Twenty‐four‐well plate miniature bioreactor high‐throughput system: Assessment for microbial cultivations

Abstract

High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (micro)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the micro-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6 +/- 2.4, 46.5 +/- 4.6, 51.6 +/- 3.7, and 56.1 +/- 1.6 h(-1) at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively.

Assessment of AMBR<sup>TM</sup> as a model for high-throughput cell culture process development strategy

“…Temperature shift was performed from 37˚C to 34˚C on day 3 in both the systems and maintained till the end of the run. One of the primary advantages of the AMBR over other known MBR systems is that the AMBR does not require agitation to pause for sampling or feed addition hence reducing interruption in temperature maintenance [14]. The mixing time of the feeds are also not impacted since the agitation is not affected.…”

“…Recent literature have demonstrated availability of various designs of micro-scale reactors from a simpler standard plate with integrated sensors called Microtiter plate based bioreactors (MTB) [6][7][8][9][10][11][12] to more advanced cell culture systems such as the Stirred Mini Bioreactors (MBR) with control capabilities close to bench-top bioreactors for high throughput cell culture screening and optimization studies [13][14][15][16][17]. MBRs possess the advantage of being able to closely mirror the bench-scale bio-reactors in their monitoring and control capabilities and hence are considered as a preferred alternative vessel over shake flasks or MTBs for early stage process development.…”

“…MBRs possess the advantage of being able to closely mirror the bench-scale bio-reactors in their monitoring and control capabilities and hence are considered as a preferred alternative vessel over shake flasks or MTBs for early stage process development. MBRs are generally equipped with optical probes to monitor pH and DO online and it has been extensively characterized for its oxygen transfer rate, mixing capabilities and gassing efficiency [13][14][15][16][17]. The above mentioned characteristics of MBRs typically make them an ideal set up for early high-throughput clonal screening efforts as part of process development.…”

High‐throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

“…Enzyme production and thermochemical pretreatment are primary economic barriers to commercial scale-up of biorefineries that require further cost reductions through reduction in enzyme loading and pre-treatment severity. Coupling high-throughput pre-treatment , hydrolysis (Berlin et al, 2005), fermentation (Isett et al, 2007;Weimer et al, 2005), and compositional analysis (Chambliss et al, 2007) will facilitate rapid screening and identification of key bottlenecks to the biomass conversion process.…”

A microwell platform for the scale‐up of a multistep bioconversion to bench‐scale reactors: Sitosterol side‐chain cleavage

“…These technological developments were also timely introduced to contribute to gaining insight on the identification of the key parameters required to allow for reproducibility between shaken vessels and bench-scale stirred reactors, a feature that only in a relatively recent period has been suitably addressed [11][12][13][14]. There are hence few examples of scaling fermentation or bioconversion processes directly from MWP to bench-scale bioreactor partially due to the lack of instrumentation in traditional MWP [5, [14][15][16]. scales, viz.…”