Modeling optimization and evaluation of tightly bound extracellular polymeric substances extraction by sonication

Tightly bound extracellular polymeric substances (TB-EPS) are important components of sludge, playing a crucial role in the behavior of activated sludge. They are located in the innermost layer of EPS which closely combine with the cell surface and are difficult to be extracted. To fully understand the role of TB-EPS, it is extremely important to find an appropriate TB-EPS extraction protocol, which provides maximum yields of TB-EPS under the premise of minimal contribution of cell lysis. Ultrasonic method has been widely applied for TB-EPS extraction due to its unique advantages, but no one has developed a systematic and scientifically optimized method for this protocol. In this study, a novel method based on response surface methodology (RSM) was successfully developed to optimize the conditions of TB-EPS extraction. The optimal conditions were determined at an ultrasound time of 1.00 min, ultrasonic density of 5.59 W/mL and a mixed liquor suspended solid (MLSS) of around 1700-1800 mg/L. Furthermore, combined analysis of microscopy, particle size, and excitation-emission matrix (EEM) was successfully applied to evaluate the optimal conditions. The result indicates that the optimal conditions are efficient, reliable, and reproducible which effectively solves the bottleneck problem of the conflict between cell viability and TB-EPS yield, and little effect on its chemical structure.