Comparison of extraction methods for the characterization of extracellular polymeric substances from aggregates of three biofilm-forming phototrophic microorganisms

This paper aims to define a robust procedure to extract extracellular polymeric substances (EPS) from aggregates of three benthic phototrophic microorganisms: the cyanobacterium Phormidium autumnale, the diatom Nitzschia palea, and the green alga Uronema confervicotum. This study focuses on the extraction efficiency of polysaccharide and protein EPS by using two physical methods (sonication, cation exchange resin) and three chemical methods (formamide, EDTA, Tween 20) with minimum cell lysis. Cell lysis was evaluated by monitoring chlorophyll a release. The results indicated that sonication or incubation of the algae aggregates with 0.25% Tween 20 induced a high level of cell lysis. A combined extraction approach, with an initial dispersing pretreatment (Ultra-Turrax, 13 500 r.min(-1) , 1 min), followed by formamide addition (0.22%) and then incubation with Dowex cation exchange resin (50 g per g of dry biomass), provided the highest amount of extracted EPS (mostly proteins), with low cell lysis. Furthermore, extracted EPS were characterized by size exclusion chromatography, and the obtained fingerprints revealed similar profiles for the three benthic microorganisms with a majority of low molecular weight polymers (400 to 11 300 Da). However, additional EPS of high (>600 000 Da) and intermediate (20 000 to 80 000 Da) molecular sizes were specifically detected in the diatom extracts.