Lamda35 uv vis spectrophotometer

The relative nitrogen concentration available in the culture medium was determined by adopting the optical method described elsewhere [39 (link)]. Briefly, 2 mL of P. tricornutum grown at different initial nitrogen levels was collected every two days. After centrifugation at 12,000× g and 4 °C for 10 min, the supernatant was collected by filtration with a 0.45 µm pore size membrane. The absorbance of culture samples and standard mixture at wavelengths of 220 nm and 275 nm was determined using a Lamda35 UV/Vis spectrophotometer (PerkinElmer, Waltham, MA, USA). Sodium nitrate solutions of at least four different concentrations (6.25, 12.5, 25, and 50 mg/L) were prepared to construct a standard curve. The difference between optical density at 220 nm and 275 nm was used to calculate the relative amount of nitrogen in the medium.

The enzyme assay was performed using commercial enzymes adopted from the total assay procedure method (AOAC Method 996.11/ AACC Method 76.13) from Megazyme (Megazyme Inc., Bray, Ireland). Thermostable α-amylase (3,000 U/mL) was diluted in MOPS buffer (Sigma-Aldrich, St. Louis, MO) at pH 7.0 followed by an incubation in C₂H₃NaO₂ buffer (200 mM) at pH 4.5 and amyloglucosidase concentrate (3,300 U/mL). Total starch content was determined by the enzymatic hydrolysis of 100 mg of fine powder from each sampled stem core. The samples were hydrolysed using 3 mL of thermostable α-amylase to extract maltodextrin from starch at 100 °C over 6 min (vortex at 2, 4 and 6 min intervals). The slurry samples were incubated at 50 °C in a water bath with 4 mL of C₂H₃NaO₂ buffer, followed by 0.1 mL of amyloglucosidase for 30 min to hydrolyse maltodextrin to glucose. The amount of glucose present was measured using an UV-VIS spectrophotometer at 510 nm (Perkin Elmer Lamda 35 UV/VIS Spectrophotometer, Perkin Elmer, MA, USA).