Impact of out-of-phase conditions on screening results in shaking flask experiments

126

125

Most experiments in screening and process development are performed in shaken bioreactors. Today, microtiter plates are the preferred vessels for small-scale microbial cultivations in high throughput, even though they have never been optimized for this purpose. To interpret the experimental results correctly and to obtain a base for a meaningful scale-up, sufficient oxygen supply to the culture liquid is crucial. For shaken bioreactors this problem can generally be addressed by the introduction of baffles. Therefore, the focus of this study is to investigate how baffling and the well geometry affect the maximum oxygen transfer capacity (OTR(max)) in microtiter plates. On a 48-well plate scale, 30 different cross-section geometries of a well were studied. It could be shown that the introduction of baffles into the common circular cylinder of a microtiter plate well doubles the maximum oxygen transfer capacity, resulting in values above 100 mmol/L/h (k(L)a > 600 1/h). To also guarantee a high volume for microbial cultivation, it is important to maximize the filling volume, applicable during orbital shaking. Additionally, the liquid height at the well bottom was examined, which is a decisive parameter for online-monitoring systems such as the BioLector. This technology performs fiber-optical measurements through the well bottom, therefore requires a constant liquid height at all shaking frequencies. Ultimately, a six-petal flower-shaped well geometry was shown to be the optimal solution taking into account all aforementioned criteria. With its favorable culture conditions and the possibility for unrestricted online monitoring, this novel microtiter plate is an efficient tool to gain meaningful results for interpreting and scaling-up experiments in clone screening and bioprocess development.

Section: Introductionmentioning

confidence: 99%

The baffled microtiter plate: Increased oxygen transfer and improved online monitoring in small scale fermentations

Funke

Diederichs

Kensy³

et al. 2009

126

125

“…In recent years, the knowledge about the conditions in shake flasks has increased. (Maier, 2002;Maier and Büchs, 2001;Maier et al, 2004) characterized the mass-transfer (Büchs et al, 2000a,b;Peter et al, 2004) characterized the power input, Peter et al (2006) the hydromechanical stress, and (Maier and Büchs, 2001;Peter et al, 2004) the fluid movement. Until lately shake flask experiments could be performed only in the batch mode of operation.…”

Section: Introductionmentioning

confidence: 99%

Scale‐up from shake flasks to fermenters in batch and continuous mode with Corynebacterium glutamicum on lactic acid based on oxygen transfer and pH

Seletzky

Noak

Fricke

et al. 2007

Scale-up from shake flasks to fermenters has been hampered by the lack of knowledge concerning the influence of operating conditions on mass transfer, hydromechanics, and power input. However, in recent years the properties of shake flasks have been described with empirical models. A practical scale-up strategy for everyday use is introduced for the scale-up of aerobic cultures from shake flasks to fermenters in batch and continuous mode. The strategy is based on empirical correlations of the volumetric mass transfer coefficient (k(L) a) and the pH. The accuracy of the empirical k(L) a correlations and the assumptions required to use these correlations for an arbitrary biological medium are discussed. To determine the optimal pH of the culture medium a simple laboratory method based on titration curves of the medium and a mechanistic pH model, which is solely based on the medium composition, is applied. The effectiveness of the scale-up strategy is demonstrated by comparing the behavior of Corynebacterium glutamicum on lactic acid in shake flasks and fermenters in batch and continuous mode. The maximum growth rate (micro(max) = 0.32 h(-1)) and the oxygen substrate coefficient (Y O2 /S= 0.0174 mol/l) of C. glutamicum on lactic acid were equal for shake flask, fermenter, batch, and continuous cultures. The biomass substrate yield was independent of the scale, but was lower in batch cultures (Y(X/S) = 0.36 g/g) than in continuous cultures (Y(X/S) = 0.45 g/g). The experimental data (biomass, respiration, pH) could be described with a simple biological model combined with a mechanistic pH model.

“…At this early stage of process development, shake flasks have proven to be an invaluable tool in research and development (Büchs, 2001). However, assignment of ''increased product titer'' to ''improved strain or medium'' requires suitable and reproducible experimental conditions (Peter et al, 2004). To date, some typical engineering parameters have been described in shake flasks to ensure defined reaction conditions: The oxygen transfer rate (Anderlei and Büchs, 2001;Anderlei et al, 2004;Auro et al, 1957;Maier and Büchs, 2001;Maier et al, 2004), specific power consumption (Büchs et al, 2000a,b;Kato et al, 1996;Sumino et al, 1972), or mixing time (Gerson and Kole, 2001;Kato et al, 1996) have been studied to provide an engineering basis for scale-up from shake flasks to ensure nonlimiting conditions at all stages of process development.…”

Section: Introductionmentioning

confidence: 99%

Hydromechanical stress in shake flasks: Correlation for the maximum local energy dissipation rate

Peter

Suzuki

Büchs

2006

Self Cite

Shake flasks are widely used in biotechnological process research. Bioprocesses for which hydromechanical stress may become the rate controlling parameter include those where oils are applied as carbon sources, biotransformation of compounds with low solubility in the aqueous phase, or processes employing animal, plant, or filamentous microorganisms. In this study, the maximum local energy dissipation rate as the measure for hydromechanical stress is characterized in shake flasks by measuring the maximum stable drop size. The theoretical basis for the method is that the maximum stable drop diameter in a coalescence inhibited liquid/liquid dispersion is only a function of the maximum local energy dissipation rate and not of the dispersing apparatus. The maximum local energy dissipation rate is obtained by comparing the drop diameters in shake flasks to those in a stirred tank reactor. At the same volumetric power consumption, the maximum energy dissipation rate in shake flasks is about 10 times lower than in stirred tank reactors explaining the common observation of considerable differences in the morphology of hydromechanically sensitive cells between these two reactor types. At the same volumetric power consumption, the maximum local energy dissipation rate in baffled and in unbaffled shake flasks is very similar. A correlation is presented to quantify the maximum local energy dissipation rate in shake flasks as a function of the operating conditions. Non-negligible drop viscosity may be considered by known literature correlations. Further, from dispersion experiments a critical Reynolds number of about 60,000 is proposed for turbulent flow in unbaffled shake flasks.