From transient to persistent foaming: Interfacial characteristics of sludge-derived proteins and humic substances under organic overloading in anaerobic digestion.

The transition from transient to persistent foaming under organic overloading in waste activated sludge anaerobic digestion (AD) remains insufficiently understood. This study investigated foam evolution under increasing organic loading rates (OLRs) in a semi-continuous mesophilic digester treating thermal-alkaline-pretreated sludge, focusing on sludge-derived amphiphilic substances and their interfacial effects. Foam initiation occurred at an OLR of 5 g COD/(L·d), whereas persistent foaming was established at 7 g COD/(L·d). Increasing OLR promoted the accumulation of soluble extracellular polymeric substances, particularly proteins and humic-like substances, accompanied by a shift in protein composition toward higher hydrophobicity. The proportion of hydrophobic amino acids, mainly alanine and proline, increased from 29.8% to 35.7% during foam development, suggesting enhanced protein affinity for the gas-liquid interface. Humic substances contributed disproportionately to foam stabilization: 0.5 g/L achieved a foaming tendency comparable to 5.0 g/L sludge-derived proteins. This strong stabilizing effect was associated with lower critical micelle concentration, reduced surface tension, higher viscosity, slower drainage, and delayed bubble coarsening. Beyond physicochemical effects, foam-microbe feedback may occur: the hydrophobic niche favors foam-associated taxa (e.g., Thermovirga and Methanofastidiosum), which, though enriched primarily as an ecological response to overloading, may in turn influence foam stability. Persistent foaming in sludge AD results from organic overloading driving a transition to interfacial stabilization, where seemingly humic substances act as efficient foam stabilizers and hydrophobic amino acids contribute to the stabilization via interfacial adsorption. This mechanism informs a basis for OLR-based early warning and targeted foam control in full-scale digesters treating thermal-alkaline-pretreated waste activated sludge.