Geobacillus thermoglucosidans DSM2542 is an industrially important microbe, however the complex nutritional requirements of Geobacilli confound metabolic engineering efforts. Previous studies have utilised semi-defined media recipes that contain complex, undefined, biologically derived nutrients which have unknown ingredients that cannot be quantified during metabolic profiling. This study used design of experiments to investigate how individual nutrients and interactions between these nutrients contribute to growth. A mathematically derived defined medium has been formulated that has been shown to robustly support growth of G . thermoglucosidans in two different environmental conditions (96-well plate and shake flask) and with a variety of lignocellulose-based carbohydrates. This enabled the catabolism of industrially relevant carbohydrates to be investigated.