Phospholipid Metabolism in an Industry Microalga Chlorella sorokiniana: The Impact of Inoculum Sizes

Chlorella sorokiniana is an important industry microalga potential for biofuel production. Inoculum size is one of the important factors in algal large-scale culture, and has great effects on the growth, lipid accumulation and metabolism of microalgae. As the first barrier of cell contents, membrane plays a vital role in algal inoculum-related metabolism. The knowledge of phospholipids, the main membrane component and high accumulation of phospholipids as the major content of total lipids mass in some microalgae, is necessary to understand the role of membrane in cell growth and metabolism under different inoculum density. Profiling of C. sorokiniana phospholipids with LC-MS led to the identification of 119 phospholipid species. To discover the phospholipid molecules most related to change of inoculum sizes, Partial Least Squares Discriminant Analysis (PLS-DA) was employed and the results revealed that inoculum sizes significantly affected phospholipid profiling. Phosphatidylglycerol (PG), phosphatidyl- ethanolamine (PE) and several phosphatidylcholine (PC) species might play an important role under our experimental conditions. Further analysis of these biomarkers indicated that cell membrane status of C. sorokiniana might play an important role in the adaption to the inoculum sizes. And the culture with inoculum size of 1×106 cells mL−1 presented the best membrane status with the highest content of PC and PG, and the lowest content of PE. We discovered that the inoculum size of 1×106 cells mL−1 might provide the best growth condition for C. sorokiniana. Also we proposed that PG, PE and several PC may play an important role in inoculum-related metabolism in C. sorokiniana, which may work through thylakoid membrane and photosynthetic pathway. Thus this study would provide more potential targets for metabolic engineering to improve biofuel production and productivity in microalgae.

[1]  Q. Hu,et al.  Occurrence and environmental stress responses of two plastid terminal oxidases in Haematococcus pluvialis (Chlorophyceae) , 2009, Planta.

[2]  Shi-jie Zhao,et al.  The unsaturation of phosphatidylglycerol in thylakoid membrane alleviates PSII photoinhibition under chilling stress , 2011, Plant Cell Reports.

[3]  Zechen Wu,et al.  Evaluation of flocculation induced by pH increase for harvesting microalgae and reuse of flocculated medium. , 2012, Bioresource technology.

[4]  M. Bakovic,et al.  Breast cancer cells adapt to metabolic stress by increasing ethanolamine phospholipid synthesis and CTP:ethanolaminephosphate cytidylyltransferase-Pcyt2 activity. , 2012, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[5]  J. Zimmerman,et al.  Biodiesel production: the potential of algal lipids extracted with supercritical carbon dioxide , 2011 .

[6]  V. Zachleder,et al.  Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations , 2011, Applied Microbiology and Biotechnology.

[7]  Jan Kern,et al.  Cyanobacterial photosystem II at 2.9-Å resolution and the role of quinones, lipids, channels and chloride , 2009, Nature Structural &Molecular Biology.

[8]  Ronan Sulpice,et al.  Genetic modification of the fatty acid unsaturation of phosphatidylglycerol in chloroplasts alters the sensitivity of tobacco plants to cold stress , 2004 .

[9]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[10]  M. Longo,et al.  Fluid-phase chain unsaturation controlling domain microstructure and phase in ternary lipid bilayers containing GalCer and cholesterol. , 2007, Biophysical journal.

[11]  C. Benning,et al.  Anionic lipids are required for chloroplast structure and function in Arabidopsis. , 2003, The Plant journal : for cell and molecular biology.

[12]  M. Ballottari,et al.  Evolution and functional properties of photosystem II light harvesting complexes in eukaryotes. , 2012, Biochimica et biophysica acta.

[13]  W. Su,et al.  Optimizing production of polyunsaturated fatty acids in Marchantia polymorpha cell suspension culture. , 2001, Journal of biotechnology.

[14]  J. Harwood,et al.  Algal lipids and effect of the environment on their biochemistry , 2009 .

[15]  Xianlin Han,et al.  Side Chain Oxygenated Cholesterol Regulates Cellular Cholesterol Homeostasis through Direct Sterol-Membrane Interactions* , 2009, Journal of Biological Chemistry.

[16]  G. Dönmez,et al.  Isolation of Dunaliella spp. from a hypersaline lake and their ability to accumulate glycerol. , 2008, Bioresource technology.

[17]  Robert Verpoorte,et al.  Renewable energy from Cyanobacteria: energy production optimization by metabolic pathway engineering , 2011, Applied Microbiology and Biotechnology.

[18]  Wei Zhang,et al.  Biodiesel production from algae oil high in free fatty acids by two-step catalytic conversion. , 2012, Bioresource technology.

[19]  Philip Owende,et al.  Biofuels from microalgae—A review of technologies for production, processing, and extractions of biofuels and co-products , 2010 .

[20]  L. Rodolfi,et al.  Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low‐cost photobioreactor , 2009, Biotechnology and bioengineering.

[21]  J. Hewson,et al.  Critical conditions for ferric chloride‐induced flocculation of freshwater algae , 2012, Biotechnology and bioengineering.

[22]  Christopher J. Howe,et al.  Influence of nitrogen-limitation regime on the production by Chlorella vulgaris of lipids for biodiesel feedstocks , 2010 .

[23]  B. Litman,et al.  Determination of membrane cholesterol partition coefficient using a lipid vesicle-cyclodextrin binary system: effect of phospholipid acyl chain unsaturation and headgroup composition. , 2002, Biophysical journal.

[24]  Jiangxin Wang,et al.  Metabolic profiling reveals growth related FAME productivity and quality of Chlorella sorokiniana with different inoculum sizes , 2012, Biotechnology and bioengineering.

[25]  J. V. Gerpen,et al.  Biodiesel processing and production , 2005 .

[26]  Yingjin Yuan,et al.  Lipidomic analysis reveals differential defense responses of Taxus cuspidata cells to two elicitors, methyl jasmonate and cerium (Ce4+). , 2008, Biochimica et biophysica acta.

[27]  Y. Liu,et al.  Lipidome profiling of Saccharomyces cerevisiae reveals pitching rate-dependent fermentative performance , 2010, Applied Microbiology and Biotechnology.

[28]  S. Tian,et al.  Peach fruit acquired tolerance to low temperature stress by accumulation of linolenic acid and N-acylphosphatidylethanolamine in plasma membrane , 2010 .

[29]  G. L. Klyachko-Gurvich,et al.  Changes in Lipid Metabolism during Adaptation of the Dunaliella salina Photosynthetic Apparatus to High CO2 Concentration , 2004, Russian Journal of Plant Physiology.

[30]  Yue Jiang,et al.  Fatty acid and lipid class composition of the eicosapentaenoic acid-producing microalga, Nitzschia laevis , 2007 .

[31]  Masahiko Morita,et al.  Photosynthetic productivity of conical helical tubular photobioreactor incorporating Chlorella sorokiniana under field conditions. , 2002, Biotechnology and bioengineering.

[32]  Perttu S. Niemelä,et al.  Influence of chain length and unsaturation on sphingomyelin bilayers. , 2006, Biophysical journal.

[33]  A. D. de Kroon.  Metabolism of phosphatidylcholine and its implications for lipid acyl chain composition in Saccharomyces cerevisiae. , 2007, Biochimica et biophysica acta.

[34]  C. Lan,et al.  Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans , 2008, Applied Microbiology and Biotechnology.

[35]  Fangxia Yang,et al.  Value-added uses for crude glycerol--a byproduct of biodiesel production , 2012, Biotechnology for Biofuels.

[36]  Robert E. Hebner,et al.  Progression of lipid profile and cell structure in a research-scale production pathway for algal biocrude. , 2013 .

[37]  Z. Wen,et al.  Production of Biodiesel Fuel from the Microalga Schizochytrium limacinum by Direct Transesterification of Algal Biomass , 2009 .

[38]  Nirbhay Kumar Singh,et al.  Microalgae as second generation biofuel. A review , 2011, Agronomy for Sustainable Development.

[39]  Yue Jiang,et al.  Variation of lipid class composition in Nitzschia laevis as a response to growth temperature change. , 2008, Food chemistry.

[40]  Steven A Arisz,et al.  The salt stress-induced LPA response in Chlamydomonas is produced via PLA2 hydrolysis of DGK-generated phosphatidic acid[S] , 2011, Journal of Lipid Research.

[41]  Qingyu Wu,et al.  Large‐scale biodiesel production from microalga Chlorella protothecoides through heterotrophic cultivation in bioreactors , 2007, Biotechnology and bioengineering.

[42]  Paul Chen,et al.  Effect of light intensity on algal biomass accumulation and biodiesel production for mixotrophic strains Chlorella kessleri and Chlorella protothecoide cultivated in highly concentrated municipal wastewater , 2012, Biotechnology and bioengineering.

[43]  A. Darzins,et al.  The promise and challenges of microalgal‐derived biofuels , 2009 .

[44]  N. Loberto,et al.  Glycosphingolipid behaviour in complex membranes. , 2009, Biochimica et biophysica acta.

[45]  Jérôme Garin,et al.  Chloroplast proteomics highlights the subcellular compartmentation of lipid metabolism. , 2010, Progress in lipid research.

[46]  S. Allakhverdiev,et al.  Genetic decrease in fatty acid unsaturation of phosphatidylglycerol increased photoinhibition of photosystem I at low temperature in tobacco leaves. , 2012, Biochimica et biophysica acta.

[47]  G. Salvador,et al.  Age‐related changes in central nervous system phosphatidylserine decarboxylase activity , 2002, Journal of neuroscience research.

[48]  E. Ünlü,et al.  Effects of Seasonal Variations on the Fatty Acid Composition of Total Lipid, Phospholipid and Triacylglicerol in the Dorsal Muscle of Mesopotamian Catfish (Silurus triostegus Heckel, 1843 ) in Tigris River (Turkey) , 2012 .

[49]  H. Wada,et al.  The Role of Phosphatidylglycerol in Photosynthesis , 2009 .

[50]  Zhiyou Wen,et al.  Development of an attached microalgal growth system for biofuel production , 2009, Applied Microbiology and Biotechnology.

[51]  C. S. Hsu,et al.  Algae Polar Lipids Characterized by Online Liquid Chromatography Coupled with Hybrid Linear Quadrupole Ion Trap/Fourier Transform Ion Cyclotron Resonance Mass Spectrometry , 2011 .

[52]  Yingjin Yuan,et al.  Comparative lipidomics analysis of cellular development and apoptosis in two Taxus cell lines. , 2007, Biochimica et biophysica acta.

[53]  R. Schneiter,et al.  Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae. , 2001, Molecular biology of the cell.

[54]  Jun Zhu,et al.  Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp. , 2010, Bioresource technology.

[55]  Yajun Li,et al.  Microalgae: A promising feedstock for biodiesel , 2009 .

[56]  T. Morinaga,et al.  Effect of high frequency of intermittent light on the growth and fatty acid profile of Isochrysis galbana , 2012 .

[57]  Yingjin Yuan,et al.  Integrated investigation of lipidome and related signaling pathways uncovers molecular mechanisms of tetramethylpyrazine and butylidenephthalide protecting endothelial cells under oxidative stress. , 2012, Molecular bioSystems.

[58]  S. Eberhard,et al.  A single mutation that causes phosphatidylglycerol deficiency impairs synthesis of photosystem II cores in Chlamydomonas reinhardtii. , 2004, European journal of biochemistry.

[59]  Lei Chen,et al.  Proteomic analysis reveals resistance mechanism against biofuel hexane in Synechocystis sp. PCC 6803 , 2012, Biotechnology for Biofuels.

[60]  S. Haas,et al.  Genome-wide comparison of cyanobacterial transposable elements, potential genetic diversity indicators. , 2011, Gene.

[61]  Z. L. Liu,et al.  Integrated phospholipidomics and transcriptomics analysis of Saccharomyces cerevisiae with enhanced tolerance to a mixture of acetic acid, furfural, and phenol. , 2012, Omics : a journal of integrative biology.

[62]  Lei Chen,et al.  Expression of fatty acid synthesis genes and fatty acid accumulation in haematococcus pluvialis under different stressors , 2012, Biotechnology for Biofuels.

[63]  Quantitative proteomic profiling reveals photosynthesis responsible for inoculum size dependent variation in Chlorella sorokiniana , 2013, Biotechnology and bioengineering.

[64]  T. Mock,et al.  Photosynthetic energy conversion under extreme conditions--II: the significance of lipids under light limited growth in Antarctic sea ice diatoms. , 2002, Phytochemistry.

[65]  Shangtian Yang,et al.  Lipidomic profiling and discovery of lipid biomarkers in snow alga Chlamydomonas nivalis under salt stress , 2012 .

[66]  S. Feng,et al.  Effects of lipid chain length on molecular interactions between paclitaxel and phospholipid within model biomembranes. , 2004, Journal of colloid and interface science.

[67]  H. Khandelia,et al.  Lipid gymnastics: evidence of complete acyl chain reversal in oxidized phospholipids from molecular simulations. , 2009, Biophysical journal.

[68]  H. Carswell,et al.  Differential effect of temperature on histamine‐ and carbachol‐stimulated inositol phospholipid breakdown in slices of guinea‐pig cerebral cortex , 1987, British journal of pharmacology.

[69]  S. Haas,et al.  Genome-wide comparison of cyanobacterial transposable elements, potential genetic diversity indicators , 2010 .

[70]  Yingjin Yuan,et al.  Cerium elicitor-induced phosphatidic acid triggers apoptotic signaling development in Taxus cuspidata cell suspension cultures. , 2009, Chemistry and physics of lipids.

[71]  Carlos Vílchez,et al.  Productivity of Chlorella sorokiniana in a short light‐path (SLP) panel photobioreactor under high irradiance , 2009, Biotechnology and bioengineering.

[72]  C. Howe,et al.  Biodiesel from algae: challenges and prospects. , 2010, Current opinion in biotechnology.

[73]  Yingjin Yuan,et al.  Integrative investigation of lipidome and signal pathways in human endothelial cells under oxidative stress. , 2011, Molecular bioSystems.

[74]  N. Sanina,et al.  The Effect of Seasonal Shifts in Temperature on the Lipid Composition of Marine Macrophytes , 2004, Russian Journal of Plant Physiology.

[75]  E. Aro,et al.  Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits. , 2010, Biochimica et biophysica acta.