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Robert D. Gardner, Gregory L. Helms b, William C. Hiscox b, Egan J. Lohman a, Brent M. Peyton a, Robin Gerlach a, and Keith E. Cooksey c Department of.

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Presentation on theme: "Robert D. Gardner, Gregory L. Helms b, William C. Hiscox b, Egan J. Lohman a, Brent M. Peyton a, Robin Gerlach a, and Keith E. Cooksey c Department of."— Presentation transcript:

1 Robert D. Gardner, Gregory L. Helms b, William C. Hiscox b, Egan J. Lohman a, Brent M. Peyton a, Robin Gerlach a, and Keith E. Cooksey c Department of Bioproducts and Biosystems Engineering West Central Research and Outreach Center University of Minnesota a Dept. of Chemical Engineering and the Center for Biofilm Engineering, Montana State University, Bozeman MT. b Center for NMR Spectroscopy, Washington State University, Pulman WA. c Evironmental Biotechnology Consultants, Manhattan MT. Insight into lipid biogenesis during TAG accumulation using stable isotope tracers coupled with NMR spectroscopy and mass spectrometry

2 Overview 1) Bicarbonate-Enhanced Growth and Bicarbonate-Induced TAG Accumulation (See poster 204 – Brent Peyton) i) Background and nitrogen dependency 2) NMR and MS to monitor inorganic carbon fixation (See poster 123 – Greg Helms) i) NMR for real time analysis ii) Verified using MS techniques on the molecular ion

3 Image from Schenk, P., et al BioEnergy Research, 1(1), Algae are biocatalysts that convert renewable sunlight into biofuels and chemical substrates Strain dependent characteristics during lipid biogenesis De novo biosynthesis vs. C-recycling in the cell Photosynthesis for Biofuels

4 Algal Carbon Concentrating Mechanisms Carbon utilization –Ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) Relatively low affinity for CO 2 Carbonic anhydrase –Reversibly convert CO 2 ↔ HCO 3 –Extra & intracellular HCO 3 Transporters –Plasmamembrane and Chloroplast membrane bound C 4 carbon utilization –Theorized in diatoms –Reversibly convert CO 2 ↔ Oxaloacetate PEPcarboxylase and PEPcarboxykinase Images from Moroney 2007, Roberts 2007

5 Bicarbonate – Enhanced Growth Optimized Growth Scenario (5 mM Bicarbonate)  50 mM Bicarbonate stops (4 d) cellular replication  Desired cell density can be achieved by “tweaking” initial nutrient concentrations  Specific Growth Rate (µ)  Increased by 69%  Biomass Productivity (g L -1 Day -1 : DCW) Increased by 27% Lohman E, Gardner R, et al Biotechnology for Biofuels (manuscript in review). 50mM NaHCO 3

6 An Optimized DIC Regime – Enhanced Growth  Optimized Growth Scenario  Optimal system had significantly higher chlorophyll content  More photosynthetically active  More efficient DIC fixation Lohman E, Gardner R, et al Biotechnology for Biofuels (manuscript in review).

7 Gardner R, et al Journal of Applied Phycology. 24(5): Gardner R, et al Biotechnology and Bioengineering 110(1): Bicarbonate Addition at Medium N-Depletion Addition of HCO 3 - stops cellular cycle and induces TAG accumulation in green algae. Addition of HCO 3 - does not stops cellular cycle but induces TAG accumulation in diatoms. Confirmed on over 20 green algae and diatom strains, both freshwater and marine. Scenedesmus sp. WC-1 C. reinhardtii ½ time required Air 5% CO 2 HCO 3 Scenedesmus sp.

8 1 H NMR Detection and Quantitation of TAGs in Live Algal Cells Directly sample culture Monitor TAG accumulation Quantitate TAG content Davey et al. Algal Research 2012 doi: /j.algal Advanced NMR Method Development

9 NMR Metabolite Signals Sucrose (Carbohydrates) Pyruvate Allylic (MUFA) Alpha C Beta C CH 2 (Bulk lipid) Choline (3 signals) Double Allylic (PUFA) Glyceraldehyde Glycerol (CH 2 and CH) MAG DAG TAG Methyl C Olefin Omega-3 Image from Schuhmann, et al Biofuels, 3(1),

10 Experimental Outline – 24 hr lighting Forward Experiment – de novo synthesis Growth in 5 mM 12 C (DIC) NaH 13 CO 3 addition at N-limitation Monitored (48 hrs) 13 C-incorperation and labeling with 1 H HR-MAS NMR Medium 13 C concentration and speciation with 13 C NMR Chlorophyll and carotenoid concentration Dry cell weight change Reverse Experiment – recycling C NaH 13 CO 3 labeled biomass NaH 12 CO 3 at N-limitation Monitored (24 hrs) 13 C-recycling with 1 H HR-MAS NMR Dry cell weight change GC-FID and GC-MS analysis

11 Experimental Results (GC analysis)

12 Chlorophyll & Carotenoid Chlorophyll and carotenoid concentration remained stable Suggests photosynthesis was maintained

13 25 mM NaH 13 CO 3 addition with N-deplete culturing

14 Quantification of the terminal methyl group

15

16 Total Correlation Spectroscopy (TOCSY) F1 decoupled TOCSY with 1D coupled spectrum Separates along the diagonal

17 Sucrose and Bulk Lipid Sucrose (>80% de novo synthesis at 24 hrs) Rapid DIC incorporation (within 15 min) Metabolic switch (steady-state) after 10 hrs Bulk CH 2 (>70% de novo synthesis at 24 hrs) DIC incorporation and recycled biomass for 8 hrs, after which increased rate of DIC incorporation

18 Allylic (MUFA) and Double Allylic (PUFA) MUFA (Allylic) (>60% de novo synthesis at 24 hrs) Initial recycling High DIC incorporation after 12 hrs PUFA (double allylic) (>60% recycled C at 24 hrs) High initial and continued incorporation of recycled carbon De novo synthesis using DIC after 6 hrs

19 Omega-3 Omega-3 (>80% recycled C at 24 hrs) Initial C-recycling and unobservable de novo synthesis from DIC C-recycling continues and de novo synthesis begins after 10 hrs

20 Mass Spectrometry Confirmation of NMR Findings Determined molecular ion values based on unlabeled standard or unlabeled extractant Fill in A-matrix diagonal and correct M+1 and M+2 values below the diagonal to correct for natural abundance of 13 C. Inherently corrects for MS ionization efficiency

21 Molecular Ion Analysis (GC-MS) High bicarbonate incorporation into C16:0 and C18:1 (primary FAs that increased) High bicarbonate incorporation into C18:3 (6,9,12), de novo synthesis High Recycling in C18:3 (9,12,15), membrane lipid reallocation

22 PUFA signal 3 signals make up the overall PUFA signal One does not incorporate bicarbonate, one ~30%, the other ~65% Total Correlation Spectroscopy (TOCSY)

23 NMR can be used as a metabolic microscope to track carbon from bicarbonate to TAG. Carbon rates of incorporation are being processed. Initial carbon source is identified (i.e., bicarbonate, CO 2, or biomass). Fundamental and controversial questions are being answered (i.e., de novo or carbon recycling in fatty acid synthesis). Additional deconvolution and fluxomic developments are in process. Summary – Key Points

24 U of MN BBE Dept. & WCROC Greg Helms & Bill Hiscox (WSU) Collaborators University of Minnesota Montana State University Washington State University Contributors & MSU Biofuels Group Members Brent Peyton (Peyton Lab Group) Robin Gerlach * Environmental and Biofilm Mass Spectrometry Facility Keith Cooksey Funding NSF IGERT Program in Geobiological Systems Church & Dwight Co., Inc. US DoE/DoD Acknowledgements


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