*4.5. Analytical determinations*

Total chlorophyll and carotenoid pigments were determined spectrophotometrically after centrifuging tubes containing samples for 6 min at 13000 rpm, heating them for 1 min, and extracting cell pellets with pure methanol. Sonication by ultrasound was also applied when necessary. After that, samples were spun down again for 5 min at 5000 rpm to eliminate cellular wastes. Calculations were done using equations according to [31]. 

For specific carotenoid analysis and quantification, separation was performed by liquid chromatography (HPLC; Merck Hitachi) using a RP-18 column with a flow rate of 1 mL/min. The applied gradient was the following (solvent A; ethyl acetate and solvent B; acetonitrile/agua, 9:1 v/v): 0–16 min, 0–60% solvent A; 16–30 min, 60% A; 30 – 35 min, 100% A. In order to quantify, pigment standards supplied by DHI-Water and Environment (Denmark) were injected. 

Nitrate was determined following the method described by Cawse *et al.* [32]. Urea was determined according to the method from Wilcox [33]. 
