Sludge-derived phytohormones produced via alkaline thermal hydrolysis enhance stress-resilient growth and biodiesel production of limnetic microalgae in seawater-containing wastewater.

Sewage sludge represents a substantial reservoir of bioactive organic matter yet remains underutilized in high-value applications. Here, we developed an upcycling strategy that transforms sewage sludge into sludge-derived phytohormones (SPs) through alkaline thermal hydrolysis treatment (ATHT), enabling stress-resilient growth and biodiesel production of limnetic microalgae in seawater-containing wastewater. Under optimized ATHT conditions (160 °C, pH 12 and 120 min), organic matter was effectively solubilized, releasing 78 identifiable phytohormones including auxin, salicylic acid, cytokinin, and jasmonic acid, with total concentrations reaching 72,601.32 μg/L. Integrated FT-ICR MS and targeted metabolite analyses established precursor-intermediate-product relationships, demonstrating that phytohormones originated from the thermochemical conversion of sludge-derived proteins, lipids, and nucleic acids. Mechanistically, sludge macromolecules underwent depolymerization and reconstruction under thermal-alkaline conditions, producing phytohormone analogues through reactions including oxidative deamination, decarboxylation, hydroxylation, cyclization, and alkylation. Application of the SPs to Scenedesmus quadricauda cultivated in seawater-diluted liquid anaerobic digestate increased biomass production by 1.45-fold and lipid accumulation by 1.68-fold compared with controls. Transcriptomic analysis indicated that SPs activated pathways related to nutrient metabolism and photosynthetic carbon fixation, enhancing photosynthetic electron transport and energy supply. Simultaneously, SPs upregulated genes encoding acetyl-CoA carboxylase, fatty acid synthase, and diacylglycerol acyltransferase, channeling carbon flux toward lipid biosynthesis. Moreover, genes involved in antioxidant defense were downregulated, indicating mitigated oxidative stress. This work provides mechanistic insights into abiotic phytohormone formation from sludge and offers an integrated strategy linking sludge upcycling with microalgae-based biodiesel production.