The refinement of biodiesel or renewable diesel from bacterial lipids has a great potential to make a contribution for energy production in the future. This study provides new data concerning suitable nutrient concentrations for cultivation of the Gram-positive Rhodococcus opacus PD630, which is able to accumulate large amounts of lipids during nitrogen limitation. Enhanced concentrations of magnesium have been shown to increase the final optical density and the lipid content of the cells. Elevated phosphate concentrations slowed down the onset of the accumulation phase, without a clear effect on the final optical density and the cell’s lipid content. A robust growth of R. opacus was possible in the presence of ammonium concentrations of up to 1.4 g l-1 and sucrose concentrations of up to 240 g l-1, with an optimum regarding growth and lipid storage observed in the range of 0.2 to 0.4 g l-1 ammonium and 20 to 40 g l-1 sucrose, respectively. Moreover, R. opacus showed tolerance to high salt concentrations.