In situ sludge reduction induced by graphene oxide: Mechanistic insights into metabolic uncoupling, maintenance energy and cryptic growth.

Nanomaterials are increasingly recognized as stressors in biological wastewater treatment systems, yet the effects on biomass yield remain poorly understood. This study systematically evaluated the effects of low-dose graphene oxide (GO; 0.1 and 1 mg/L) on pollutant removal and sludge yield in activated sludge systems. At day 40, sludge yields were 0.356, 0.298, and 0.237 g VSS/g COD in the Control, 0.1 and 1 mg/L GO systems, respectively, corresponding to reductions of 16.29% (p = 0.055) and 33.43% (p < 0.05) without compromising nitrogen removal. GO exposure also loosened floc structure and increased mean intrafloc dissolved oxygen concentrations by 23.88% and 32.84%, respectively (both p < 0.01). Activities of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and the electron transport system increased, indicating intensified endogenous oxidative metabolism. Despite enhanced respiration, ATP production decreased by 19.48% (p < 0.05) and 27.95% (p < 0.01), suggesting uncoupling between oxidation and phosphorylation. Intracellular reactive oxygen species increased by 65.46% and 145.60% (both p < 0.01), respectively. The resulting oxidative stress increased maintenance energy demand and promoted cell death and cryptic growth. Metagenomic analysis further revealed enrichment of genes related to oxidative stress responses, macromolecular repair, and extracellular polymeric substance secretion, together with decrease of genes involved in cell replication and division. Collectively, enhanced intrafloc oxygen transfer and endogenous respiration, oxidation-phosphorylation uncoupling, increased maintenance energy demand, and cryptic growth jointly drove GO-induced sludge reduction.