Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 1

Sufficient quantities of cells of consistent characteristics are needed for studying biologicalprocesses (at the population level ) in many areas of applied microbiology. However, generating the requisite biomass by cell culture is usually the rate-limiting step of a project given the relatively low biomass yield of many commercial culture media in shake flask culture systems. This work reports the formulation of a semi-defined medium that enabled aerobic high cell density cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. The formulated medium (FM) comprises: a buffer system (K₂HPO₄ : 12.54 g/L and KH₂ PO₄ : 2.31 g/L); vitamins and trace elements (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 are: high buffer capacity (89 mM phosphate), 1:1 molar ratio between D-Glucose and NH₄Cl, and yeast extract providing trace elements and a secondary source of carbon and nitrogen. Preliminary data revealed an OD _600nm of 9 after 24 hours of cultivation at 37 ^oC, presumably with glucose and NH₄Cl as the main nutrients. At 48 hours, an OD _600nm of 11 was attained with yeast extract providing the necessary nutrients for cell growth and biomass formation. The broth’s pH varied between 5.5 and 7.8 during cultivation. On the other hand, the maximum OD _600nm of E. coli grown in three commonly used complex media: Nutrient Broth, LB Lennox, and Tryptic Soy Broth (TSB) were 1.4, 3.2 and 9.2, respectively, under identical culture conditions. Finally, FM maintained the viability of a larger population of cells for three days, compared to a population collapse in TSB broth after one day. Collectively, the results suggested that the formulated medium might find use as a high cell density aerobic growth medium for E. coli in shake flasks. Part 2 of this work describes improvements in medium performance ; specifically, higher cell yield as well as a shorter diauxic lag phase and total culture period achieved through a small reduction in D-Glucose and NH₄Cl concentrations in the medium composition. An abstract preprint of Part 2 is available at https://peerj.com/preprints/117/