Combined lysine and cobalt supplementation improves semi-thermophilic anaerobic digestion performance with enhanced Methanosarcina-associated methylotrophic potential.

Protein-rich food waste challenges anaerobic digestion (AD) through rapid acidification and chronic ammonia stress. Semi-thermophilic AD (STAD, 41-49℃) offers a promising balance between mesophilic stability and thermophilic conversion efficiency, but further improvement may depend on strengthening methylotrophic methanogenesis, a route better aligned with the methylamine-forming potential of this substrate. l-lysine and cobalt were therefore selected as targeted additives to support its key methyl-transfer step. Their enhancement effects were evaluated through a series of experiments. Initial tests across different temperatures showed that STAD outperformed mesophilic and thermophilic digestion in both methane production and process stability, and combined supplementation gave the strongest enhancement. Under STAD, combined addition increased methane production by 58.0%, reduced volatile fatty acids (VFAs) and free ammonia by 24.7% and 21.9%, respectively, and strengthened Methanosarcina-linked methylotrophic signatures. Further optimization under STAD showed that intermediate doses performed best, and the predicted optimum, 45 mg·L-1l-lysine and 3.5 mg·L-1 cobalt, was validated in a continuous reactor. This combination increased methane yield (314.21 ± 42.35 mL·gVS-1·d-1) by 33.3%, reduced VFAs and residual soluble chemical oxygen demand, by 29.6% and 44.8%, respectively, without aggravating ammonia stress. It also showed favorable preliminary economic potential, with a benefit-cost ratio of ∼15.0 during subsequent maintenance dosing. Mechanistically, these effects were linked to enrichment of hydrolytic, fermentative, and syntrophic bacteria, reduced competition from non-methylotrophic taxa, and more favorable conditions for Methanosarcina-centered, potentially methylamine-utilizing pathways. Overall, this study provides a practical strategy with clear engineering potential to further strengthen STAD for food-waste treatment.