scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 2

2017 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
Defined Medium ◽  
Lag Phase ◽  
Overflow Metabolism ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
Batch Mode ◽  
Shake Flasks ◽  
Diauxic Lag

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium (comprising salts, buffer components and small amount of carbon and nitrogen sources) help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains a comprehensive medium with all necessary nutrients for converting cells into biomass, and as a repository for secreted metabolites, some of which detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake flask is an optimization process with the aim to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximum optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth at 37 oC. After a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease together with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism (and associated negative effects on growth); thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration, which suggested that an optimum exist, beyond which occurrence of overflow metabolism would reduce cell yield and biomass formation. Part 1 of this work can be found at: https://peerj.com/preprints/115/

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 2

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
Defined Medium ◽  
Lag Phase ◽  
Overflow Metabolism ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
Batch Mode ◽  
Shake Flasks ◽  
Diauxic Lag

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium (comprising salts, buffer components and small amount of carbon and nitrogen sources) help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains, at the outset, a comprehensive medium with all necessary nutrients for conversion into biomass, and also serves as a repository for secreted metabolites - some of which are detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake flask is an optimization process with the aim to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximum optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth on glucose at 37 oC. After a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease along with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism (and associated negative effects on growth); thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration - which suggested that an optimum exist, beyond which occurrence of overflow metabolism would reduce cell yield and biomass formation.

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 2

2017 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
Defined Medium ◽  
Lag Phase ◽  
Overflow Metabolism ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
Batch Mode ◽  
Shake Flasks ◽  
Diauxic Lag

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium (comprising salts, buffer components and small amount of carbon and nitrogen sources) help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains a comprehensive medium with all necessary nutrients for converting cells into biomass, and as a repository for secreted metabolites, some of which detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake flask is an optimization process with the aim to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximum optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth at 37 oC. After a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease together with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism (and associated negative effects on growth); thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration, which suggested that an optimum exist, beyond which occurrence of overflow metabolism would reduce cell yield and biomass formation. Part 1 of this work can be found at: https://peerj.com/preprints/115/

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 2

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
Defined Medium ◽  
Lag Phase ◽  
Overflow Metabolism ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
Batch Mode ◽  
Shake Flasks ◽  
Diauxic Lag

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium – comprising salts, buffer components and small amount of carbon and nitrogen sources - help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains, at the outset, a comprehensive medium with all necessary nutrients for conversion into biomass, and also serves as a repository for secreted metabolites - some of which are detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake flask is an optimization process with the aim to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximal optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth on glucose at 37 oC. Following a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease along with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism - and associated negative effects on growth - thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration - which suggested that an optimum exist, beyond which occurrence of overflow metabolism would reduce cell yield and biomass formation.

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system: Part 2

2013 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Cell Density ◽  
Shake Flask ◽  
High Cell Density ◽  
Defined Medium ◽  
Lag Phase ◽  
Overflow Metabolism ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
Batch Mode ◽  
Diauxic Lag

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium – comprising salts, buffer components and small amount of carbon and nitrogen sources - help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains, at the outset, a comprehensive medium with all necessary nutrients for conversion into biomass and, also serves as a repository for secreted metabolites - some of which are detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake-flask is an optimization process aiming to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium (Wenfa Ng, 2013, https://peerj.com/preprints/115v1) for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flask. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximal optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth on glucose at 37 oC. Following a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease along with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism - and associated negative effects on growth - thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration - which suggested that an optimum exist, beyond which, occurrence of overflow metabolism would reduce cell yield and biomass formation.

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system: Part 2

2015 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Cell Density ◽  
Shake Flask ◽  
High Cell Density ◽  
Defined Medium ◽  
Lag Phase ◽  
Overflow Metabolism ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
Batch Mode ◽  
Diauxic Lag

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium – comprising salts, buffer components and small amount of carbon and nitrogen sources - help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains, at the outset, a comprehensive medium with all necessary nutrients for conversion into biomass and, also serves as a repository for secreted metabolites - some of which are detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake-flask is an optimization process aiming to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flask. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximal optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth on glucose at 37 oC. Following a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease along with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism - and associated negative effects on growth - thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration - which suggested that an optimum exist, beyond which, occurrence of overflow metabolism would reduce cell yield and biomass formation.

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

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Yeast Extract ◽  
High Cell Density ◽  
Defined Medium ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Shake Flasks

Sufficient quantities of cells of consistent characteristics are needed for studying biological processes (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 flasks. 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 KH2PO4: 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 that an OD600nm of 9 was attained after 24 hours of cultivation at 37 oC, with glucose and NH4Cl as the main nutrients. At 48 hours, the OD600nm reached a maximum value of 11 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. For comparison, the maximum OD600nm 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 observed in TSB after one day. Collectively, the present findings 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/

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

2017 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Yeast Extract ◽  
High Cell Density ◽  
Defined Medium ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Shake Flasks

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/

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system: Part 1

2013 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Cell Density ◽  
Yeast Extract ◽  
Shake Flask ◽  
High Cell Density ◽  
Defined Medium ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Shake Flasks

Sufficient quantities of cells of consistent characteristics are needed for studying processes - at the population level and beyond - in many areas of applied microbiology research. Nevertheless, given the relatively low biomass yield of many commercial culture media in shake flasks, producing the requisite biomass by cell culture is generally the rate-limiting step. 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 KH2PO4: 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 that an OD600nm of 9 was attained after 24 hours of cultivation at 37 oC – most probably fuelled by glucose and NH4Cl. At 48 hours, the OD600nm reached a maximal value of 11 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. For comparison, the maximum OD600nm 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 for three days - compared to a population collapse observed in TSB after one day. Taken together, the present findings suggest that the formulated medium may find use as a high cell density aerobic growth medium for E. coli in shake flask.

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system: Part 1

2015 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Cell Density ◽  
Yeast Extract ◽  
Shake Flask ◽  
High Cell Density ◽  
Defined Medium ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Shake Flasks

Sufficient quantities of cells of consistent characteristics are needed for studying processes - at the population level and beyond - in many areas of applied microbiology research. Nevertheless, given the relatively low biomass yield of many commercial culture media in shake flasks, producing the requisite biomass by cell culture is generally the rate-limiting step. 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 KH2PO4: 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 that an OD600nm of 9 was attained after 24 hours of cultivation at 37 oC – most probably fuelled by glucose and NH4Cl. At 48 hours, the OD600nm reached a maximal value of 11 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. For comparison, the maximum OD600nm 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 for three days - compared to a population collapse observed in TSB after one day. Taken together, the present findings suggest that the formulated medium may find use as a high cell density aerobic growth medium for E. coli in shake flask. 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. A preprint of the work is available at https://peerj.com/preprints/117v1.

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