Nitrogen Starvation Induced Oxidative Stress in an Oil-Producing Green Alga Chlorella sorokiniana C3

Microalgal lipid is one of the most promising feedstocks for biodiesel production. Chlorella appears to be a particularly good option, and nitrogen (N) starvation is an efficient environmental pressure used to increase lipid accumulation in Chlorella cells. The effects of N starvation of an oil-producing wild microalga, Chlorella sorokiniana C3, on lipid accumulation were investigated using thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM) and flow cytometry (FCM). The results showed that N starvation resulted in lipid accumulation in C. sorokiniana C3 cells, oil droplet (OD) formation and significant lipid accumulation in cells were detected after 2 d and 8 d of N starvation, respectively. During OD formation, reduced photosynthetic rate, respiration rate and photochemistry efficiency accompanied by increased damage to PSII were observed, demonstrated by chlorophyll (Chl) fluorescence, 77K fluorescence and oxygen evolution tests. In the mean time the rate of cyclic electron transportation increased correspondingly to produce more ATP for triacylglycerols (TAGs) synthesis. And 0.5 d was found to be the turning point for the early stress response and acclimation of cells to N starvation. Increased level of membrane peroxidation was also observed during OD formation, and superoxide dismutase (SOD), peroxide dismutase (POD) and catalase (CAT) enzyme activity assays suggested impaired reactive oxygen species (ROS) scavenging ability. Significant neutral lipid accumulation was also observed by artificial oxidative stress induced by H2O2 treatment. These results suggested coupled neutral lipid accumulation and oxidative stress during N starvation in C. sorokiniana C3.

[1]  A. Ahmad,et al.  Microalgae as a sustainable energy source for biodiesel production: A review , 2011 .

[2]  N. Stanietsky,et al.  The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity , 2009, Proceedings of the National Academy of Sciences.

[3]  A. Meister,et al.  Cytometry of freshwater phytoplankton. , 2004, Methods in cell biology.

[4]  C. Somerville,et al.  Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl coenzyme A reductase , 1997, Journal of bacteriology.

[5]  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.

[6]  D. Kell,et al.  Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. , 1996, Microbiological reviews.

[7]  K. Paek,et al.  Differential responses of anti-oxidants enzymes, lipoxygenase activity, ascorbate content and the production of saponins in tissue cultured root of mountain Panax ginseng C.A. Mayer and Panax quinquefolium L. in bioreactor subjected to methyl jasmonate stress , 2005 .

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

[9]  A. Murakami.  Quantitative analysis of 77K fluorescence emission spectra in Synechocystis sp. PCC 6714 and Chlamydomonas reinhardtii with variable PS I/PS II stoichiometries , 1997, Photosynthesis Research.

[10]  Y. Chisti.  Biodiesel from microalgae. , 2007, Biotechnology advances.

[11]  Arief Widjaja,et al.  Study of increasing lipid production from fresh water microalgae Chlorella vulgaris , 2009 .

[12]  K. Yamaberi,et al.  Limited feeding of potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp. UTEX LB1999 , 2000, Applied Microbiology and Biotechnology.

[13]  Qingfang He,et al.  The High Light-Inducible Polypeptides Stabilize Trimeric Photosystem I Complex under High Light Conditions in Synechocystis PCC 68031[C][W][OA] , 2008, Plant Physiology.

[14]  R. Ruan,et al.  Mixotrophic cultivation of Chlorella vulgaris and its potential application for the oil accumulation from non-sugar materials , 2011 .

[15]  A. Converti,et al.  EFFECT OF TEMPERATURE AND NITROGEN CONCENTRATION ON THE GROWTH AND LIPID CONTENT OF NANNOCHLOROPSIS OCULATA AND CHLORELLA VULGARIS FOR BIODIESEL PRODUCTION , 2009 .

[16]  H. Lichtenthaler.  CHLOROPHYLL AND CAROTENOIDS: PIGMENTS OF PHOTOSYNTHETIC BIOMEMBRANES , 1987 .

[17]  T. G. Truscott,et al.  The carotenoids as anti-oxidants--a review. , 1997, Journal of photochemistry and photobiology. B, Biology.

[18]  Julian N. Rosenberg,et al.  A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution. , 2008, Current opinion in biotechnology.

[19]  P. Pribyl,et al.  Flow cytometry for the development of biotechnological processes with microalgae. , 2013, Biotechnology advances.

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

[21]  Jian-Guo Jiang,et al.  Toxic effects of chemical pesticides (trichlorfon and dimehypo) on Dunaliella salina. , 2011, Chemosphere.

[22]  Wen-Teng Wu,et al.  Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. , 2009, Bioresource technology.

[23]  C. Brussaard,et al.  AUTOLYSIS KINETICS OF THE MARINE DIATOM DITYLUM BRIGHTWELLII (BACILLARIOPHYCEAE) UNDER NITROGEN AND PHOSPHORUS LIMITATION AND STARVATION 1 , 1997 .

[24]  Ana Cristina Oliveira,et al.  Microalgae as a raw material for biofuels production , 2009, Journal of Industrial Microbiology & Biotechnology.

[25]  A Anandraj,et al.  PAM fluorometry as a tool to assess microalgal nutrient stress and monitor cellular neutral lipids. , 2011, Bioresource technology.

[26]  Xudong Xu,et al.  Depletion of Vipp1 in Synechocystis sp. PCC 6803 affects photosynthetic activity before the loss of thylakoid membranes. , 2009, FEMS microbiology letters.

[27]  Georgi Petkov,et al.  Which are fatty acids of the green alga Chlorella , 2007 .

[28]  David M. Kramer,et al.  New Fluorescence Parameters for the Determination of QA Redox State and Excitation Energy Fluxes , 2004, Photosynthesis Research.

[29]  Jo‐Shu Chang,et al.  Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. , 2011, Bioresource technology.

[30]  J. Briantais,et al.  The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence , 1989 .

[31]  Yongding Liu,et al.  Effects of an Algicidal Bacterium Pseudomonas mendocina on the Growth and Antioxidant System of Aphanizomenon flos-aquae , 2009, Current Microbiology.

[32]  D. Vaulot,et al.  Cell cycle distributions of prochlorophytes in the north western Mediterranean Sea , 1992 .

[33]  Julian N. Rosenberg,et al.  Physiological evaluation of a new Chlorella sorokiniana isolate for its biomass production and lipid accumulation in photoautotrophic and heterotrophic cultures , 2012, Biotechnology and bioengineering.

[34]  Nick D. Tsihlis,et al.  Isopropylamine NONOate (IPA/NO) moderates neointimal hyperplasia following vascular injury. , 2010, Journal of vascular surgery.

[35]  J. Morgan,et al.  Heterotrophic growth and lipid production of Chlorella protothecoides on glycerol , 2011, Bioprocess and biosystems engineering.

[36]  Yajun Li,et al.  Novel molecular insights into nitrogen starvation-induced triacylglycerols accumulation revealed by differential gene expression analysis in green algae Micractinium pusillum , 2012 .

[37]  Ayhan Demirbas,et al.  Biodiesel from oilgae, biofixation of carbon dioxide by microalgae: A solution to pollution problems , 2011 .

[38]  Helena M. Amaro,et al.  Advances and perspectives in using microalgae to produce biodiesel , 2011 .

[39]  D. W. Dhar,et al.  Biochemical modulation of growth, lipid quality and productivity in mixotrophic cultures of Chlorella sorokiniana , 2012, SpringerPlus.

[40]  H. Hirt,et al.  Reactive oxygen species: metabolism, oxidative stress, and signal transduction. , 2004, Annual review of plant biology.

[41]  Andrew D. Jones,et al.  Supporting Online Material for: Ethanol Can Contribute To Energy and Environmental Goals , 2006 .

[42]  E. Elstner.  Mechanisms of oxygen activation in different compartments of plant cells , 1991 .

[43]  C. Remacle,et al.  Isolation and partial characterization of mutants with elevated lipid content in Chlorella sorokiniana and Scenedesmus obliquus. , 2012, Journal of biotechnology.

[44]  Elisa Berdalet,et al.  Effect of nitrogen or phosphorus starvation on pigment composition of cultured Heterocapsa sp , 1994 .

[45]  Yasuyuki Yamada,et al.  Directed disruption of the tobacco ndhB gene impairs cyclic electron flow around photosystem I. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[46]  M. S. Cooper,et al.  Visualizing "green oil" in live algal cells. , 2010, Journal of bioscience and bioengineering.

[47]  B. Demmig‐Adams,et al.  Chlorophyll and Carotenoid Composition in Leaves of Euonymus kiautschovicus Acclimated to Different Degrees of Light Stress in the Field , 1996 .

[48]  Teresa M. Mata,et al.  Microalgae for biodiesel production and other applications: A review , 2010 .