Growth and Flocculation of Heterosigma akashiwo on Industrial Emissions Jennifer J. Stewart1 Colleen M. Bianco1 Mark E. Warner1 Katherine R. Miller2 Kathryn J. Coyne1 1University of Delaware CEOE 2Salisbury University Algae Biomass Summit 2013 Heterosigma akashiwo Raphidophyte Globally prevalent Forms dense blooms in excess of 107 cells * L-1 Growth Rate (m) up to 1 No rigid cell wall 10-15 mm Mixotrophic Vertical Migrator Can accumulate high amounts of lipids (up to 73% dry weight under N- limitation; Fuentes- www.eweb.furman.edu Grünewald et al. 2012) Heterosigma akashiwo Optimum growth maintained over a wide range of salinity (10-30 psu) and temperature (16-30°C) Tolerates nutrient limitation and high light stress Resistant to crashing Exhibits no strong preference for nitrogen - - + source (NO3 , NO2 , NH4 ) Photo by L. Salvitti Nitrate Metabolism in Microalgae Nitrate Reductase NO - catalyzes the rate- 3 limiting step in nitrate NO - NO - NH + assimilation 3 2 4 NR NiR e- Rubisco Amino Acids Proteins Highly conserved in CO2 PGA plants and algae PSII Carbohydrates Lipids hv >2200 eukaryotic NR (Modified from Lomas & Glibert, 1999) sequences in the NCBI Entrez Protein Database Novel Nitrate Reductase (NR2-2/2HbN) NR1 NR2 Found in Heterosigma akashiwo (4 strains) and Chattonella subsalsa Stewart & Coyne. 2011. Analysis of raphidophyte assimilatory nitrate reductase reveals unique domain architecture incorporating a 2/2 hemoglobin. Plant Mol Biol 77:565–575 Theoretical Mechanism of NR2-2/2HbN Currently Under Investigation Dual NO dioxygenase and Nitrate Reductase Activities 5 e- when fully reduced: 2 e- accepted by FAD, 1e- by heme- Fe, and 2e- by Mo-MPT Univalent reduction of both heme-Fe centers possible Nitrate captured Experimental Addition of Nitric Oxide to Cell Cultures Cultures reduce the NR2-2/2HbN transcript concentration of dissolved expression increases in NO in liquid medium response to NO H. akashiwo Assimilates Nitric Oxide for Cellular Nitrogen Saturated solution of 15 N-NO in O2 free H2O added to cultures at 20 mM final concentration (n=4) Controls spiked with O2 free H2O Incubated 18 hours Filtered, dried, and sent to stable isotope facility Note Log Scale Nitrogen Oxides (NOx) in Industrial Emissions – The Other Big Problem Toxic Gas > 90% Nitric Oxide (NO) Contributes to Acid Rain and Ground Level Ozone Damages human health and destroys food crops Accounts for 27% of nitrogen entering Chesapeake Bay Earthobservatory.nasa.gov Combustion of fossil fuels for commercial, industrial, and electricity generation processes produces 40% of total NOx emissions released into the atmosphere Well controlled plants still emit 10-50% of Major Impacts to Mid-Atlantic NOx after emissions States from Upwind Sources are scrubbed H. akashiwo Growth on Model Flue Gas Batch Culture (Nutrient Replete Log Phase Growth) >3.7-fold increase in biomass productivity 6.3 g/m2/day for air grown cultures 23.0 g/m2/day for flue gas grown cultures H. akashiwo on Air Semi-Continuous Growth Up to 57.9 g/m2/day for flue gas grown cultures H. akashiwo on Gas Cell size significantly increased (10.5 to 12.4 mm) Chl a content significantly increased Photochemistry Flue Gas treatment does not affect overall photosynthetic efficiency. Air Flue Gas p-value Fv/Fm 0.528 (± 0.008) 0.483 (±0.012) p<0.001 Fq'/Fm' 0.457 (±0.015) 0.442 (± 0.023) NS ETRmax 159 (± 14) 167 (± 24) NS Expression of NR2-2/2HbN 8 7 Expression of the NR2- 6 2/2HbN transcript was 5.7x 5 higher in Flue Gas versus Air 4 cultures (p<0.003) 3 2 Relative Expression Relative 1 0 Air Flue Gas Resource Partitioning Increased productivity of biofuel intermediates by: 1.7X for lipids 3.0X for proteins 17.8X for carbohydrates Novel Cell Flocculation Method A C B Relative expression of NR2-2/2HbN in Autoflocculation of H. akashiwo in response to H. akashiwo cultures before the gas containing 12% CO2 and 150 ppm NO. induction of flocculation (Control) and Flocculation occurs in stages: (a) Settling, (b) during the migration stage of Migration, and (c) Flocculation. A) Cells during the flocculation (Migration). Error bars settling phase accumulate on the bottom of the represent standard deviation (n=3). flask. B) Arrows show cells forming a path to the gas source during the migration phase. C) An arrow shows a compact cell pellet has flocculated at the gas source in a dense culture. Acknowledgements • UD: Kathy Coyne, Mark Warner, & Tom Hanson • SU: Katherine Miller • Lab Members: Colleen Bianco, Chris Main, Kaytee P., Josee Nina Bouchard • DNREC Division of Air Quality: Ali Mirzakhalili & Mark Lutrzykowski • The following funding sources: R83R83-3221 .