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 %
Lipid
29%
Glucan
20%
Glucan
31%
Lipid
48%
Membrane
Carbs
10%
Membrane
Carbs
6%
Protein
15%
Protein
41%
Results
 Overall lipid increase after resuspension:
Total Lipid Content
0.18
T = 3 days
0.16
0.14
mg/ml
0.12
0.1
NN+
0.08
0.06
T=0
0.04
0.02
0
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