Background

Carbohydrates play a number of roles in the metabolism of algae and cyanobacteria. As nicely summarized by Raven and Beardall (Raven and Beardall, 2003), carbohydrates represent major sink of intermediates in carbon reduction/oxidation pathways (photosynthesis and photorespiration), they provide skeletons required for growth (e.g., for amino-acid or cell wall biosynthesis), they represent sources of ATP and reducing equivalents (through respiration pathways), they serve as compatible solutes, they are essential for maintaining cellular turgor (by securing rigid structure of the cell wall) and they can scavenge free radicals. Storage polysaccharides (of which the main forms in algae and cyanobacteria are starch and glycogen) that serve as both energy and carbon source, buffer the disproportion between the carbohydrates production and consumption rates, and allow for active metabolism (e.g., nitrogen fixation) in the dark periods.

Carbohydrates are routinely analyzed in many life science laboratories. The saccharides content can be quantified by a wide range of chemical (e.g., chromatographic), biochemical (e.g. gravimetric, colorimetric, enzymatic) or physical (e.g., polarimetry) methods. The estimation of storage carbohydrates as described in this protocol represents a combination of starch/glycogen purification according to the previous studies (Schneegurt et al., 1994; Bandyopadhyay et al., 2010; Sinetova et al., 2012), starch/glycogen decomposition in acidic environment and free glucose determination by the classical phenol-sulfuric acid method (Dubois et al., 1956; Masuko et al., 2005). Total carbohydrates estimation consists of simple resuspension of the cellular pellet (after extraction of chlorophyll and carotenoids) in phenol solution and hydrolyzation of cellular saccharides by sulfuric acid.

The advantages of this protocol are simplicity, sensitivity and quickness. The carbohydrates/glycogen/starch estimation requires only several standard reagents, which makes this protocol much simpler and cheaper when compared to protocols that include enzymatic cleavage of glycogen (De Porcellinis et al., 2017; Khan et al., 2018), enzymatic determination of free glucose (Bandyopadhyay et al., 2010; Sinetova et al., 2012; Khan et al., 2018) or extensive amounts of chemicals (Khan et al., 2018). Another advantage of this protocol is that only 1 ml of diluted culture suspension is needed for the analysis (for further details see Note 1) which is significantly lower amount than required in other protocols (De Porcellinis et al., 2017). Additionally, saccharides/glycogen/starch measurement can be performed on the same samples that were originally used for determination of chlorophyll and carotenoids content (Sinetova et al., 2012).

On the other hand, this protocol is not as specific for determination of storage polysaccharides as the enzymatic assays (De Porcellinis et al., 2017) since glycogen or starch are distinguished only partially from other cellular polysaccharides (e.g., from polysaccharides of the cell wall). Another limitation is using D-glucose as a calibration standard since various carbohydrates that are present in the cells differ in the absorption spectra (Dubois et al., 1956; Masuko et al., 2005). Nevertheless, even with these limitations, this cheap, simple and fast protocol is suitable for rough estimation of carbohydrates content in algae and cyanobacteria.