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Phenotyping the quality of complex medium components by simple online-monitored shake flask experiments.

Diederichs S, Korona A, Staaden A, Kroutil W, Honda K, Ohtake H, Büchs J - Microb. Cell Fact. (2014)

Bottom Line: In cultivations with parallel offline analysis, the highest volumetric activity was found at different cultivation times.This work proves that cultivations conducted in complex media may be prone to significant variation in final product quality and quantity if the quality of the raw material for medium preparation is not thoroughly checked.In this study, the RAMOS technique enabled a reliable and reproducible screening and phenotyping of complex raw material lots by online measurement of the respiration activity.

View Article: PubMed Central - PubMed

Affiliation: AVT - Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany. sylvia.diederichs@avt.rwth-aachen.de.

ABSTRACT

Background: Media containing yeast extracts and other complex raw materials are widely used for the cultivation of microorganisms. However, variations in the specific nutrient composition can occur, due to differences in the complex raw material ingredients and in the production of these components. These lot-to-lot variations can affect growth rate, product yield and product quality in laboratory investigations and biopharmaceutical production processes. In the FDA's Process Analytical Technology (PAT) initiative, the control and assessment of the quality of critical raw materials is one key aspect to maintain product quality and consistency. In this study, the Respiration Activity Monitoring System (RAMOS) was used to evaluate the impact of different yeast extracts and commercial complex auto-induction medium lots on metabolic activity and product yield of four recombinant Escherichia coli variants encoding different enzymes.

Results: Under non-induced conditions, the oxygen transfer rate (OTR) of E. coli was not affected by a variation of the supplemented yeast extract lot. The comparison of E. coli cultivations under induced conditions exhibited tremendous differences in OTR profiles and volumetric activity for all investigated yeast extract lots of different suppliers as well as lots of the same supplier independent of the E. coli variant. Cultivation in the commercial auto-induction medium lots revealed the same reproducible variations. In cultivations with parallel offline analysis, the highest volumetric activity was found at different cultivation times. Only by online monitoring of the cultures, a distinct cultivation phase (e.g. glycerol depletion) could be detected and chosen for comparable and reproducible offline analysis of the yield of functional product.

Conclusions: This work proves that cultivations conducted in complex media may be prone to significant variation in final product quality and quantity if the quality of the raw material for medium preparation is not thoroughly checked. In this study, the RAMOS technique enabled a reliable and reproducible screening and phenotyping of complex raw material lots by online measurement of the respiration activity. Consequently, complex raw material lots can efficiently be assessed if the distinct effects on culture behavior and final product quality and quantity are visualized.

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Detailed characterization of selected auto-induction media withE. coliHBD. Characteristic growth parameters of E. coli BL21 (DE3) expressing 3-hydroxybutyryl-CoA dehydrogenase (HBD) in Overnight Express auto-induction medium (OnEx_1 and OnEx_2) and in auto-inducing TB+lactose medium (Roth_4). Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C. (A-C) Oxygen transfer rate (OTR), cell dry weight (CDW), and optical densitiy (OD). The respiration behavior is categorized into two patterns, as described in the caption of Figure 4. (D-F) pH-value and glycerol, lactose, glucose, and acetate concentrations. To improve data visualization, lactose concentration was multiplied with a factor of 5 and glucose concentration with a factor of 10, respectively. (G-I) Volumetric activity and protein content of recombinant protein per total protein.
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Fig6: Detailed characterization of selected auto-induction media withE. coliHBD. Characteristic growth parameters of E. coli BL21 (DE3) expressing 3-hydroxybutyryl-CoA dehydrogenase (HBD) in Overnight Express auto-induction medium (OnEx_1 and OnEx_2) and in auto-inducing TB+lactose medium (Roth_4). Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C. (A-C) Oxygen transfer rate (OTR), cell dry weight (CDW), and optical densitiy (OD). The respiration behavior is categorized into two patterns, as described in the caption of Figure 4. (D-F) pH-value and glycerol, lactose, glucose, and acetate concentrations. To improve data visualization, lactose concentration was multiplied with a factor of 5 and glucose concentration with a factor of 10, respectively. (G-I) Volumetric activity and protein content of recombinant protein per total protein.

Mentions: For an in-depth characterization of cultivation and enzyme expression in E. coli, additional RAMOS experiments were conducted in OnEx_1 medium, OnEx_2 medium, and TB+lactose medium with Roth_4 yeast extract. E. coli HBD was examined as representative of the four E. coli variants. The results for two other variants (E. coli GDH and E. coli ADH-A) are included as Additional files 2 and 3 in the appendix. Offline analysis was performed measuring biomass formation, carbon source consumption, acetate formation, pH, and expression of recombinant protein in conventional shake flasks under the same conditions. The results are presented in Figure 6.Figure 6


Phenotyping the quality of complex medium components by simple online-monitored shake flask experiments.

Diederichs S, Korona A, Staaden A, Kroutil W, Honda K, Ohtake H, Büchs J - Microb. Cell Fact. (2014)

Detailed characterization of selected auto-induction media withE. coliHBD. Characteristic growth parameters of E. coli BL21 (DE3) expressing 3-hydroxybutyryl-CoA dehydrogenase (HBD) in Overnight Express auto-induction medium (OnEx_1 and OnEx_2) and in auto-inducing TB+lactose medium (Roth_4). Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C. (A-C) Oxygen transfer rate (OTR), cell dry weight (CDW), and optical densitiy (OD). The respiration behavior is categorized into two patterns, as described in the caption of Figure 4. (D-F) pH-value and glycerol, lactose, glucose, and acetate concentrations. To improve data visualization, lactose concentration was multiplied with a factor of 5 and glucose concentration with a factor of 10, respectively. (G-I) Volumetric activity and protein content of recombinant protein per total protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4230760&req=5

Fig6: Detailed characterization of selected auto-induction media withE. coliHBD. Characteristic growth parameters of E. coli BL21 (DE3) expressing 3-hydroxybutyryl-CoA dehydrogenase (HBD) in Overnight Express auto-induction medium (OnEx_1 and OnEx_2) and in auto-inducing TB+lactose medium (Roth_4). Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C. (A-C) Oxygen transfer rate (OTR), cell dry weight (CDW), and optical densitiy (OD). The respiration behavior is categorized into two patterns, as described in the caption of Figure 4. (D-F) pH-value and glycerol, lactose, glucose, and acetate concentrations. To improve data visualization, lactose concentration was multiplied with a factor of 5 and glucose concentration with a factor of 10, respectively. (G-I) Volumetric activity and protein content of recombinant protein per total protein.
Mentions: For an in-depth characterization of cultivation and enzyme expression in E. coli, additional RAMOS experiments were conducted in OnEx_1 medium, OnEx_2 medium, and TB+lactose medium with Roth_4 yeast extract. E. coli HBD was examined as representative of the four E. coli variants. The results for two other variants (E. coli GDH and E. coli ADH-A) are included as Additional files 2 and 3 in the appendix. Offline analysis was performed measuring biomass formation, carbon source consumption, acetate formation, pH, and expression of recombinant protein in conventional shake flasks under the same conditions. The results are presented in Figure 6.Figure 6

Bottom Line: In cultivations with parallel offline analysis, the highest volumetric activity was found at different cultivation times.This work proves that cultivations conducted in complex media may be prone to significant variation in final product quality and quantity if the quality of the raw material for medium preparation is not thoroughly checked.In this study, the RAMOS technique enabled a reliable and reproducible screening and phenotyping of complex raw material lots by online measurement of the respiration activity.

View Article: PubMed Central - PubMed

Affiliation: AVT - Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany. sylvia.diederichs@avt.rwth-aachen.de.

ABSTRACT

Background: Media containing yeast extracts and other complex raw materials are widely used for the cultivation of microorganisms. However, variations in the specific nutrient composition can occur, due to differences in the complex raw material ingredients and in the production of these components. These lot-to-lot variations can affect growth rate, product yield and product quality in laboratory investigations and biopharmaceutical production processes. In the FDA's Process Analytical Technology (PAT) initiative, the control and assessment of the quality of critical raw materials is one key aspect to maintain product quality and consistency. In this study, the Respiration Activity Monitoring System (RAMOS) was used to evaluate the impact of different yeast extracts and commercial complex auto-induction medium lots on metabolic activity and product yield of four recombinant Escherichia coli variants encoding different enzymes.

Results: Under non-induced conditions, the oxygen transfer rate (OTR) of E. coli was not affected by a variation of the supplemented yeast extract lot. The comparison of E. coli cultivations under induced conditions exhibited tremendous differences in OTR profiles and volumetric activity for all investigated yeast extract lots of different suppliers as well as lots of the same supplier independent of the E. coli variant. Cultivation in the commercial auto-induction medium lots revealed the same reproducible variations. In cultivations with parallel offline analysis, the highest volumetric activity was found at different cultivation times. Only by online monitoring of the cultures, a distinct cultivation phase (e.g. glycerol depletion) could be detected and chosen for comparable and reproducible offline analysis of the yield of functional product.

Conclusions: This work proves that cultivations conducted in complex media may be prone to significant variation in final product quality and quantity if the quality of the raw material for medium preparation is not thoroughly checked. In this study, the RAMOS technique enabled a reliable and reproducible screening and phenotyping of complex raw material lots by online measurement of the respiration activity. Consequently, complex raw material lots can efficiently be assessed if the distinct effects on culture behavior and final product quality and quantity are visualized.

Show MeSH
Related in: MedlinePlus