1. Differential Gene Expression in Heterosigma akashiwo in Response to Model Flue Gas: Where Does the Carbon Go? 8 th Annual Algae Biomass Summit Algal Strain Development October 2, 2014 Jennifer J. Stewart, Ph.D. Scientist, NSF SEES Fellow 302-645-4371 Jen@udel.edu R83-3221

2. Koonin et al. (2010) Genome Biology 11:209 What is a Raphidophyte? Hara & Chihara 1987

3. 2 Heterosigma akashiwo  Raphidophyte  Metabolizes NO gas  Optimum growth maintained over a wide range of salinity (10-30 psu) and temperature (16-30°C)  Survives nutrient limitation and high light stress  Exhibits no strong preference for nitrogen source (NO 3 -, NO 2 -, NH 4 + ) Photos by Elif Demir-Hilton and Kirk Czymmek Jennifer J. Stewart, Ph.D. Scientist, NSF SEES Fellow 302-645-4371 Jen@udel.edu

4. Theoretical Mechanism of NR2-2/2HbN  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 (Stewart & Coyne 2011) Jennifer J. Stewart, Ph.D. Scientist, NSF SEES Fellow 302-645-4371 Jen@udel.edu

5. 4 Air 10.5  M Flue Gas 12.4  M 12% CO 2, 150 ppm NO, N 2 balance Heterosigma akashiwo on a Model Flue Gas Jennifer J. Stewart, Ph.D. Scientist, NSF SEES Fellow 302-645-4371 Jen@udel.edu Compositional Breakdown-Per Cell Basis

6. 5  1641 Transcripts Were Significantly Differentially Expressed  524 Received KEGG Orthology (KO) Identifiers  274 Received Enzyme Commission (EC) Numbers  149 Mapped to KEGG Reference Pathways Global Gene Expression Analysis

7. 6 Metabolic Overview of Differentially Expressed Genes

8. 7 Photosynthesis Carbohydrate TCA Lipid Syn N-Metabolism AA Isoprenoids Abiotic Stress Redox Nucleotides RNA Processing DNA Synthesis Protein Synthesis Signaling Cell Cycle Transporters Unknowns Metabolic Overview of Differentially Expressed Genes

9. 8  For NR, Only NR2 sequences were Found to be Differentially Expressed  The Highest Up-regulated Transporter Genes were Phosphate Transporters  Flue Gas is a Significant Source of Nitrogen for Protein Synthesis Nitrogen Uptake and Utilization

10. 9 Fatty Acid Biosynthesis  Increase in FAs involved in plastid membrane composition

11. 10 Fatty Acid Biosynthesis

12. 11 Carbonate Chemistry

13. 12 CO 2 Fixation During Growth on Model Flue Gas

14.  -D-Glucose-6P  -D-Glucose-6P  -D-Fructose-6P  -D-Fructose-1,6P 2 Glyceraldehyde-3PGlyceraone-P Glycerate-1,3P 2 Glycerate-3P Glycerate-2P PhosphoenolpyruvatePyruvate Carbon Fixation AA SynthesisFatty Acid Synthesis Up-Regulation Seen Throughout the Glycolysis Pathway

15. Pathway for Storage Carbohydrates? 12%CO 2 + 150 ppm NO 2%CO 2 – Day 5 Batch Growth

16. Pathway for Storage Carbohydrates? 15 Green algae = Starch Diatoms = Chrysolaminarin Jennifer J. Stewart, Ph.D. Scientist, NSF SEES Fellow 302-645-4371 Jen@udel.edu

17. 16 Glycolytic and Glucan Biosynthesis in the Diatom Phaeodactylum tricornutum Chauton et al. (2013) Plant Physiology 161:1034

18. 17 All Roads Lead to  -D-Fructose-6P

19. 18 Storage Carbohydrate metabolism of Ectocarpus siliculosus. Michel et al. (2010) New Phytologist 188:67 Dittami et al. (2011) Plant Signaling & Behavior 6:8 Fate of  -D-Fructose-6P in Brown Macro-Algae

20. Acknowledgements 19 R83-3221 UD: Kathy Coyne, Mark Warner, & Tom Hanson SU: Katherine Miller ASU: John McGowen, Tom Dempster, Hank Gerken, and Crew Lab Members: Colleen Bianco, Chris Main, Kaytee P., Josee Nina Bouchard DNREC Division of Air Quality: Ali Mirzakhalili & Mark Lutrzykowski The following funding sources: Jennifer J. Stewart, Ph.D. Scientist, NSF SEES Fellow 302-645-4371 Jen@udel.edu