Lactose Utilization and Growth Kinetics

Pure cultures were grown under static conditions at 30°C in 10 mL liquid YEL. After 72 h cells were harvested by centrifugation (14,000 rpm for 3 min), resuspended in sterile water to final OD₆₀₀ = 0.2. For qualitative evaluation of lactose utilization, 24 well microplates containing 990 μL of modified API50CH medium were inoculated with 10 μL of cell suspension prepared as above described. Microplates were incubated under anaerobic conditions (Anaerocult A system, Merck, Darmstadt, Germany) for 7 days at 30°C. The production of acid from lactose was determined based on bromocresol purple color change (de Freitas et al., 2015 (link)). For the evaluation of the kinetics of growth on lactose, cells were inoculated to a final OD₆₅₀ = 0.01 in 200 μL YELactose within 96 well microplates. For the assessment of growth kinetics on lactose plus lactate YELactose was added with increasing concentration of Na lactate (0.0, 1.5, 3.0, and 6.0 g L⁻¹). Growth was measured automatically every hour at OD₆₅₀ using a SPECTROstar nano microplate spectrophotometer (BMG Labtech, Ortenberg, Germany). Carrying capacity (final OD₆₅₀), specific growth rate (μ), lag phase duration (λ), and area under the curve (AUC) were determined by fitting the growth curves with the Bayesian non-parametric model developed by Tonner et al. (2020) (link).