Spectrophotometric quantification of cyanobacterial extracellular polymeric substances (EPS) using safranin

Abstract

EPS are synthesized by various microorganisms and they play crucial roles in microorganism's growth and survival within their ecosystem. Quantification of EPS in culture liquid is generally done through gravimetric analysis after precipitation using large quantities of ethanol or acetone. Such analyses with a small volume of culture filtrate are error-prone. This study introduces a straightforward spectrophotometer-based technique for quantifying dissolved EPS secreted by cells into the growth medium. Utilizing EPS extracted from Synechocystis sp. IMMT47, the method involves staining and precipitating cyanobacterial EPS secreted from cells, with safranin. Subsequently, the precipitated pellet after centrifugation was reconstituted in water and the absorbance of the filtrate solution was measured at 519 nm. The obtained linear relationship between the concentration of EPS and absorbance from this method is compared with the earlier reported alcian blue-based quantification approach. Conventional gravimetric quantification of EPS using acetone was used as a standard to compare the efficacy of analysis. The R2 value was found to be 0.97 with a p-value of 0.33, showing no significant variation among the quantification methods. The presented assay proficiently quantifies dissolved extracellular polysaccharides by evaluating reconstituted absorbance. Compared to the conventional gravimetric and alcian blue-based methods, the safranin assay exhibited higher sensitivity and reproducibility for small-volume samples. This approach not only streamlines EPS quantification but also minimizes sample loss and reagent consumption. Given its simplicity, cost-effectiveness, and accessibility, this method holds promise for advancing microbial EPS research, particularly in ecological and biotechnological studies. This innovative method has significant potential for advancing research on the biogeochemistry of EPS in microorganisms.