Fructose-Induced bioenergetic surplus Unlocks fatty acid biosynthesis pathway dominance over reverse β-Oxidation: Mechanistic insights into High-Caproate production from food waste

Chain elongation (CE) is an effective strategy for converting organic wastes into value-added medium-chain fatty acids (MCFAs), wherein electron donors (EDs) dictate process efficiency. However, beyond substrate toxicity and limited reducing power, conventional EDs such as ethanol and lactate impose a chronic bioenergetic constraint: their minimal net ATP yield thermodynamically restricts CE strictly to the energy-neutral reverse β-oxidation (RBO) pathway. To overcome this bioenergetic bottleneck, this study investigated fructose as a high-energy-yielding multidimensional ED to drive n-caproate production from food waste in a mixed-culture system. Herein, the results demonstrated a dose-dependent enhancement of n-caproate, peaking at 12.38 g/L with a remarkable selectivity of 63.0 % (50 g/L fructose dosage). Mechanistically, fructose fermentation established an in-situ synergistic multi-ED microenvironment (fructose, ethanol, and lactate) that buffered toxicity and sustained robust reducing power. More critically, intensive glycolytic flux induced a hyper-energetic intracellular state characterized by abundant ATP and elevated NADH/NAD⁺ ratio. Meanwhile, the activities of key enzymes (e.g., phosphofructokinase and butyrate kinase) were significantly stimulated, redirecting carbon flow toward butyrate and n-caproate. This favorable energetic and metabolic environment further selectively enriched Limosilactobacillus spp., which glycolyzed fructose into essential carbon intermediates for CE. Finally, metagenomic profiling revealed that the fructose-induced ATP surplus profoundly enriched genes associated with the ATP-dependent fatty acid biosynthesis (FAB), while suppressing RBO-related genes. This uncovers a paradigm shift from the RBO-dominated route to a FAB-driven mechanism. These findings unravel how a targeted carbohydrate structurally rewires the thermodynamic hierarchy of CE pathways, providing novel mechanistic blueprints for upgrading complex organic wastes into high-value biochemicals.