Comparison and optimization of extraction methods of extracellular polymeric substances in anammox granules: From maintaining protein secondary structure perspective

Extracellular proteins are crucial for the formation and structural maintenance of anammox granules. However, the influence of extracellular polymeric substances (EPS) extraction methods on the secondary structure of extracellular proteins has not been studied fully. In this work, ten EPS extraction methods were applied to anammox granules and compared systematically. The EPS extraction methods were then applied to bovine serum albumin (BSA), a representative of extracellular proteins, to investigate their influence on protein secondary structure by circular dichroism (CD) spectroscopy. The ten EPS extraction methods yielded EPS concentration varying from 5.2 +/- 0.1 to 84.8 +/- 2.0 mg/g volatile suspended solid (VSS). Tween-20 treatment resulted in the most similar CD spectrum and protein secondary structure to the control. Treatment 0.5% Tween-20 for 4 h was found to be the best condition to preserve protein secondary structure while achieving a high EPS yield (44.4 +/- 1.4 mg/g VSS) and limited cell lysis for anammox granules. This study provides an optimized protocol to extract granular EPS when the role of the secondary structure of extracellular proteins in regulating aggregation, adsorption and biofilm formation of microbes is to be elucidated. (C) 2020 Elsevier Ltd. All rights reserved.