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Biomass and Bioproducts from Nannochloropsis Sp Peter Lammers, Professor Chemistry & Biochemistry Biomass and Bioproducts from Nannochloropsis sp. New Mexico State University Nannochloropsis as a candidate first-generation biofuel production organism • Nannochloropsis salina – CCMP Strain 1776 • Lipid Biochemistry: – Assay development, – Biomass and algal oil content management – Polar lipid identification – Single cell imaging (SNL) New Mexico State University Deep Divergence Between Various Phytoplankton New Mexico State University How Will Production Strains Compete with Predators / Pathogens? Ciliates, Rotifers Heterotrophic flagellates Chytrid fungi Bacteria, Viruses Other algae: green, yellow, diatoms, cyanobacteria New Mexico State University Nannochloropsis Mar 2008 - Jan 2009 Diatoms Pond 1 1.E+09 Tetraselmis Ciliates 1.E+08 1.E+07 1.E+06 1.E+05 1.E+04 Cells/mL Cells/mL 1.E+03 1.E+02 1.E+01 1.E+00 3/18 4/15 5/13 6/10 7/8 8/5 9/2 9/30 10/28 11/25 12/23 1/20 Two frequently quoted conclusions from the DOE Aquatic Species Report are: i) local strains will overtake introduced strains and ii) Southeast New Mexico is Too Cold for Growing Algae Not necessarily so… New Mexico State University Nitrate and Bicarbonate Levels Alter Lipid Content in Nannochloropsis Marijn de Jong Jan Winkler Isaac Rhodes New Mexico State University Excitation and Emission Data for Nile Red in Triacylglycerol and Phospholipids. Nile Red Assay 1. Samples counted on Flow Cytometer and diluted to 200 cells per microliter 2. Frozen at -80C and stored for assay when convenient 3. Thawed and incubated with Nile Red reagent for 30 minutes 4. Count >5,000 events and capture the mean yellow- channel flourescence 1.2 Lipid accumulation is a 1 mM NaNO3 1.0 stationary phase response 1 mM NaNO3 + 0.8 5 mM NaHCO3 (A750) 0.6 in Nannochloropsis sp. 20 mM NaNO3 0.4 Absorbance 20 mM NaNO3 + 5 mM NaHCO3 Growth of Nannochloropsis in 0.2 0.0 0 2 4 6 8 10 12 14 16 18 high/low nitrate and Time (Days) bicarbonate (top panel) 2200 2100 2000 1900 1800 1700 1600 1 mM NaNO3 Lipid content determination 1500 1400 1300 1 mM NaNO3 + 1200 5 mM NaHCO3 by Nlle Red Fluorescence in 1100 1000 900 20 mM NaNO3 800 the same cultures as above Shift in Fluorescence 700 600 20 mM NaNO3 + 500 5 mM NaHCO3 400 (bottom panel) 300 200 100 0 1 3 5 7 9 16 Time (Days) New Mexico State University Linear increase in total lipid determined gravimetrically after growth in 1 mM nitrate + 5 mM NaCO3 Correlation of gravimetric total lipid and NR dependant fluorescent shift plotted semi-log. Total extractable Lipids were Fractionated on Silica Column Eluent Series Increasing Polarity 1. Chloroform 2. Acetone 3. and Methanol Carotenoids TAGs Chlorophyll and Polar lipids New Mexico State University TAG and Carotenoid Levels Peak at 21 Days 1 mM nitrate, 5 mM bicarbonate treatment; Day CHCl3 Acetone Methanol 14 3.1 6.8 7.3 18.0% 39.5% 42.4% 21 28.8 17.6 8.9 52.1% 31.8% 16.1% 35 20.7 19.8 10.9 40.3% 38.5% 21.2% New Mexico State University Polar Lipid FA Composition Field – grown Nannochloropsis Fatty Acid as Methyl Ester Percent composition C14:0 6% C16:0 28% C16:1 26% C18:1 8% C20:5 32% Neutral Lipid FA Composition Field – grown Nannochloropsis Fatty Acid as Methyl Ester Percent composition C16:0 35% C16:1 30% C18:0 6% C18:1 21% C20:5 8% Ion Cyclotron Resonance Mass Spectrometry Schaub, T.M.; Hendrickson, C.L.; Horning, S.; Quinn, J.P.; Senko, M.W. and Marshall, A.G., High Performance Mass Spectrometry: Fourier Transform Ion Cyclotron Resonance at 14.5 Tesla, Anal. Chem., 80, 3985-3990 (2008) New Mexico State University 60 N1O7 = probable betaine lipids O6 = Triacylglycerols 50 40 30 20 10 Relative Abundance Abundance Relative 0 Hetero-atom composition of major peaks in positive ion mode Ion- Cycloton-Resonance (ICR) Mass Spectrometry New Mexico State University Betaine Lipids Phosphatidyl Choline versus DiacylGlycerylTrimethylhomoSerine Riekhof, W.R., Andre, C. and Benning, C. Two enzymes, BtaA and BtaB, are sufficient for betaine lipid biosynthesis in bacteria. Arch. Biochem. Biophys., 441, 96-105 (2005). New Mexico State University O6 Type Distribution 150 Day 1 100 Day 7 Day 14 50 Day 21 0 Relative Abundance Relative 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Double Bond Equivalents New Mexico State University Next Stop: SOLIX Biofuels Two Testbeds: Fort Collins – 6,000 Liter system and ~1.5 Kg/day dry weight algae biomass with CO2 enrichment Coyote Gulch – 120,000 Liters in three basins, CO2 from amine plant Test Strain: Nannochloropsis New Mexico State University Acknowledgements • NMSU P.I.s – Lammers - Chemistry & Biochemistry – Van Voorhies - Molecular Biology – Boeing - Fisheries and Wildlife – Tanner Schaub, ICR-MS • Center of Excellence for Hazardous Materials Management – Carlsbad, NM • Sandia National Lab – Grant Heffelfinger – Howland Jones – Seema Singh • Funding: State of New Mexico Energy Innovation Fund, Sandia National Lab (LDRD), Gift from Sapphire Energy New Mexico State University .
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