The results of the characterisation of anaerobic sludge-derived extracellular polymeric substances (EPS) under different extraction conditions and their effects on the fouling in submerged hollow fibre membranes are presented. A wide range of EPS extraction results was obtained depending on the extraction conditions. Proteins, polysaccharides, and humic substances represented 80–99% of the total organic matter extracted, with 54–60% identified as humic substances. Ultrasonication was more effective than vortex agitation, ensuring higher EPS extraction yields in a shorter contact time. The increase in temperature from 30 °C to 50 °C and the decrease in NaCl concentration favoured the EPS diffusion, but this positive temperature effect was negligible for an ultrasonic contact. Linear, quadratic, and combined effects of ultrasonication time and solvent salinity were statistically significant effects for ultrasonication-assisted EPS extraction. The highest extraction yield was achieved using ultrasonication at 30 ºC for at least 60 min with salt-free water as the solvent, and an EPS extract with 138.8 mg TOC/L was obtained. Batch ultrafiltration-backwash tests at different permeation rates allowed to determine the specific resistance to filtration of the EPS extracts, which increased from 103.8·1012 m/kg TOC to 169.4·1012 m/kg TOC in presence of 1.8 wt% of NaCl. Salinity also provided rigidity to the EPS gel layer, reducing the compressibility index from 0.23 to 0.03.
