Evaluation of Phenol Degradation

Cultures grown in LB test tubes were gradiently diluted from 0.1 to 10^–6. Then 5 μL cultures were spotted onto MM plates (1.5% agar) containing phenol at concentrations ranging from 0 to 1.1 g/L. The growth phenotype of A. lwoffii NL1 was observed after 2 days to assess phenol tolerance.

Phenol degradation under different culture conditions was evaluated in liquid MM medium with phenol as the sole carbon source. Single colonies were first activated in LB test tubes at 28°C and 200 rpm for 36 h. Cells were washed and appropriately diluted using sterile K₃PO₄ buffer and then incubated in 50 mL of MM medium with phenol as the sole carbon source. Cultivation parameters, including initial phenol concentration (0, 0.2, 0.5, 0.6, and 0.7 g/L), inoculum volume (v/v, 2%, 5%, 8%, 10%, 12%, and 15%), initial pH (5, 6, 7, 8, 9, and 10), and temperature (25°C, 38°C, 30°C, 33°C, 35°C, 37°C, and 40°C) were analyzed separately to observe the influence on cell growth and phenol utilization in strain NL1. Bacterial growth was assessed by the optical density (OD) of biomass measured at 600 nm using a spectrophotometer, and phenol concentrations were monitored by the colorimetric assay using 4-aminoantipyrine (King et al., 1991). The results were expressed as the means standard error of the mean from three repetitions using the Microsoft Office Excel’s data analysis tool (2019 version).