Table 1.
Culture conditions and measurements for each culture.
Fig 1.
Flow cytometry measurements for Chlorella vulgaris cells labeled with BODIPY 505/515.
All axes are logarithmic scale. (a) Flow cytogram of BODIPY fluorescence vs. forward scatter of cells after four days culturing in replete or nitrogen-limited medium. (b) Histogram of forward scatter for cells after four days culturing in replete or nitrogen-limited medium. (c) Histogram of BODIPY fluorescence for cells after four days culturing in replete or nitrogen-limited medium. (d) Box plot of medians and interquartile ranges (25th and 75th percentiles) for BODIPY fluorescence of cells after three and four days culturing in replete or nitrogen-limited medium.
Fig 2.
Representative measured NMR spectra for suspensions of intact Chlorella vulgaris cells cultured in (A) nitrogen-limited and (B) replete media.
Samples resuspended in an inorganic NMR analysis solution, and characterized with a coaxial insert containing a TMSP-d4 reference as described in the Materials and Methods section. Peak assigments taken from Davey et al. [4]. Asterisks indicate peaks due to residual tris(hydroxymethyl)aminomethane (δ = 3.7 ppm) and acetate (δ = 1.9 ppm) from the TAP medium. For scale, note that the side peaks located at δ = ±0.1 ppm are 13C satellite peaks for the TMSP-d4 reference. The combined integral intensity of these peaks is 1.1% of total TMSP-d4 intensity, corresponding to approximately 120 μM effective concentration.
Fig 3.
Background spectra for (A) the TAP medium that the algae were cultivated in and (B) the inorganic NMR analysis solution that the algae were resuspended in for 1H NMR measurements.
Asterisks in the TAP medium spectrum indicate peaks from tris(hydroxymethyl)aminomethane (δ = 3.7 ppm) and acetate (δ = 1.9 ppm) that are also visible in our algae spectra due to residual tris(hydroxymethyl)aminomethane and acetate, and are similarly indicated in those spectra. As in Fig 2, the side peaks located at δ = ±0.1 ppm are 13C satellite peaks for the TMSP-d4 reference, with a combined integral intensity of approximately 120 μM effective concentration.
Fig 4.
Mean BODIPY fluorescence per cell of each Chlorella vulgaris culture as measured via flow cytometry, plotted with respect to mean triacylglyceride (TAG) concentration per cell as measured via liquid-state 1H NMR.
Error bars for TAG concentration by 1H NMR are calculated via propagation of uncertainty from the measured variation in estimated initial cell concentration and NMR quantification described in the evaluation of measurement precision section. Error bars for BODIPY fluorescence are taken directly from the measured variation in BODIPY fluorescence described in that section. Linear fit is ⟨BODIPY⟩ = 3.609[TAGs]+1682 (r2 = 0.9974, p = 0.0013) for a linear regression of mean BODIPY fluorescence ⟨BODIPY⟩ in fluorescence intensity units (FIU) as a function of mean TAGs per cell by NMR [TAGs] in fg/cell, with 95% confidence intervals of [3.050, 4.168] for the slope and [1308, 2056] for the intercept.