Phenotyping the quality of complex medium components by simple online-monitored shake flask experimentsReport as inadecuate




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Microbial Cell Factories

, 13:149

First Online: 07 November 2014Received: 26 June 2014Accepted: 15 October 2014

Abstract

BackgroundMedia 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.

ResultsUnder 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.

ConclusionsThis 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.

KeywordsRAMOS OTR Yeast extract Complex medium E. coli Auto-induction Screening Lactose PAT AbbreviationsFDAFood and Drug Administration

PATProcess Analytical Technology

E. coliEscherichia coli

LB mediumLysogeny broth medium

TB mediumTerrific broth medium

RAMOSRespiration Activity Monitoring System

OTROxygen transfer rate

HBD3-hydroxybutyryl-CoA dehydrogenase

GDHGlucose 1-dehydrogenase

ADH-AAlcohol dehydrogenase A

OnEx mediumOvernight Express auto-induction medium

CDWCell dry weight

ODOptical density

Electronic supplementary materialThe online version of this article doi:10.1186-s12934-014-0149-5 contains supplementary material, which is available to authorized users.

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Author: Sylvia Diederichs - Anna Korona - Antje Staaden - Wolfgang Kroutil - Kohsuke Honda - Hisao Ohtake - Jochen Büchs

Source: https://link.springer.com/



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