Modelling concentration gradients in fed‐batch cultivations of E. coli – towards the flexible design of scale‐down experiments

Abstract

BACKGROUND The impact of concentration gradients in large industrial‐scale bioreactors on microbial physiology can be studied in scale‐down bioreactors. However, scale‐down systems pose several challenges in construction, operation and footprint. Therefore, it is challenging to implement them in emerging technologies for bioprocess development, such as in high throughput cultivation platforms. In this study, a mechanistic model of a two‐compartment scale‐down bioreactor is developed. Simulations from this model are then used as bases for a pulse‐based scale‐down bioreactor suitable for application in parallel cultivation systems. RESULTS As an application, the pulse‐based system model was used to study the misincorporation of non‐canonical branched‐chain amino acids into recombinant pre‐proinsulin expressed in Escherichia coli, as a response to oscillations in glucose and dissolved oxygen concentrations. The results show significant accumulation of overflow metabolites, up to 18.3% loss in product yield and up to 10‐fold accumulation of the non‐canonical amino acids norvaline and norleucine in the product in the pulse‐based cultivation, compared with a reference cultivation. CONCLUSIONS Results indicate that the combination of a pulse‐based scale‐down approach with mechanistic models is a very suitable method to test strain robustness and physiological constraints at the early stages of bioprocess development. © 2018 Society of Chemical Industry

A model‐based framework for parallel scale‐down fed‐batch cultivations in mini‐bioreactors for accelerated phenotyping

“…The linear enzyme feed resulted in an approximately exponential glucose release, which then switched to a near‐constant release rate when enzyme feeding was stopped (Panula‐Perälä et al, ). C1–C3: These were fed with a glucose pulse every 5 min during the fed‐batch phase to mimic concentration gradients experienced in a larger bioreactor with a mixing time of approximately 40 s (Anane, Sawatzki et al, ). In the first 3 hr of feeding, the pulse size was increased exponentially to maintain a specific growth rate of 0.35 hr −1 in this phase, after which the pulse size was kept constant, upon IPTG induction.…”

“…The residual glucose supply was calculated to be less than 10% of the glucose requirements to maintain the specific growth rate at the set value. This was to ensure that the culture in these bioreactors does not fall into acute starvation conditions between the pulses. The last set of nine minibioreactors was similar to the previous set of nine, with the only exception that the glucose pulses were administered every 10 min, producing approximate glucose and dissolved oxygen gradients experienced in bioreactors with mixing times of approximately 90 s (Anane, Sawatzki et al, ). Thus, C1–C3 F1–F3, D1–D3 G1–G3 and E1–E3 H1–H3, with the exception of a glucose pulse frequency of 10 min in the latter sets, as shown in Table .…”

“…The cell pellets were re‐suspended in 500 µl phosphate‐buffered saline (PBS) and normalized to OD 600 = 5 in 1 ml. After centrifugation, the pellet from the normalized samples was lysed using Bugbuster® reagent, according to a previously described procedure (Anane, Sawatzki et al, ). Further inclusion body purification was carried out using the Bugbuster protocol, also as described previously (Anane, Sawatzki et al, ).…”

“…After centrifugation, the pellet from the normalized samples was lysed using Bugbuster® reagent, according to a previously described procedure (Anane, Sawatzki et al, ). Further inclusion body purification was carried out using the Bugbuster protocol, also as described previously (Anane, Sawatzki et al, ). From this procedure, three sample fractions were isolated for further processing: the raw cell lysate, intracellular soluble fraction, and purified inclusion bodies.…”

“…A platform of 21 parallel mini‐bioreactors was used, that was coupled to a robotic liquid handling station (LHS) for automated operation of the cultivations. The operation of the LHS was based on mechanistic model outputs which describe both the dynamic physiological behavior of the strain (Anane, López C, Neubauer, & Cruz Bournazou, ) and gradient profiles of scale‐down bioreactors determined from typical mixing times of large‐scale bioreactors (Anane, Sawatzki, Neubauer, & Cruz‐Bournazou, ). As a demonstration, the platform was used to study the effects of the oscillating glucose/dissolved oxygen concentrations on the amount and quality of recombinant proinsulin expressed in E. coli as inclusion bodies.…”