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

2. 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

3. 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

4. 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).

5. 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

6. 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

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

8. Results
Overall lipid increase after resuspension:

9. 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

10. 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