Different chemical extractants (NaCl, EDTA, HCl and NaOH) and physical methods (ultrasonication and heating) were examined by their efficacies of extracting "attached" exopolymeric substances (EPS) secreted by marine bacterium Sagittula stellata (SS) and terrestrial bacterium Pseudomonas fluorescens Biovar II (PF). Extraction by 0.5 N HCl for 3 In was best for SS while extraction by 0.05 N NaCl for 3-5 h was regarded as optimal for PF. Improvements in EPS purification included a pre-diafiltration step to remove the broth material and reduce the solution volume, thus the usage of ethanol, and time. The EPS harvested at the optimal time and purified by the improved method were enriched in polysaccharides, with smaller amounts of proteins, thus having amphiphilic properties. Isoelectric focusing of (234)Th or (240)Pu labeled EPS showed both actinides were strongly bound to macromolecules with low pl, similar to reported marine or soil colloidal natural organic matter (NOM). (C) 2009 Elsevier Ltd. All rights reserved.