Reassessing the out‐of‐phase phenomenon in shake flasks by evaluating the angle‐dependent liquid distribution relative to the direction of the centrifugal acceleration

Abstract

Shake flasks are still the most relevant experimental tool in the development of viscous fermentation processes. The phase number, which defines the onset of the unfavorable out‐of‐phase (OP) phenomenon in shake flasks, was previously defined via specific power input measurements. In the OP state, the bulk liquid no longer follows the orbital movement of the imposed centrifugal force, which is for example, detrimental to oxygen transfer. In this study, an optical fluorescence technique was used to measure the three‐dimensional liquid distribution in shake flasks. Four new optically derived evaluation criteria for the phase transition between the in‐phase and OP condition were established: (a) thickness of the liquid film left on the glass wall by the rotating bulk liquid, (b) relative slope of the leading edge of bulk liquid (LB) lines, (c) trend of the angular position of LB, and (d) very high angular position of the leading edge. In contrast to the previously applied power input measurements, the new optical evaluation criteria describe the phase transition in greater detailed. Instead of Ph = 1.26, a less conservative value of Ph = 0.91 is now suggested for the phase transfer, which implies a broader operating window for shake flask cultivations with higher viscosities.

Understanding and controlling filamentous growth of fungal cell factories: novel tools and opportunities for targeted morphology engineering

“…Due to the high number of genes to be analysed and the heterogeneous character of fungal macromorphological populations in a bioreactor, there is an increased need for the development of parallel multibioreactor systems at the millilitre scale that allow for high-throughput and that generate reproducible cultivation data with low variance. The development and commercialization of several online monitoring tools for shaken flasks (Respiration Activity MOnitoring System, RAMOS) and microtiter plates (µRAMOS, BioLector) are suitable techniques that allow deep insight into the metabolic state of microbial cultivations and are suitable as scaling parameters that can be determined at an early stage of process development with filamentous microorganisms [ 53 – 55 ].…”

Characterization of hydrodynamics and volumetric power input in microtiter plates for the scale‐up of downstream operations

“…These are met when the Froude number is below 0.4 and the Phase number, Ph, is below 1.26. The Ph was later adjusted to 0.91 (Azizan et al, 2019 ). It can be calculated via: …”

“…Büchs et al ( 2000 ) established the dimensionless Phase number (Ph), the related Reynolds number ( Re ), and a minimum Froude number (Fr) of 0.4 as engineering characteristics for mixing in shaken flasks. Ph considers the ratio of the shaking diameter and the working volume to the inner diameter of the reactor and gives Ph>0.91 as the characteristic for the “in‐phase” conditions which verifies homogenous flow and optimal mixing (Azizan et al, 2019 ). However, it does not seem to be the only criterion to ensure in‐phase conditions, as Ducci and Weheliye ( 2014 ) considered that the in‐phase conditions are dependent on the positioning of the velocity vector rather than the Ph number.…”

Implementation of Perforated Concentric Ring Walls Considerably Improves Gas-Liquid Mass Transfer of Shaken Bioreactors

“…This even includes pH control and, thus, by reducing buffer concentrations, similar osmotic conditions as commonly used in stirred tank bioreactors can be applied ( Scheidle et al, 2011 ; Philip et al, 2018 ). 4) The extensive characterization with respect to engineering parameters such as power input ( Büchs et al, 2000a ; Büchs et al, 2000b ; Büchs et al, 2001 ; Peter et al, 2006a ; Peter et al, 2006b ), mixing times ( Tan et al, 2011 ), hydrodynamics ( Büchs et al, 2001 ; Peter et al, 2004 ; Büchs et al, 2007 ; Tianzhong et al, 2010 ; Liu et al, 2016 ; Palacios-Morales et al, 2016 ; Azizan and Büchs, 2017 ; Azizan et al, 2019 ), and gas-liquid mass transfer ( Van Suijdam et al, 1978 ; Henzler and Schedel, 1991 ; Maier and Büchs, 2001 ; Maier et al, 2004 ; Giese et al, 2014 ) enables good and reliable experimental designs. 5) Due to surface aeration, complicating phenomena that usually come along with bubble aeration are avoided.…”

“…4) The extensive characterization with respect to engineering parameters such as power input ( Büchs et al, 2000a ; Büchs et al, 2000b ; Büchs et al, 2001 ; Peter et al, 2006a ; Peter et al, 2006b ), mixing times ( Tan et al, 2011 ), hydrodynamics ( Büchs et al, 2001 ; Peter et al, 2004 ; Büchs et al, 2007 ; Tianzhong et al, 2010 ; Liu et al, 2016 ; Palacios-Morales et al, 2016 ; Azizan and Büchs, 2017 ; Azizan et al, 2019 ), and gas-liquid mass transfer ( Van Suijdam et al, 1978 ; Henzler and Schedel, 1991 ; Maier and Büchs, 2001 ; Maier et al, 2004 ; Giese et al, 2014 ) enables good and reliable experimental designs.…”