2.5. Lubricant Oil Biodegradation Tests

Starter cultures and inocula were prepared according to Laczi et al. [54]. Cells were inoculated in 1% (v v⁻¹) into 160 mL serum vials (Merck KGaA, Germany) containing fresh minimal medium (MM: 0.68 g L⁻¹ KH₂PO₄, 0.87 g L⁻¹ K₂HPO₄, 0.58 g L⁻¹ NaCl, 0.125 g L⁻¹ MgSO₄ × 7H₂O, 0.044 g L⁻¹ CaCl₂ × 2H₂O, 1.2 g L⁻¹ NH₄NO₃, 0.014 g L⁻¹ FeSO₄, 0.0093 g L⁻¹ EDTA, 0.0002 g L⁻¹ ZnSO₄ × 7H₂O, 0.00006 g L⁻¹ MnCl₂ × 7H₂O, 0.0006 g L⁻¹ H₃BO₄, 0.0004 g L⁻¹ CoCl₂ × 6H₂O, 0.00002 g L⁻¹ CuCl₂ × 2H₂O, 0.00004 g L⁻¹ NiCl₂ × 6H₂O and 0.000046 g L⁻¹ NaMoO₄ × 6H₂O) [53] supplemented with 1% (m v⁻¹) fresh MK8 lubricant oil at a final volume of 20 mL aqueous phase and a 140 mL headspace. Cell-free samples were used as controls. All vials were capped with butyl rubber septa, then incubated in a rotary shaker (160 rpm at 28 °C) for 16 days. Since MK8 lubricant oil was used as the sole carbon source in each vial, increasing the CO₂ level was considered as an indirect measure for LO biodegradation. Microbial respiration was measured with gas chromatography as described below. At the end of the experiment, remaining LO was extracted using 10 mL diethyl ether and measured gravimetrically [57].