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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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Related in: MedlinePlus

Cultivation ofE. coliunder non-induced conditions. Oxygen transfer rate during cultivation of two E. coli BL21 (DE3) variants carrying the genes for (A) 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD) and (B) glucose 1-dehydrogenase (E. coli GDH) in non-inducing TB medium with different yeast extracts as specified in Table 1. Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C.
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Fig2: Cultivation ofE. coliunder non-induced conditions. Oxygen transfer rate during cultivation of two E. coli BL21 (DE3) variants carrying the genes for (A) 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD) and (B) glucose 1-dehydrogenase (E. coli GDH) in non-inducing TB medium with different yeast extracts as specified in Table 1. Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C.

Mentions: Cultivations were conducted in the RAMOS device under non-inducing conditions in TB medium to evaluate the influence of the eight yeast extract lots (Table 1) on growth behavior of E. coli. Figure 2 shows the OTR of two recombinant E. coli variants harboring the gene for the 3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus (E. coli HBD, Figure 2A) and for the glucose 1-dehyrogenase from Sulfolobus solfataricus (E. coli GDH, Figure 2B). The experiments were performed in 250-mL RAMOS flasks containing 10 mL TB medium with different yeast extract lots. The shapes of all depicted OTR curves as function of time were similar reaching a maximum OTR between 70 and 80 mmol/L/h after an exponential increase. After a cultivation time of 5 to 7.5 h, the OTR dropped to about 5 to 10 mmol/L/h. At this time, all available carbon sources were depleted and no further respiration was possible. Cultivations in TB medium prepared with Merck yeast extract reached the lowest maximum OTR and the shortest duration of respiration activity for both investigated strains. Since the TB medium differed only in yeast extract lot, Merck yeast extract exhibited inferior growth properties as compared to the other lots tested.Table 1


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)

Cultivation ofE. coliunder non-induced conditions. Oxygen transfer rate during cultivation of two E. coli BL21 (DE3) variants carrying the genes for (A) 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD) and (B) glucose 1-dehydrogenase (E. coli GDH) in non-inducing TB medium with different yeast extracts as specified in Table 1. 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

Fig2: Cultivation ofE. coliunder non-induced conditions. Oxygen transfer rate during cultivation of two E. coli BL21 (DE3) variants carrying the genes for (A) 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD) and (B) glucose 1-dehydrogenase (E. coli GDH) in non-inducing TB medium with different yeast extracts as specified in Table 1. Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C.
Mentions: Cultivations were conducted in the RAMOS device under non-inducing conditions in TB medium to evaluate the influence of the eight yeast extract lots (Table 1) on growth behavior of E. coli. Figure 2 shows the OTR of two recombinant E. coli variants harboring the gene for the 3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus (E. coli HBD, Figure 2A) and for the glucose 1-dehyrogenase from Sulfolobus solfataricus (E. coli GDH, Figure 2B). The experiments were performed in 250-mL RAMOS flasks containing 10 mL TB medium with different yeast extract lots. The shapes of all depicted OTR curves as function of time were similar reaching a maximum OTR between 70 and 80 mmol/L/h after an exponential increase. After a cultivation time of 5 to 7.5 h, the OTR dropped to about 5 to 10 mmol/L/h. At this time, all available carbon sources were depleted and no further respiration was possible. Cultivations in TB medium prepared with Merck yeast extract reached the lowest maximum OTR and the shortest duration of respiration activity for both investigated strains. Since the TB medium differed only in yeast extract lot, Merck yeast extract exhibited inferior growth properties as compared to the other lots tested.Table 1

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