Reducing self-shading effects in Botryococcus braunii cultures: effect of Mg²⁺ deficiency on optical and biochemical properties, photosynthesis and lipidomic profile

Abstract

Microalgae biomass exploitation as a carbon–neutral energy source is currently limited by several factors, productivity being one of the most relevant. Due to the high absorption properties of light-harvesting antenna, photosynthetic cells tend to capture an excessive amount of energy that cannot be entirely channeled through the electron transfer chain that ends up dissipated as heat and fluorescence, reducing the overall light use efficiency. Aiming to minimize this hurdle, in this work we studied the effect of decreasing concentrations of Magnesium (Mg²⁺) on the chlorophyll a content, photosynthetic performance, biomass and lipid production of autotrophic cultures of Botryococcus braunii LB 572. We also performed, for the first time, a comparative lipidomic analysis to identify the influence of limited Mg²⁺ supply on the lipid profile of this algae. The results indicated that a level of 0.0037 g L⁻¹ MgSO₄ caused a significant decline on chlorophyll a content with a concomitant 2.3-fold reduction in the biomass absorption coefficient. In addition, the Mg²⁺ limitation caused a decrease in the total carbohydrate content and triggered lipid accumulation, achieving levels of up to 53% DCW, whereas the biomass productivity remained similar for all tested conditions. The lipidome analysis revealed that the lowest Mg²⁺ concentrations also caused a differential lipid profile distribution, with an enrichment of neutral lipids and an increase of structural lipids. In that sense, we showed that Mg²⁺ limitation represents an alternative optimization approach that not only enhances accumulation of neutral lipids in B. braunii cells but also may potentially lead to a better areal biomass productivity due to the reduction in the cellular light absorption properties of the cells.

Keywords

Biofuels / Botryococcus braunii / Lipidomics / Mg²⁺ limitation / Photosynthesis / Self-shading effect

References

[1] Åkerman KEO. Inhibition and stimulation of respiration-linked Mg2+ efflux in rat heart mitochondria. J Bioenerg Biomembr, 1981, 13: 133-139.

[2] Alemán-Nava GS, Cuellar-Bermudez SP, Cuaresma M, . How to us Nile Red, a selective fluorescent stain for microalgal neutral lipids. J Microbiol Methods, 2016, 128: 74-79.

[3] Algranati ID. Inhibition of polypeptide synthesis initiation by a change of Mg++ concentration in wheat germ cell-free systems. Top Catal, 1980, 96: 54-60.

[4] Barros MP, Pedersén M, Colepicolo P, Snoeijs P. Self-shading protects phytoplankton communities against H2O2-induced oxidative damage. Aquat Microb Ecol, 2003

[5] Ben Amor-Ben Ayed H, Taidi B, Ayadi H, . Effect of magnesium ion concentration in autotrophic cultures of Chlorella vulgaris. Algal Res, 2015, 9: 291-296.

[6] Bromke MA, Hochmuth A, Tohge T, . Liquid chromatography high-resolution mass spectrometry for fatty acid profiling. Plant J, 2015, 81: 529-536.

[7] Burlacot A, Peltier G, Li-Beisson Y. Subcellular Energetics and Carbon Storage in Chlamydomonas. Cells, 2019, 8: 1154.

[8] Butler EI. A manual of chemical and biological methods for sea water analysis. Deep Sea Res Part A Oceanogr Res Pap, 1984

[9] Cagliari A, Margis R, Dos Santos MF, . Biosynthesis of triacylglycerols (TAGs) in plants and algae. Int J Plant Biol, 2011, 2: 40-52.

[10] Çakmak ZE, Ölmez TT, Çakmak T, . Induction of triacylglycerol production in Chlamydomonas reinhardtii: Comparative analysis of different element regimes. Bioresour Technol, 2014, 155: 379-387.

[11] Cataldo DA, Maroon M, Schrader LE, Youngs VL. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun Soil Sci Plant Anal, 1975, 6: 71-80.

[12] Cheng P, Wang J, Liu T. Effects of nitrogen source and nitrogen supply model on the growth and hydrocarbon accumulation of immobilized biofilm cultivation of B. braunii. Bioresour Technol, 2014

[13] Chen D, Yuan X, Liang L, Liu K, Ye H, Liu Z, Liu Y, Huang L, He W, Chen Y, Zhang Y, Xue T. Overexpression of acetyl-CoA carboxylase increases fatty acid production in the green alga Chlamydomonas reinhardtii. Biotechnol Lett, 2019, 41(10): 1133-1145.

[14] Choi GG, Kim BH, Ahn CY, Oh HM. Effect of nitrogen limitation on oleic acid biosynthesis in Botryococcus braunii. J Appl Phycol, 2011, 23: 1031-1037.

[15] Choi YY, Patel AK, Hong ME, . Microalgae Bioenergy with Carbon Capture and Storage (BECCS): an emerging sustainable bioprocess for reduced CO2 emission and biofuel production. Bioresour Technol Reports, 2019

[16] Chong J, Wishart DS, Xia J. Using MetaboAnalyst 4.0 for comprehensive and integrative metabolomics data analysis. Curr Protoc Bioinforma, 2019

[17] Cosgrove J, Borowitzka MA (2010) Chlorophyll fluorescence terminology: an introduction. In: Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications

[18] Deng X, Fei X, Li Y. The effects of nutritional restriction on neutral lipid accumulation in Chlamydomonas and Chlorella. African J Microbiol Res, 2011, 5: 260-270.

[19] Esakkimuthu S, Krishnamurthy V, Govindarajan R, Swaminathan K. Augmentation and starvation of calcium, magnesium, phosphate on lipid production of Scenedesmus obliquus. Biomass Bioenerg, 2016, 88: 126-134.

[20] Ferreira GF, Ríos Pinto LF, Carvalho PO, . Biomass and lipid characterization of microalgae genera Botryococcus, Chlorella, and Desmodesmus aiming high-value fatty acid production. Biomass Convers Biorefinery, 2019

[21] Figueroa FL, Bonomi-Barufi J, Celis-Plá PSM, . Short-term effects of increased CO₂, nitrate and temperature on photosynthetic activity in Ulva rigida (Chlorophyta) estimated by different pulse amplitude modulated fluorometers and oxygen evolution. J Exp Bot, 2021

[22] Figueroa FL, Domínguez-González B, Korbee N. Vulnerability and acclimation to increased UVB radiation in three intertidal macroalgae of different morpho-functional groups. Mar Environ Res, 2014

[23] Finkle BJ, Appleman D. The Effect of magnesium concentration on growth of chlorella. Plant Physiol, 1953, 28: 664-673.

[24] Finkle BJ, Appleman D. The effect of magnesium concentration on chlorophyll and catalase development in chlorella. Plant Physiol, 1953, 28: 652-663.

[25] Friedland N, Negi S, Vinogradova-Shah T, . Fine-tuning the photosynthetic light harvesting apparatus for improved photosynthetic efficiency and biomass yield. Sci Rep, 2019

[26] Fu W, Gudmundsson O, Feist AM, . Maximizing biomass productivity and cell density of Chlorella vulgaris by using light-emitting diode-based photobioreactor. J Biotechnol, 2012

[27] Gifuni I, Pollio A, Safi C, . Current bottlenecks and challenges of the microalgal biorefinery. Trends Biotechnol., 2019, 37: 242-252.

[28] Gollan PJ, Lima-Melo Y, Tiwari A, . Interaction between photosynthetic electron transport and chloroplast sinks triggers protection and signalling important for plant productivity. Philos Trans R Soc B Biol Sci, 2017

[29] Gorain PC, Bagchi SK, Mallick N. Effects of calcium, magnesium and sodium chloride in enhancing lipid accumulation in two green microalgae. Environ Technol, 2013, 34: 1887-1894.

[30] Griffiths MJ, Garcin C, van Hille RP, Harrison STL. Interference by pigment in the estimation of microalgal biomass concentration by optical density. J Microbiol Methods, 2011

[31] Grima EM, Camacho FG, Pérez JAS, . A mathematical model of microalgal growth in light-limited chemostat culture. J Chem Technol Biotechnol, 1994

[32] Grubbs RD, Maguire ME. Magnesium as a regulatory cation: criteria and evaluation. Magnesium, 1987, 6: 113-127.

[33] Guarnieri MT, Nag A, Smolinski SL, . Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga. PLoS ONE, 2011, 6: 25851.

[34] Gupta SS, Bhartiya S, Shastri Y. The practical implementation of microalgal biodiesel: challenges and potential solutions. CAB Rev., 2014, 9: 1-2.

[35] Hanifzadeh MM, Garcia EC, Viamajala S. Production of lipid and carbohydrate from microalgae without compromising biomass productivities: role of Ca and Mg. Renew Energy, 2018, 127: 989-997.

[36] Hawkesford M, Horst W, Kichey T, . Marschner P, . Functions of macronutrients. Mineral nutrition of higher plants, 2012, 3, Amsterdam: Elsevier Ltd., 135-189.

[37] Hendrickson L, Furbank RT, Chow WS. A simple alternative approach to assessing the fate of absorbed light energy using chlorophyll fluorescence. Photosynth Res, 2004

[38] International Renewable Energy Agency-IRENA (2019) Future of solar photovoltaic Deployment, investment, technology, grid integration and socio-economic aspects

[39] Katsuda T, Shimahara K, Shiraishi H, . Effect of flashing light from blue light emitting diodes on cell growth and astaxanthin production of Haematococcus pluvialis. J Biosci Bioeng, 2006

[40] Klok AJ, Martens DE, Wijffels RH, Lamers PP. Simultaneous growth and neutral lipid accumulation in microalgae. Bioresour Technol, 2013, 134: 233-243.

[41] Kramer DM, Avenson TJ, Edwards GE. Dynamic flexibility in the light reactions of photosynthesis governed by both electron and proton transfer reactions. Trends Plant Sci., 2004, 9: 349-357.

[42] Malapascua JRF, Jerez CG, Sergejevová M, . Photosynthesis monitoring to optimize growth of microalgal mass cultures: application of chlorophyll fluorescence techniques. Aquat Biol, 2014, 22: 123-140.

[43] Masuko T, Minami A, Iwasaki N, . Carbohydrate analysis by a phenol–sulfuric acid method in microplate format. Anal Biochem, 2005, 339: 69-72.

[44] Mitra M, Melis A. Optical properties of microalgae for enhanced biofuels production. Opt Express, 2008, 16: 21807.

[45] Nikkanen L, Solymosi D, Jokel M, Allahverdiyeva Y. Regulatory electron transport pathways of photosynthesis in cyanobacteria and microalgae: recent advances and biotechnological prospects. Physiol Plant ppl, 2021

[46] Perrine Z, Negi S, Sayre RT. Optimization of photosynthetic light energy utilization by microalgae. Algal Res, 2012, 1: 134-142.

[47] Pessarakli M (2016) Handbook of photosynthesis, Third Edition

[48] Polat E, Yüksel E, Altınbaş M. Mutual effect of sodium and magnesium on the cultivation of microalgae Auxenochlorella protothecoides. Biomass Bioenerg, 2020, 132: 105441.

[49] Ren HY, Liu BF, Kong F, . Enhanced lipid accumulation of green microalga Scenedesmus sp. by metal ions and EDTA addition. Bioresour Technol, 2014

[50] Rodolfi L, Zittelli GC, Bassi N, . Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol Bioeng, 2009, 102: 100-112.

[51] Rubin H. Central role for magnesium in coordinate control of metabolism and growth in animal cells. Proc Natl Acad Sci USA, 1975, 72: 3551-3555.

[52] Sasaki Y, Kozaki A, Hatano M. Link between light and fatty acid synthesis: thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase. Proc Natl Acad Sci U S A, 1997, 94: 11096-11101.

[53] Schlesinger A, Eisenstadt D, Bar-Gil A, . Inexpensive non-toxic flocculation of microalgae contradicts theories; overcoming a major hurdle to bulk algal production. Biotechnol Adv, 2012, 30: 1023-1030.

[54] Schulze PSC, Brindley C, Fernández JM, . Flashing light does not improve photosynthetic performance and growth of green microalgae. Bioresour Technol Reports, 2020

[55] Shifrin NS, Chisholm SW. Phytoplankton lipids: interspecific differences and effects of nitrate, silicate and light-dark cycles. J Phycol, 1981, 17: 374-384.

[56] Simionato D, Block MA, La Rocca N, . The response of Nannochloropsis gaditana to nitrogen starvation includes de novo biosynthesis of triacylglycerols, a decrease of chloroplast galactolipids, and reorganization of the photosynthetic apparatus. Eukaryot Cell, 2013, 12: 665-676.

[57] Smith BT, Davis RH. Sedimentation of algae flocculated using naturally-available, magnesium-based flocculants. Algal Res, 2012, 1: 32-39.

[58] Sydney EB, Sturm W, de Carvalho JC, . Potential carbon dioxide fixation by industrially important microalgae. Bioresour Technol, 2010, 101: 5892-5896.

[59] Takahashi T. Usefulness of a microalgal biorefinery in conversion to chemical products and recent technology in automatic evaluation of microalgae. Curr Opin Green Sustain Chem., 2021, 39: 101692.

[60] Terasaki M, Rubin H. Evidence that intracellular magnesium is present in cells at a regulatory concentration for protein synthesis. Proc Natl Acad Sci USA, 1985, 82: 7324-7326.

[61] Ummalyma SB, Gnansounou E, Sukumaran RK, . Bioflocculation: an alternative strategy for harvesting of microalgae–an overview. Bioresour Technol, 2017, 242: 227-235.

[62] Vandamme D, Beuckels A, Markou G, . Reversible flocculation of microalgae using magnesium hydroxide. Bioenergy Res, 2015, 8: 716-725.

[63] Vecchi V, Barera S, Bassi R, Dall’osto L. Potential and challenges of improving photosynthesis in algae. Plants, 2020, 9(1): 67.

[64] Vejrazka C, Janssen M, Streefland M, Wijffels RH. Photosynthetic efficiency of Chlamydomonas reinhardtii in flashing light. Biotechnol Bioeng, 2011

[65] Vernon WB, Wacker WEC. Alberti KGMM. Magnesium metabolism. Recent advances in clinical biochemistry, 1978, Edinburgh: Churchill-Livingstone, 39.

[66] Vishwakarma R, Dhar DW, Saxena S. Influence of nutrient formulations on growth, lipid yield, carbon partitioning and biodiesel quality potential of Botryococcus sp. and Chlorella sp. Environ Sci Pollut Res, 2019, 26: 7589-7600.

[67] Volgusheva A, Kukarskikh G, Krendeleva T, . Hydrogen photoproduction in green algae Chlamydomonas reinhardtii under magnesium deprivation. RSC Adv, 2015

[68] Walker GM. The roles of magnesium in biotechnology. Crit Rev Biotechnol, 1994, 14: 311-354.

[69] Walker GM, Birch-Andersen A, Hamburger K, Kramhøft B. Magnesium-induced mitochondrial polymorphism and changes in respiratory metabolism in the fission yeast, Schizosaccharomyces pombe. Carlsberg Res Commun, 1982, 47: 205-214.

[70] Xia L, Ge H, Zhou X, . Photoautotrophic outdoor two-stage cultivation for oleaginous microalgae Scenedesmus obtusus XJ-15. Bioresour Technol, 2013, 144: 261-267.

[71] Yoshimura T, Okada S, Honda M. Culture of the hydrocarbon producing microalga Botryococcus braunii strain Showa: optimal CO2, salinity, temperature, and irradiance conditions. Bioresour Technol, 2013, 133: 232-239.

[72] Zhang X, Wang L, Sommerfeld M, Hu Q. Harvesting microalgal biomass using magnesium coagulation-dissolved air flotation. Biomass Bioenerg, 2016, 93: 43-49.

[73] Zhila NO, Kalacheva GS, Volova TG. Effect of nitrogen limitation on the growth and lipid composition of the green alga Botryococcus braunii Kütz IPPAS H-252. Russ J Plant Physiol, 2005, 52: 311-319.

[74] Zhila NO, Kalacheva GS, Volova TG. Influence of nitrogen deficiency on biochemical composition of the green alga Botryococcus. J Appl Phycol, 2005, 17: 309-315.

[75] Zhou X, Yuan S, Chen R, Song B. Modelling microalgae growth in nitrogen-limited continuous culture. Energy, 2014, 73: 575-580.

Funding

Agencia Nacional de Hidrocarburos (ANH)(721-2015)

Ministerio de Ciencia, Tecnología e Innovación (Minciencias)(721-2015)

Universidad de Antioquia(721-2015)