Preliminary Analysis of the Source and Destination of Carbon in Phaeodactlyum tricornutum under Nitrogen Deprivation Steven Baldassano

AMOPs in Biofuels AMOP = Aquatic Microbial Oxygenic Photoautotroph Includes cyanobacteria, algae, diatoms High neutral lipid yield (20-50% of dry weight) Potentially more useful for biofuels than current sources such as corn grain ethanol More efficient conversion of solar energy Use of little or no land Easier conversion of lipids to useful biofuels Useful byproducts such as protein for animal feed

Purpose Determination of the effects of nitrogen deprivation on Phaeodactylum tricornutum Inability to create proteins will change carbon composition Measurement of carbohydrate/protein/lipid composition Use of C13 labeling to determine source and distribution of carbon

Experimental Setup Cultures of Phaeodactylum tricornutum grown for 4 days in F/2 media with sufficient N and C12 On day 4, cells were pelleted and resuspended in C13 labeled bicarbonate in sealed flasks 3 in F/2 media 3 in F/2 media without NO3- N2 bubbling used to evacuate flasks Assays performed on day 4 (t=0 timepoint) and day 7 (t=3 days after resuspension).

Methods pH Cell count by microscopy Optical density at 625 nm Chlorophyll content β-1,3-glucan (carbohydrate storage) C13 content by mass spectroscopy Membrane carbohydrates Protein content Lipid Content Fractionation and C13 content using LCMS Ash weight percent

Results N- cells became much larger and more ovoid Significantly lower cell count in N- samples due to arrested cell division Decreased chlorophyll content in N- samples Decrease in pH relative to N+ samples Optical density readings correlate well with cell count and dry weight for N+ samples only

Results N-, ash-free dry weight % N+, ash-free dry weight %

Results Overall lipid increase after resuspension:

Ongoing Research Completion of lipid content analysis Fractionation C13 labeling Mass spectroscopy for carbohydrate C13 content C13 content will provide information as to the source of carbon for glucan and lipid increase rearrangement vs de novo synthesis

Summary Nitrogen deprivation resulted in changes in composition Increase in cell size and mass Increase in glucan content Increase in lipid content (per cell and dry weight) Slight decrease in overall lipids per ml Decrease in membrane carbohydrate content Decrease in protein content Source of carbon for lipid and glucan increase to be determined soon Provide insights as to the mechanisms by which these diatoms produce lipids under environmental stress, thereby aiding in the overall goal of someday understanding this process well enough to use AMOPs as a major source of biofuels