High Cell Density Cultivation of Escherichia coli DH5α in Shake Flasks with a New Formulated Medium

2021 ◽  
Vol 2 (10) ◽  
pp. 01-11
Author(s):  
Wenfa Ng
Keyword(s):  
Cell Density ◽  
Optical Density ◽  
Yeast Extract ◽  
Glucose Concentration ◽  
High Cell Density ◽  
Lag Phase ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
Biomass Formation ◽  
E Coli

High cell density cultivation necessitates cell division and biomass formation, the mechanisms of which remain poorly understood, especially from the cellular energetics perspective. Specifically, the sensing of energy abundance and the channelling of nutritional energy into biomass formation and cell maintenance remains enigmatic at the sensory, effector and decision levels. Thus, optimization of cell growth remains an iterative trial and error process where the principal parameters are growth medium composition and incubation temperature. In this study, a new semidefined formulated medium was shown to be useful for high cell density cultivation of Escherichia coli DH5α (ATCC 53868). Comprising K2HPO4, 12.54; KH2PO4, 2.31; D-Glucose, 4.0; NH4Cl, 1.0; Yeast extract, 12.0; NaCl, 5.0; MgSO4, 0.24; the medium possessed a high capacity phosphate buffer able to moderate pH fluctuations during cell growth known to be detrimental to biomass formation. With glucose and NH4Cl providing the nutrients for initial growth, followed by a lag phase of 3 hours, a maximal optical density of 12.0 was obtained after 27 hours of cultivation at 37 oC and 230 rpm. Yeast extract provides a secondary source of carbon and nitrogen. Maximal optical density obtained in formulated medium was higher than the 10.1, 4.2, and 3.4 obtained in Tryptic Soy Broth, M9 with 1 g/L of yeast extract, and LB Lennox, respectively. Cultivation of E. coli DH5α in formulated medium with 6 g/L of glucose resulted in a longer lag phase of 8 hours and a longer time (68 hours) to attainment of maximal optical density, which marked the upper limit of glucose concentration beyond which biomass formation would be reduced. Specifically, glucose concentration above 6 g/L markedly reduced biomass formation possibly due to the environmental stress arising from low pH in the culture broth. Glucose concentration below 4 g/L, on the other hand, reduced biomass formation through a smaller pool of nutrients serving as biomass building blocks. Deviation from 1:1 molar ratio between glucose and NH4Cl was not detrimental to biomass formation and growth rates. Collectively, a semi-defined formulated medium could increase optical density of E. coli DH5α beyond that of LB Lennox and Tryptic Soy Broth, and may find use in cultivation of cells for applied microbiology research.

scholarly journals High cell density cultivation of Escherichia coli DH5α in shake flasks with a new formulated medium

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Cell Density ◽  
Optical Density ◽  
Yeast Extract ◽  
Glucose Concentration ◽  
High Cell Density ◽  
Lag Phase ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
Biomass Formation ◽  
E Coli

High cell density cultivation necessitates cell division and biomass formation, the mechanisms of which remain poorly understood, especially from the cellular energetics perspective. Specifically, the sensing of energy abundance and the channelling of nutritional energy into biomass formation and cell maintenance remains enigmatic at the sensory, effector and decision levels. Thus, optimization of cell growth remains an iterative trial and error process where the principal parameters are growth medium composition and incubation temperature. In this study, a new semi-defined formulated medium was shown to be useful for high cell density cultivation of Escherichia coli DH5α (ATCC 53868). Comprising K2HPO4, 12.54; KH2PO4, 2.31; D-Glucose, 4.0; NH4Cl, 1.0; Yeast extract, 12.0; NaCl, 5.0; MgSO4, 0.24; the medium possessed a high capacity phosphate buffer able to moderate pH fluctuations during cell growth known to be detrimental to biomass formation. With glucose and NH4Cl providing the nutrients for initial growth, followed by a lag phase of 3 hours, a maximal optical density of 12.0 was obtained after 27 hours of cultivation at 37 oC and 230 rpm. Yeast extract provides a secondary source of carbon and nitrogen. Maximal optical density obtained in formulated medium was higher than the 10.1, 4.2, and 3.4 obtained in Tryptic Soy Broth, M9 with 1 g/L of yeast extract, and LB Lennox, respectively. Cultivation of E. coli DH5α in formulated medium with 6 g/L of glucose resulted in a longer lag phase of 8 hours and a longer time (68 hours) to attainment of maximal optical density, which marked the upper limit of glucose concentration beyond which biomass formation would be reduced. Specifically, glucose concentration above 6 g/L markedly reduced biomass formation possibly due to the environmental stress arising from low pH in the culture broth. Glucose concentration below 4 g/L, on the other hand, reduced biomass formation through a smaller pool of nutrients serving as biomass building blocks. Deviation from 1:1 molar ratio between glucose and NH4Cl was not detrimental to biomass formation and growth rates. Collectively, a semi-defined formulated medium could increase optical density of E. coli DH5α beyond that of LB Lennox and Tryptic Soy Broth, and may find use in cultivation of cells for applied microbiology research.

scholarly journals High cell density cultivation of Escherichia coli DH5α in shake flasks with a new formulated medium

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Cell Density ◽  
Optical Density ◽  
Yeast Extract ◽  
Glucose Concentration ◽  
High Cell Density ◽  
Lag Phase ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
Biomass Formation ◽  
E Coli

High cell density cultivation necessitates cell division and biomass formation, the mechanisms of which remain poorly understood, especially from the cellular energetics perspective. Specifically, the sensing of energy abundance and the channelling of nutritional energy into biomass formation and cell maintenance remains enigmatic at the sensory, effector and decision levels. Thus, optimization of cell growth remains an iterative trial and error process where the principal parameters are growth medium composition and incubation temperature. In this study, a new semi-defined formulated medium was shown to be useful for high cell density cultivation of Escherichia coli DH5α (ATCC 53868). Comprising K2HPO4, 12.54; KH2PO4, 2.31; D-Glucose, 4.0; NH4Cl, 1.0; Yeast extract, 12.0; NaCl, 5.0; MgSO4, 0.24; the medium possessed a high capacity phosphate buffer able to moderate pH fluctuations during cell growth known to be detrimental to biomass formation. With glucose and NH4Cl providing the nutrients for initial growth, followed by a lag phase of 3 hours, a maximal optical density of 12.0 was obtained after 27 hours of cultivation at 37 oC and 230 rpm. Yeast extract provides a secondary source of carbon and nitrogen. Maximal optical density obtained in formulated medium was higher than the 10.1, 4.2, and 3.4 obtained in Tryptic Soy Broth, M9 with 1 g/L of yeast extract, and LB Lennox, respectively. Cultivation of E. coli DH5α in formulated medium with 6 g/L of glucose resulted in a longer lag phase of 8 hours and a longer time (68 hours) to attainment of maximal optical density, which marked the upper limit of glucose concentration beyond which biomass formation would be reduced. Specifically, glucose concentration above 6 g/L markedly reduced biomass formation possibly due to the environmental stress arising from low pH in the culture broth. Glucose concentration below 4 g/L, on the other hand, reduced biomass formation through a smaller pool of nutrients serving as biomass building blocks. Deviation from 1:1 molar ratio between glucose and NH4Cl was not detrimental to biomass formation and growth rates. Collectively, a semi-defined formulated medium could increase optical density of E. coli DH5α beyond that of LB Lennox and Tryptic Soy Broth, and may find use in cultivation of cells for applied microbiology research.

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

2015 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Yeast Extract ◽  
High Cell Density ◽  
Growth Media ◽  
Buffer System ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen

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.

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

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Yeast Extract ◽  
High Cell Density ◽  
Environmental Research ◽  
Buffer System ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen

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 OD600nm 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 OD600nm 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 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) 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.

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.

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. 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.

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/

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