Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system
- Subject Areas
- Biochemistry, Bioengineering, Biotechnology, Environmental Sciences, Microbiology
- High cell density, Gram-negative, aerobic, shake flask, culture medium, bacteria
- © 2015 Ng et al.
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
- Cite this article
- 2015. Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system. PeerJ PrePrints 3:e1406v1 https://doi.org/10.7287/peerj.preprints.1406v1
Microbes in environmental studies should be cultured in growth media with characteristics as close to their original habitat as possible, and which also allows a high cell density to be attained for providing enough cells in subsequent experiments. This in-progress report describes the formulation of a medium with an environmentally-relevant composition, and which also 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 OD600nm of 9 was attained after 24 hours of cultivation at 37 oC. This phase of growth was largely fuelled by glucose and NH4Cl. After 48 hours, the OD600nm reached 11, which was fuelled by the 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 OD600nm 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) relative 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.
This is an extended abstract preprint describing research presented as an oral presentation at the 17th Regional Symposium on Chemical Engineering, 22-23 November 2010, Bangkok, Thailand.
Figure 1 and 2 of main text
Figure 1: Growth curve of E. coli in different media.Figure 2: Viable cell population of E. coli at various time points when cultivated in high cell density medium and Tryptic Soy Broth.