Synergistic effects of glycerol and lactose on propionic acid production by Cutibacterium acnes ZW-1: Insights from untargeted metabolomics

Biodiesel production from dairy wastewater yields crude glycerol containing residual lactose, forming co-substrates suitable for microbial fermentation. This work converts these residues into propionic acid (PA) using Cutibacterium acnes ZW-1 and clarifies how a small lactose fraction reshapes metabolism. ZW-1 was cultivated anaerobically on a 90 % glycerol-10 % lactose co-substrate and on a 100 % glycerol control. The co-substrate system reached 4.36 g/L PA after 14 days, 1.20 g/L higher than the control, and doubled the maximum production rate from 0.39 to 0.80 g/L/d. Lactose addition increased biomass (OD 600 from 0.62 to 0.74), lowered the final pH (5.14–4.68) and raised the intracellular NADH/NAD⁺ ratio from 2.9 to 4.0. Activities of pyruvate carboxylase and lactate dehydrogenase increased by 1.8- and 2.1-fold, indicating strengthened anaplerotic and redox-balancing routes. Untargeted metabolomics identified 454 differential metabolites; galactose metabolism and the pentose phosphate pathway were markedly upregulated, while biotin derivatives, pyrimidine nucleotides, pyridine compounds and fatty acyls showed positive correlations with PA yield. Overall, a modest lactose supplement unlocks more efficient glycerol fermentation and provides a mechanistically supported route for upgrading dairy-related byproducts to value-added PA.