Cells were dark acclimated for 15 min and gently

filtered onto 13-mm diameter Millipore AP20 glass fiber filters. These filters were placed into the manufacturer’s leaf clip Entospletinib in vitro and an actinic light intensity of 217 μmol photons m−2 s−1 was used to probe the photo-physiology of the algal cells. Chlorophyll a fluorescence parameters were assayed and calculated according to the definitions of Baker (2008). Results Growth of photoheterotrophic versus phototrophic Chlamydomonas To determine the impact of photoheterotrophic versus phototrophic conditions on the growth of Chlamydomonas, wild-type cells were grown in various concentrations of iron with either acetate or CO2 supplied as a carbon source. Within carbon source treatments, iron-replete (20-μM Fe) and iron-deficient (1-μM Fe) cultures grew at the same rate, while iron-limited (≤0.2-μM Fe) cultures grew at a slower rate. The difference in growth rate as a function of iron nutrition was more pronounced in photoheterotrophic conditions where the growth rate in iron limitation was about half (57%) of the rate in the replete situation when compared to phototrophic conditions where the rate in iron limitation was 75% of that in the replete situation (Table 1). In the presence of acetate, iron-replete and -deficient cultures reached a final density of 1.5 × 107 cells/ml

after CHIR98014 6 days of growth, while iron-limited cultures reached stationary phase in 8 days, achieving a final density of only 5–9 × 106 cells/ml (Fig. 1). In contrast,

phototrophic iron-replete and -deficient phototrophic cultures reached a density of only 9 × 106 cells/ml, comparable to the final cell density of iron-limited photoheterotrophic cultures (Fig. 1). Table 1 Growth rate of photoheterotrophic versus phototrophic cells in response to iron nutrition Fe (μM) Acetate μ (day−1) CO2 μ (day−1) 0.1 0.96 ± 0.12 0.56 ± 0.04 0.2 0.90 ± 0.04 0.59 ± 0.07 1 1.44 ± 0.15 0.68 ± 0.11 20 1.68 ± 0.08 0.74 ± 0.06 Osimertinib Standard deviation based on biological triplicates Fig. 1 Growth in photoheterotrophic versus phototrophic growth conditions in response to iron nutrition. Cells were grown in the presence (A) and absence (B) of acetate in various concentrations of iron. Cultures lacking acetate were bubbled with air. Various concentrations of iron represented by empty triangles (0.1-μM Fe), filled triangles (0.2-μM Fe), empty circles (1-μM Fe), and filled circles (20-μM Fe). Standard deviation based on biological triplicates. Dotted line indicates cell density at which cells were collected for analysis Phototrophic cells accumulate more Fe than photoheterotrophic cells In order to relate the growth rate to iron nutrition, the iron content of cells in the presence and in the absence of acetate was determined by inductively coupled plasma-mass spectroscopy.