Excess sludge contains large amounts of protein, which accounts for about 20%-60% of dry sludge content (Chen et al., 2007; Vinay and Shang-Lien, 2013; Wang et al., 2019). Protein extracted from excess sludge can be used to prepare protein foaming agents (Su et al., 2015), feed supplements (Chirwa and Lebitso, 2014; Jiyeon et al., 2008) and foliar fertilizers (Liu et al., 2009) and thus allow the effective utilization of sludge with high added value.
Enzymatic hydrolysis (EH) is a potential technology for protein recovery from excess sludge. EH offers advantages of mild reaction conditions and prevention of protein denaturation, and it is an environmentally friendly alternative to the popular alkali thermal process. To enhance the protein extraction efficiency, ultrasonic pretreatment was introduced before EH and successfully achieved significant improvements in protein extraction. Yan et al. (2020) used 1 W/mL ultrasonication for 20 min to pretreat sludge protein before extraction by alkaline protease, and the protein recovery rate was approximately 20% higher than that of enzymatic hydrolysis alone.
The application of sludge protein is closely related to the contents of peptides (e.g., foaming agents) and amino acids (e.g., foliar fertilizer) in protein recovery. Ultrasound-assisted enzymatic hydrolysis (UEH) can not only promote the hydrolysis of sludge protein extraction but also affect the further hydrolysis of extracted protein into peptides and amino acids. Therefore, it is necessary to understand the changes in protein and its hydrolysed products during UEH.
Most of the proteins are found inside the sludge cell, and only approximately 20% of the proteins exist in the extracellular polymeric substance (EPS) (Yixin et al., 2022); thus, sludge wall breaking is critical for protein extraction. Ultrasonic stimulation of sludge hydrolysis has two main functions. Lu et al. (2015) believed that the strong shear forces and cavitation of ultrasonic irradiation effectively destroy the EPS in sludge (Tyagi et al., 2014) so that the enzyme mixes with sludge more thoroughly, thus improving the efficiency of enzyme action. Tian (2017) argued that ultrasonic shear forces and sonochemical action could break the cells and release their contents. To quantitatively understand the mechanism of UEH, it is necessary to study the extraction of intracellular proteins by EH with and without ultrasonic assistance.
The work of this study are to: i) the protein transformation process during EH and UEH and ultrasonic pretreatment was compared, and the kinetic equation of the important intermediate peptide during hydrolysis was elucidated; ii) EPS protein and intracellular protein were separately extracted, and the proteins extracted by separate hydrolysis and integral hydrolysis were compared to analyse the influence of ultrasound on EPS barrier effects; and iii) the intracellular protein contents of EH and UEH and their hydrolysates of peptide and amino acid were detected to analyse the influence of ultrasound on intracellular protein extraction and proteolysis. The findings of this study are expected to provide a theoretical foundation and application basis for the green and efficient UEH method for sludge protein extraction.