Enhancement of waste activated sludge hydrolysate digestion efficiency via promotion of direct interspecies electron transfer.

Anaerobic digestion (AD) of waste activated sludge (WAS) yields low methane (CH₄) because extracellular polymeric substances hinder hydrolysis and limit its biodegradability. Pretreatment methods such as alkaline or mechanical disruption can enhance solubilization; however, the resulting hydrolysate often contains recalcitrant compounds that inhibit further degradation. In this study, a combination of alkali along with ultrasonication was applied to enhance the solubilization of WAS, followed by CH₄ production under AD. Pretreated results showed alkaline (pH = 12) + ultrasonication (30 min) showed 300 % higher solubilization compared to ultrasonication (60 min) alone. Batch experiments (with and without Fe₃O₄) were conducted, and the results showed that pretreated hydrolysate supplemented Fe₃O₄ showed higher CH₄ yield than their control counterparts (up to 85 %). To validate the batch results of pretreated hydrolysate, a continuous operation was conducted without (Control) and with an electric voltage reactor (EVR) at different organic loading rates (OLR) up to 4 g chemical oxygen demand (COD)/L/d. The results showed that EVR enhanced the CH₄ production by 28 % and COD removal by 19 % at 4 g COD/L/d compared to the control. Microbial community analysis highlighted the dominance of Syntrophomonas zehnderi (a fatty acid oxidizer) in EVR, which increased by 27 %, suggesting stronger syntrophic partnerships with methanogens. Genetic profiling further supported these findings, showing a 25 % upregulation in Adenosine triphosphatease related genes and a striking 69 % increase in pili-associated genes, both critical for direct interspecies electron transfer. These results demonstrated that the combined pretreatment (alkali + ultrasonication) offers a promising alternative for enhanced AD of WAS.