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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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Comparison of oxygen transfer rates of allE. colivariants in selected auto-induction media. Oxygen transfer rate during cultivation of E. coli BL21 (DE3) variants expressing different recombinant target proteins (3-hydroxybutyryl-CoA dehydrogenase (HBD), glucose 1-dehydrogenase (GDH), HBD-GDH, and alcohol dehydrogenase A (ADH-A)) in Overnight Express auto-induction medium ((A) OnEx_1 and (B) OnEx_2) and (C) auto-inducing TB+lactose medium (Roth_4). The respiration behavior is categorized into two patterns, as described in the caption of Figure 4. Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C.
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Fig5: Comparison of oxygen transfer rates of allE. colivariants in selected auto-induction media. Oxygen transfer rate during cultivation of E. coli BL21 (DE3) variants expressing different recombinant target proteins (3-hydroxybutyryl-CoA dehydrogenase (HBD), glucose 1-dehydrogenase (GDH), HBD-GDH, and alcohol dehydrogenase A (ADH-A)) in Overnight Express auto-induction medium ((A) OnEx_1 and (B) OnEx_2) and (C) auto-inducing TB+lactose medium (Roth_4). The respiration behavior is categorized into two patterns, as described in the caption of Figure 4. Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C.

Mentions: To further confirm the impact of complex medium components on other E. coli expression systems, the trend of respiration behavior as function of time is compared for all recombinant E. coli variants (listed in Figure 1) in three different auto-induction media in Figure 5. It needs to be added that the ADH-A is mainly produced in inclusion bodies under the investigated cultivation conditions [45]. In Figure 5A, OTR curves in OnEx_1 medium are depicted. Besides E. coli ADH-A, the respiration behavior of all variants could be assigned to the second pattern, since the OTR was exponentially increasing until the maximum oxygen transfer capacity of the system was reached. E. coli ADH-A showed essentially the same respiration behavior except for the first OTR increase to only 55 mmol/L/h. Figure 5B depicts the results of cultivation in OnEx_2 medium. The OTR curves of all variants clearly correlate to the first pattern of respiration behavior having a first OTR plateau between 35 to 50 mmol/L/h followed by a second increase to approx. 70 mmol/L/h. In Figure 5C, the respiration behavior is shown for TB+lactose medium prepared with Roth_4 yeast extract. All OTR curves exhibited respiration behavior belonging to the second pattern.Figure 5


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)

Comparison of oxygen transfer rates of allE. colivariants in selected auto-induction media. Oxygen transfer rate during cultivation of E. coli BL21 (DE3) variants expressing different recombinant target proteins (3-hydroxybutyryl-CoA dehydrogenase (HBD), glucose 1-dehydrogenase (GDH), HBD-GDH, and alcohol dehydrogenase A (ADH-A)) in Overnight Express auto-induction medium ((A) OnEx_1 and (B) OnEx_2) and (C) auto-inducing TB+lactose medium (Roth_4). The respiration behavior is categorized into two patterns, as described in the caption of Figure 4. Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Comparison of oxygen transfer rates of allE. colivariants in selected auto-induction media. Oxygen transfer rate during cultivation of E. coli BL21 (DE3) variants expressing different recombinant target proteins (3-hydroxybutyryl-CoA dehydrogenase (HBD), glucose 1-dehydrogenase (GDH), HBD-GDH, and alcohol dehydrogenase A (ADH-A)) in Overnight Express auto-induction medium ((A) OnEx_1 and (B) OnEx_2) and (C) auto-inducing TB+lactose medium (Roth_4). The respiration behavior is categorized into two patterns, as described in the caption of Figure 4. Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C.
Mentions: To further confirm the impact of complex medium components on other E. coli expression systems, the trend of respiration behavior as function of time is compared for all recombinant E. coli variants (listed in Figure 1) in three different auto-induction media in Figure 5. It needs to be added that the ADH-A is mainly produced in inclusion bodies under the investigated cultivation conditions [45]. In Figure 5A, OTR curves in OnEx_1 medium are depicted. Besides E. coli ADH-A, the respiration behavior of all variants could be assigned to the second pattern, since the OTR was exponentially increasing until the maximum oxygen transfer capacity of the system was reached. E. coli ADH-A showed essentially the same respiration behavior except for the first OTR increase to only 55 mmol/L/h. Figure 5B depicts the results of cultivation in OnEx_2 medium. The OTR curves of all variants clearly correlate to the first pattern of respiration behavior having a first OTR plateau between 35 to 50 mmol/L/h followed by a second increase to approx. 70 mmol/L/h. In Figure 5C, the respiration behavior is shown for TB+lactose medium prepared with Roth_4 yeast extract. All OTR curves exhibited respiration behavior belonging to the second pattern.Figure 5

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