Extracellular polymeric substances extraction and recovery from anammox granules: Evaluation of methods and protocol development

Selection of appropriate extraction methods of extracellular polymeric substances (EPSs)is crucial, affecting the yield, composition and the properties of EPSs. However, there is still a lack of standard methods for EPS extraction. In this study, seven methods were compared to extract EPS from anaerobic ammonium oxidation (anammox)granules, and the effectiveness and cell lysis of these methods were evaluated. The heating-Na₂CO₃ based method obtained a higher EPS yield with limited cell lysis. Proteins (PN)and polysaccharides (PS)were predominant in extractable EPSs. Particularly, PN-related substances were substantially enriched and high PN/PS ratios resulted in most methods. Different methods resulted in EPSs with a different size distribution. By analysis of Attenuated total reflectance-Fourier transform infrared spectra (ATR-FTIR), amide- and polysaccharide-associated bands were dominant. ATR-FTIR and fluorescence density of excitation-emission matrix spectra indicated EPS compositions and structures varied with the extraction methods. Moreover, the extraction/recovery protocol based on the heating-Na₂CO₃ method was remarkably improved, by varying heating time and using a centrifugal filter device with membrane (CFDM)to replace concentration and purification steps. Eventually, the thermal method with an extraction time of 45 mins was identified to be most effective. The application of CFDM was demonstrated as an alternative approach to concentrate and purify for EPS recovery. To our best knowledge, this is the first original study to systematically evaluate the different EPS extraction methods from anammox granules.