Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system

Microbes for environmental research should be cultured in growth media with characteristics as close to their original habitat as possible and, which also allows high cell density to be obtained - needed for providing sufficient cells in subsequent experiments. This report describes the formulation of a medium with an environmentally-relevant composition, and that affords aerobic high cell density cultivation of Escherichia coli DH5α in shake flasks. The formulated medium comprises four components: a buffer system (K₂HPO₄: 12.54 g/L and KH₂PO₄: 2.31 g/L), vitamins (yeast extract: 12.0 g/L), salts (NaCl: 5.0 g/L and MgSO₄: 0.24 g/L), and carbon and nitrogen sources (D-Glucose: 6.0 g/L and NH₄Cl: 1.5 g/L). Notable characteristics of this medium were: high capacity phosphate buffer system (89 mM phosphate); 1:1 molar ratio between D-Glucose and NH₄Cl; and yeast extract providing trace elements and a secondary carbon and nitrogen source. Growth experiments revealed that an OD_600nm of 9 was attained after 24 hours of cultivation at 37 ^oC. This phase of growth was most probably fuelled by glucose and NH₄Cl. After 48 hours, the OD_600nm reached 11, which was likely fuelled by a mixture of carbohydrates, lipids and proteins in yeast extract. Broth’s pH varied between 5.5 and 7.8 during cultivation, which was in the range conducive for growth of E. coli. In comparison, the OD_600nm of E. coli reached 1.4, 3.2, and 9.2 for three commonly used complex media; Nutrient Broth, LB Lennox, and Tryptic Soy Broth, respectively, over 48 hours under identical culture conditions. In addition, the formulated medium was able to maintain a large viable cell population for a longer period of time (three days) compared to Tryptic Soy Broth. Thus, preliminary data suggested that the formulated medium holds potential for use as a high cell density aerobic growth medium for Gram-negative bacteria.