Alcian Blue-based quantification of soluble extracellular polymeric substances in microalgal and cyanobacterial cultures: optimisation, validation, and matrix-effect assessment.

The accurate quantification of soluble extracellular polymeric substances (S-EPS) in microalgal and cyanobacterial cultures requires methods that remain reliable across different culture media. This study optimised and validated an Alcian Blue-based assay strategy for S-EPS determination in culture media. Post-preparation dye mixing was optimised at 60 rpm for 60 min to ensure stable residual absorbance and reproducible readout. A base Alcian Blue concentration of 0.80 g/L (pH 2.5) was selected based on dye stability and dye-only linearity. The dye-only response remained stable across pH 3-12 and salinity 0-37 ‰, whereas Ca2+, Mg2+, and Fe2+ significantly reduced the dye response. Alcian Blue remained stable for up to 61 days at 4 °C, but only 7 days at room temperature. Spike-recovery assessment in the A. minutissima/BG11 culture system showed deviations within the 95 % prediction interval and recovery values close to 100 %, supporting quantitative application under this matrix condition. Two calibration levels were distinguished: dye-only calibration for instrumental quality control (R2 = 0.9999; 0.06-1.0 g/L; LOD = 0.006 g/L; LOQ = 0.021 g/L) and matrix-matched S-EPS calibrations to evaluate medium-dependent effects on quantification. No detectable matrix interference was observed in the BG11-based system, whereas Spirulina and ASP-12 media required matrix-matched calibration and 1.0 g/L Alcian Blue. After medium-specific optimisation and calibration, the assay provides a rapid, low-cost, low-sample-volume approach for repeated S-EPS quantification, supporting laboratory studies and routine monitoring of culture-based production systems, while reinforcing that reliable application requires matrix-effect assessment before quantitative use.