Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum

Abstract

Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post-resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.

Fig. 1

P. tricornutum growth parameters under control conditions, no nutrient resupplementation. a Growth curve cells per milliliters (filled square) and Nile Red fluorescence intensity (cross). b DIC (cross), NO₃ ⁻ (filled square), PO₄ ³⁻ (filled circle) throughout growth, (phosphate was multiplied by a scaling factor of 10). c Chlorophyll a (cross) concentrations and Nile Red fluorescence intensity (filled diamond)

Fig. 2

P. tricornutum growth parameters during continued N replete conditions (solid lines) and N resupplemented (dashed lines), meaning phosphorus stress conditions. The dashed vertical line indicates where N was resupplemented at 189 h. a Growth curve cells per milliliter (unfilled triangle, filled diamond) and Nile Red fluorescence intensity (filled triangle, filled diamond). b DIC (cross), NO₃ ⁻ (filled square), PO₄ ³⁻ (filled circle) throughout growth (phosphate was multiplied by a scaling factor of 10). c Chlorophyll a (cross) concentrations and Nile Red fluorescence intensity (filled diamond)

Fig. 3

P. tricornutum growth parameters during continued P replete conditions (solid lines) and P resupplemented (dashed lines), meaning nitrogen stress conditions. The dashed vertical line indicates where N was resupplemented at 189 h. a Growth curve cells per milliliters (unfilled square, filled circle) and Nile Red fluorescence intensity (unfilled square, filled circle). b DIC (cross), NO₃ ⁻ (filled square), PO₄ ³⁻ (filled circle) throughout growth (phosphate was multiplied by a scaling factor of 10). c Chlorophyll a (cross) concentrations and Nile Red fluorescence intensity (filled diamond)

Fig. 4

P. tricornutum growth parameters during continued N + P replete conditions (solid lines) and N + P resupplemented (dashed lines), meaning no nutrient stress conditions. The dashed vertical line indicates where N was resupplemented at 189 h. a Growth curve cells per milliliter (filled triangle, plus) and Nile Red fluorescence intensity (filled triangle, plus). b DIC (cross), NO₃ ⁻, (filled square), PO₄ ³⁻ (filled circle) throughout growth; phosphate was multiplied by a scaling factor of 10. c Chlorophyll a (cross) concentrations and Nile Red fluorescence intensity (filled diamond)

Fig. 5

P. tricornutum lipid accumulation. a Total moles of FAMEs per liter of culture in control (N + P-depleted cells) compared to (N + P-resupplemented cells). b Cells stained with Nile Red during N + P-resupplemented conditions. c Cells stained with Nile Red under N + P-depleted conditions, showing lipid bodies in yellow. Scale bars, 10μm

Fig. 6

P. tricornutum fatty acid profiles and abundance (mg/liter of culture) from control cells during hours 119, 189, and 263 of growth. Nutrient depletion occurred at hour 189. Results are shown on a logarithmic scale