorespiratory pathway affecting astaxanthin accumulation of H.pluvialis is proposed.This study provides the first metabolomic evidence that photorespiration enhances astaxanthin accumulation.5.The inoculation proportion had a great influence on both cell growth during the green motile stage and astaxanthin accumulation during the subsequent red non-motile stage.The biomass and cell density were higher when inoculation proportion was higher,as well as astaxanthin content.However,the specific growth rate and the of ratio of astaxanthin/chlorophyll were higher when inoculation proportion was lower,and the color of algal cultures in lower inoculation proportion was redder.At the green stage,the effect of inoculation proportion on photosynthetic electron transfer chain?OJIP curve?decreased over time,while at the red stage,initial inoculation proportion had significant effects on photosynthetic electron transfer chain?OJIP curve?.The photosynthetic performance during the green stage was significantly higher than that during the red stage,however,when the inoculation proportion was 2:1,the algal cells still had high photosynthetic activity than other groups of lower inoculation proportion during the red stage.Therefore,in the large-scale commercial cultivation of H.pluvialis,it is necessary to select the appropriate inoculation proportion,in order to achieve better production efficiency of astaxanthin. |
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