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Development, parallelization, and automation of a gas-inducing milliliter-scale bioreactor for high-throughput bioprocess design (HTBD)
Abstract
A novel milliliter-scale bioreactor equipped with a gas-inducing impeller was developed with oxygen transfer coefficients as high as in laboratory and industrial stirred-tank bioreactors. The bioreactor reaches oxygen transfer coefficients of >0.4 s(-1). Oxygen transfer coefficients of >0.2 s(-1) can be maintained over a range of 8- to 12-mL reaction volume. A reaction block with integrated heat exchangers was developed for 48-mL-scale bioreactors. The block can be closed with a single gas cover spreading sterile process gas from a central inlet into the headspace of all bioreactors. The gas cover simultaneously acts as a sterile barrier, making the reaction block a stand-alone device that represents an alternative to 48 parallel-operated shake flasks on a much smaller footprint. Process control software was developed to control a liquid-handling system for automated sampling, titration of pH, substrate feeding, and a microtiter plate reader for automated atline pH and atline optical density analytics. The liquid-handling parameters for titration agent, feeding solution, and cell samples were optimized to increase data quality. A simple proportional pH-control algorithm and intermittent titration of pH enabled Escherichia coli growth to a dry cell weight of 20.5 g L(-1) in fed-batch cultivation with air aeration. Growth of E. coli at the milliliter scale (10 mL) was shown to be equivalent to laboratory scale (3 L) with regard to growth rate, mu, and biomass yield, Y(XS).
... Metabolite-protein interactions are also widely unknown 7 and only recently starting to be systematically mapped 8-analytical workflows for obtaining metabolomics, lipidomics, proteomics, and fluxomics data require several millions of cells per sample. Commercial automated cultivation platforms that fall into this range include 2Mag [13][14][15] and BioLector [16][17][18] . The 2Mag reaction block supports 48 [8][9][10][11][12] mL continuously stirred cultures grown aerobically or anaerobically with online pH and optical density (OD) sensors. ...
... The BioLector was designed to operate 48 well microtiter plates (MTPs) that can range from 800 to 2400 uL with online pH and dissolved oxygen (dO2) sensors and online OD measurements. Both the 2Mag reaction block and BioLector have been integrated with liquid handling robots to enable online pH control, substrate feeding, and sampling for other online or offline measurements [13][14][15][16][17][18] . While both commercial 2Mag and BioLector platforms provide tight control of physiological conditions such as temperature, aeration, etc., several major limitations exist that limit their use for high throughput omics experiments. ...
... Uniformly labeled 13 solution contains 68 g/L Na 2 HPO4 anhydrous, 30 g/L KH 2 PO4, 5 g/L NaCl, and 10 g/L NH 4 Cl dissolved in ddH 2 O and autoclaved. Saccharomyces cerevisiae CEN.PK113-7D was grown in Delft media supplemented with 20g/L glucose 24 . ...
Multi-omics datasets are becoming of key importance to drive discovery in fundamental research as much as generating knowledge for applied biotechnology. However, the construction of such large datasets is usually time-consuming and expensive. Automation is needed to overcome these issues by streamlining workflows from sample generation to data analysis. Here, we describe the construction of a complex workflow for the generation of high-throughput microbial multi-omics datasets. The workflow comprises a custom-built platform for automated cultivation and sampling of microbes, sample preparation protocols, analytical methods for sample analysis and automated scripts for raw data processing. We demonstrate possibilities and limitations of such workflow in generating data for three biotechnologically relevant model organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Pseudomonas putida.
... [7][8][9] Next to microtiter plate (MTP) based systems, [10,11] microscale bubble-column and stirred tank reactor-based MBR technologies with reaction volumes in the mL-range were developed. [12,13] In order to ensure a fair comparison of microbial strains among different test conditions, MBR systems have been validated toward controlled environmental conditions, e.g., temperature, mass transfer, power input [11,[14][15][16][17] and, most importantly, scalability of results. [18][19][20] Current MBR technologies enable data acquisition at high temporal resolution by means of non-invasive measurements of various culture parameters. ...
... [24] Here, the integration of MBR systems with liquid handling robots provides the possibility to sample microbial cultivations in an automated way. [13,[24][25][26][27][28] However, for the determination of concentration changes in a time-resolved manner as well as specific yields and rates derived thereof, transient sampling of cultures is typically conducted in MBR systems with several milliliters of working volume (e.g., 10 mL in bioREACTOR MBR device). [13,29] In contrast, in MBR systems with a working volume of up to 1 mL (e.g., BioLector MBR device) usually a total harvest of replicate cultures is performed to obtain sufficient sampling volumes, [24,27,28] i.e., one culture replicate is needed for one data point. ...
... [13,[24][25][26][27][28] However, for the determination of concentration changes in a time-resolved manner as well as specific yields and rates derived thereof, transient sampling of cultures is typically conducted in MBR systems with several milliliters of working volume (e.g., 10 mL in bioREACTOR MBR device). [13,29] In contrast, in MBR systems with a working volume of up to 1 mL (e.g., BioLector MBR device) usually a total harvest of replicate cultures is performed to obtain sufficient sampling volumes, [24,27,28] i.e., one culture replicate is needed for one data point. Clearly, for the investigation of process kinetics this is a drawback compared to classical lab-scale bioreactor setups because the originally intended elevated throughput by miniaturization is lost. ...
With modern genetic engineering tools, high number of potentially improved production strains can be created in a short time. This results in a bottleneck in the succeeding step of bioprocess development, which can be handled by accelerating quantitative microbial phenotyping. Miniaturization and automation are key technologies to achieve this goal. In this study, a novel workflow for repeated low‐volume sampling of BioLector‐based cultivation setup is presented. Six samples of 20 µL each can be taken automatically from shaken 48‐well microtiter plates without disturbing cell population growth. The volume is sufficient for quantification of substrate and product concentrations by spectrophotometric‐based enzyme assays. From transient concentration data and replicate cultures, valid performance indicators (titers, rates, yields) are determined through process modelling and random error propagation analysis.
Practical relevance of the workflow is demonstrated with a set of five genome‐reduced C. glutamicum strains that were engineered for Sec‐mediated heterologous cutinase secretion. Quantitative phenotyping of this strain library led to the identification of a strain with a 1.6‐fold increase in cutinase yield. The prophage‐free strain carries combinatorial deletions of three gene clusters (Δ3102‐3111, Δ 3263‐3301 and Δ 3324‐3345) of which the last two likely contain novel target genes to foster rational engineering of heterologous cutinase secretion in C. glutamicum.
... Furthermore, the engineering characteristic of miniature bioreactors has been studied in term of energy dissipation rate using computational fluid dynamic (CFD) analysis (Lamping et al., 2003), k L a values (Puskeiler et al., 2005), and mixing time . ...
... The specific power input of the miniature bioreactor was compared with a conventional 7 L bioreactor. It was concluded that the performance using an equivalent P/V is almost similar between these two bioreactors with regard of growth and product kinetics Furthermore, Puskeiler et al. (2005) has developed a millilitre size bioreactor with a gas inducing impeller that has k L a value as high as over 0.45 s -1 (for 8 mL volume) agitated at 2300 rpm. The key feature of the 48 ml miniature bioreactor developed by Puskeiler et al. (2005) is a standalone reaction block with integrated heat exchangers. ...
... It was concluded that the performance using an equivalent P/V is almost similar between these two bioreactors with regard of growth and product kinetics Furthermore, Puskeiler et al. (2005) has developed a millilitre size bioreactor with a gas inducing impeller that has k L a value as high as over 0.45 s -1 (for 8 mL volume) agitated at 2300 rpm. The key feature of the 48 ml miniature bioreactor developed by Puskeiler et al. (2005) is a standalone reaction block with integrated heat exchangers. ...
... We used a combination of MTPs, MBRs, and STRs to avoid the limitations of a single cultivation platform and demonstrate the advantages of an automated platform-based approach. Our initial MTP experiments showed that strains with 20 g L −1 glucose went into oxygen limitation due to limitations in the OTRs of 30 mmol O2 L −1 h −1 under the applied conditions [30]. This highlighted the need for lower glucose concentrations to prevent anoxic conditions and bias in the analysis. ...
... This highlighted the need for lower glucose concentrations to prevent anoxic conditions and bias in the analysis. Overall, our study provides valuable insights into the early phase of microbial bioprocess development and the trade-offs between throughput, limited OTRs, and analytics required for a comprehensive strain characterization [15,30]. ...
The bacterium Escherichia coli is a widely used organism in biotechnology. For high space-time yields, glucose-limited fed-batch technology is the industry standard; this is because an overflow metabolism of acetate occurs at high glucose concentrations. As an interesting alternative, various strains with limited glucose uptake have been developed. However, these have not yet been characterized under process conditions. To demonstrate the efficiency of our previously developed high-throughput robotic platform, in the present work, we characterized three different exemplary E. coli knockout (KO) strains with limited glucose uptake capacities at three different scales (microtiter plates, 10 mL bioreactor system and 100 mL bioreactor system) under excess glucose conditions with different initial glucose concentrations. The extensive measurements of growth behavior, substrate consumption, respiration, and overflow metabolism were then used to determine the appropriate growth parameters using a mechanistic mathematical model, which allowed for a comprehensive comparative analysis of the strains. The analysis was performed coherently with these different reactor configurations and the results could be successfully transferred from one platform to another. Single and double KO mutants showed reduced specific rates for substrate uptake qSmax and acetate production qApmax; meanwhile, higher glucose concentrations had adverse effects on the biomass yield coefficient YXSem. Additional parameters compared to previous studies for the oxygen uptake rate and carbon dioxide production rate indicated differences in the specific oxygen uptake rate qOmax. This study is an example of how automated robotic equipment, together with mathematical model-based approaches, can be successfully used to characterize strains and obtain comprehensive information more quickly, with a trade-off between throughput and analytical capacity.
... Hence, intermittent feeding leads to frequent changes in metabolic states [1] and transitional oxygen limitation, which can negatively affect cell physiology and growth [1,14]. That is why the oxygen supply is a critical process parameter in aerobic cultivations [18]. Insufficient oxygen transfer to the liquid phase to satisfy the oxygen needs of the cell is a known issue for MBRs [5,[19][20][21]. ...
... 7 216-396 --10-100 Impeller speed up to 100-7000 rpm, dry cell weight up to 10 g/L [38,39] The value of OUR and K La obtained from the DOT signal analysis (Table 3) are comparable to the literature values of experiments with a similar volume and biomass concentration. q o , the oxygen specific uptake rate value, seems slightly lower than the value reported by [18]. ...
Intermittent bolus feeding for E. coli cultivations in minibioreactor systems (MBRs) profoundly affects the cell metabolism. Bolus feeding leads to temporal substrate surplus and transient oxygen limitation, which triggers the formation of inhibitory byproducts. Due to the high oxygen demand right after the injection of the substrate, the dissolved oxygen tension (DOT) signal exhibits a negative pulse. This contribution describes and analyzes this DOT response in E. coli minibioreactor cultivations. In addition to gaining information on culture conditions, a unique response behavior in the DOT signal was observed in the analysis. This response appeared only at a dilution ratio per biomass unit higher than a certain threshold. The analysis highlights a plausible relationship between a metabolic adaptation behavior and the newly observed DOT signal segment not reported in the literature. A hypothesis that links particular DOT segments to specific metabolic states is proposed. The quantitative analysis and mechanistic model simulations support this hypothesis and show the possibility of obtaining cell physiological and growth parameters from the DOT signal.
... Most analytical workflows for obtaining metabolomic, lipidomic, proteomic, and fluxomic data require several millions of cells per sample. Commercial automated cultivation platforms that fall into this range include 2Mag [13][14][15] and BioLector [16][17][18] . The 2Mag reaction block supports 48 8-12 mL continuously stirred cultures grown aerobically or anaerobically with online pH and optical density (OD) sensors. ...
... The BioLector was designed to operate 48 well microtiter plates (MTPs) that can range from 800 to 2400 μL with online pH and dissolved oxygen (dO2) sensors and online OD measurements. Both the 2Mag reaction block and BioLector have been integrated with liquid handling robots to enable online pH control, substrate feeding, and sampling for other online or offline measurements [13][14][15][16][17][18] . While both commercial 2Mag and BioLector platforms provide tight control of physiological conditions such as temperature, aeration, etc., several major limitations exist that limit their use for high throughput omics experiments involving thousands of different strains. ...
Multi-omics datasets are becoming of key importance to drive discovery in fundamental research as much as generating knowledge for applied biotechnology. However, the construction of such large datasets is usually time-consuming and expensive. Automation might enable to overcome these issues by streamlining workflows from sample generation to data analysis. Here, we describe the construction of a complex workflow for the generation of high-throughput microbial multi-omics datasets. The workflow comprises a custom-built platform for automated cultivation and sampling of microbes, sample preparation protocols, analytical methods for sample analysis and automated scripts for raw data processing. We demonstrate possibilities and limitations of such workflow in generating data for three biotechnologically relevant model organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Pseudomonas putida.
... Because of the need to perform time-consuming and laborintensive experiments for bioprocess development, miniaturized and automated bioreactor systems have been developed with which a variety of process parameters can be screened rapidly [1,2]. Parallel microbioreactor systems are often coupled with a pipetting robot (liquid handling station, LHS) to use the flexibility of the LHS for at-line process analysis [3][4][5]. It has been shown that microbioreactor systems can yield scalable results for both biomass growth and product formation [4,6,7]. ...
... Parallel microbioreactor systems are often coupled with a pipetting robot (liquid handling station, LHS) to use the flexibility of the LHS for at-line process analysis [3][4][5]. It has been shown that microbioreactor systems can yield scalable results for both biomass growth and product formation [4,6,7]. Heterologous proteins are usually over expressed by cloning the encoding gene downstream from a regulated promoter in a suitable host to allow for cheap and simple protein production [8]. ...
Autonomously operated parallelized mL-scale bioreactors are considered the key to reduce bioprocess development cost and time. However, their application is often limited to products with very simple analytics. In this study, we investigated enhanced protein expression conditions of a carboxyl reductase from Nocardia otitidiscaviarum in E. coli. Cells were produced with exponential feeding in a L-scale bioreactor. After the desired cell density for protein expression was reached, the cells were automatically transferred to 48 mL-scale bioreactors operated by a liquid handling station where protein expression studies were conducted. During protein expression, the feed rate and the inducer concentration was varied. At the end of the protein expression phase, the enzymatic activity was estimated by performing automated whole-cell biotransformations in a deep-well-plate. The results were analyzed with hierarchical Bayesian modelling methods to account for the biomass growth during the biotransformation, biomass interference on the subsequent product assay, and to predict absolute and specific enzyme activities at optimal expression conditions. Lower feed rates seemed to be beneficial for high specific and absolute activities. At the optimal investigated expression conditions an activity of [Formula: see text] was estimated with a [Formula: see text] credible interval of [Formula: see text]. This is about 40-fold higher than the highest published data for the enzyme under investigation. With the proposed setup, 192 protein expression conditions were studied during four experimental runs with minimal manual workload, showing the reliability and potential of automated and digitalized bioreactor systems.
... advantages of MBR systems have in the past been used for high throughput screening, 1-4 strain engineering 5,6 and bioprocess development. [7][8][9][10] To closely mimic the microenvironment of cells, the design of MBR can be tailored for specific applications, including cytotoxicity testing and drug development. 11,12 In a microfluidic setup, they are also used as cell culturing and analytical devices, in clinical embryology or tissue engineering. ...
... Mimicking stirred tank reactors, micro-reactors can be homogenized using rotating stirrers. 10,[22][23][24][25] Avoiding an extended rotary shaft, miniaturized magnetic stirrer bars have been implemented as active mixing elements by Zhang et al. 26 and are widely used in other MBR systems. 27,28 Batch cultivations of E. coli and S. cerevisiae in the aforementioned stirred MBR systems showed good correspondence with 500 mL lab-scale reactors and shaking flasks proving the significance of these MBR systems. ...
Micro‐bioreactors (MBRs) have become an indispensable part for modern bioprocess development enabling automated experiments in parallel while reducing material cost. Novel developments aim to further intensify the advantages as dimensions are being reduced. However, one factor hindering the scale‐down of cultivation systems is to provide adequate mixing and mass transfer. Here, vertical oscillation is demonstrated as an effective method for mixing of MBRs with a reaction volume of 20 μL providing adequate mass transfer. Electrodynamic exciters are used to transduce kinetic energy onto the cultivation broth avoiding additional moving parts inside the applied model MBR. The induced vertical vibration leads to oscillation of the liquid surface corresponding to the frequency and displacement. On this basis, the resonance frequency of the fluid was identified as the most decisive factor for mixing performance. Applying this vertical oscillation method outstanding mixing times below 1 s and exceptionally high oxygen transport with volumetric mass transfer coefficients (kLa) above 1,000/hr can be successfully achieved and controlled. To evaluate the applicability of this vertical oscillation mixing for low volume MBR systems, cultivations of Escherichia coli BL21 as proof‐of‐concept were performed. The dissolved oxygen was successfully online monitored to assure any avoidance of oxygen limitations during the cultivation. The here presented data illustrate the high potential of the vertical oscillation technique as a flexible measure to adapt mixing times and oxygen transfer according to experimental demands. Thus, the mixing technique is a promising tool for various biological and chemical micro‐scale applications still enabling adequate mass transfer.
... Microplate systems can also be equipped with individual impellers for enhancement of the oxygen transfer coefficient (K L a) values (Micheletti e Lye, 2006). Puskeiler, Kaufmann, and Weuster-Botz (2005) developed a 48-well microliter plate with incorporated impellers, either free floating or supported by a hollow shaft, reaching K L a values of around > 0.4 s -1 . Commercially available microplate systems with automatic stirrers are also available. ...
... Mixing methods in microbioreactors can be divided into active and passive. Active methods usually use either free floating or supported impellers inserted into the microbioreactor (Puskeiler et al., 2005;Tan et al., 2015). This application requires extra caution when designing the bioreactor because the appearance of dead zones might reduce the mixing profile over the fermentation chamber. ...
Within the engineering and development of industrial bioprocesses, microbioreactors are microfluidic tools able to provide high-throughput screening tests in a fast and cheap manner by using small amounts of reagents. In addition, such tools are versatile and may allow a better controllability of parameters when compared to conventional bench-scale reactors. Consequentially, this technology has been gaining attention from the scientific community over the past years. In such scenario, this work provides a review study of the microbioreactor technology, outlining the origin, main concepts and principles of such technology, aiming to elucidate general questions that may emerge when studying such an approach. Past and current approaches are discussed aiming at drawing a comparative picture about such technology regarding future developments.
... They are attractive as they combine easy handling with the ability to control the bioprocess in a similar way as in standard bioreactors [35,126,127]. To fulfill the demand for high throughput and more advanced sample analysis at-line or as endpoint samples, MBR platforms with 8-48 parallelized reactors or microtiterplates, often supported by automated sampling, have been designed [118,[128][129][130]. MBRs are advantageous concerning space requirements, process costs and material [126]. ...
... To fulfill the demand for high throughput and more advanced sample analysis at-line or as endpoint samples, MBR platforms with 8-48 parallelized reactors or microtiterplates, often supported by automated sampling, have been designed [118,[128][129][130]. MBRs are advantageous concerning space requirements, process costs and material [126]. Special to these reactors is that due to the small volume, mixing, mostly achieved by miniaturized versions of impellers or magnetic stirrers, is close to ideal so that cells do not encounter significant gradients of process parameters [127][128][129]131]. Therefore, single cell dynamics using reporter strains or fluorescent dyes using online sensors or a fluorescence reader can be investigated in a wellcontrolled environment, e.g. the fiber-optic online monitoring BioLector system was used to monitor GFP expression as a measure for recombinant protein product [132,133]. ...
Population heterogeneity is omnipresent in all bioprocesses even in homogenous environments. Its origin, however, is only so well understood that potential strategies like bet-hedging, noise in gene expression and division of labour that lead to population heterogeneity can be derived from experimental studies simulating the dynamics in industrial scale bioprocesses. This review aims at summarizing the current state of the different parts of single cell studies in bioprocesses. This includes setups to visualize different phenotypes of single cells, computational approaches connecting single cell physiology with environmental influence and special cultivation setups like scale-down reactors that have been proven to be useful to simulate large-scale conditions. A step in between investigation of populations and single cells is studying subpopulations with distinct properties that differ from the rest of the population with sub-omics methods which are also presented here. Moreover, the current knowledge about population heterogeneity in bioprocesses is summarized for relevant industrial production hosts and mixed cultures, as they provide the unique opportunity to distribute metabolic burden and optimize production processes in a way that is impossible in traditional monocultures. In the end, approaches to explain the underlying mechanism of population heterogeneity and the evidences found to support each hypothesis are presented. For instance, population heterogeneity serving as a bet-hedging strategy that is used as coordinated action against bioprocess-related stresses while at the same time spreading the risk between individual cells as it ensures the survival of least a part of the population in any environment the cells encounter.
... Puskeiler et al. 65 devised a unique self-inducing impeller milli-upgrade bioreactor with a high volumetric mass transfer coefficient. Batch culture experiments were performed by utilizing Escherichia coli, yielding noteworthy outcomes. ...
Packed tower reactors, mechanically stirred reactors, airlift reactors, and gas-self-inducing reactors are frequently utilized among the various types of reactors. Self-inducing reactors exhibit notable advantages owing to their simple structure, effective gas–liquid intermixing, and low energy requirements, rendering them highly suitable for bioengineering endeavors. The purpose of this analysis is to shed light on the use of self-inducing reactors in bioengineering by examining the following five parameters: critical speed, suction rate, volumetric mass transfer coefficient, power characteristics, and gas hold-up. Through a comprehensive analysis of the advancements achieved in these domains, it is possible to determine the challenges and opportunities that lie ahead in the realm of bioengineering.
... Deviations from ideal reactor behavior, such as changing levels of dissolved oxygen due to varying substrate availability resulting from intermittent feeding, have been linked to population heterogeneity [26,27]. Moving between scales, non-ideality may vary in kind and magnitude, resulting in differences in product, metabolite, or inhibitor concentration or process variables like optimal harvesting time to achieve high space-time yield [28]. The proposed scheduling software enables researchers to further investigate and quantify these deviations by setting defined feeding intervals. ...
The shift towards high-throughput technologies and automation in research and development in industrial biotechnology is highlighting the need for increased automation competence and specialized software solutions. Within bioprocess development, the trends towards miniaturization and parallelization of bioreactor systems rely on full automation and digital process control. Thus, mL-scale, parallel bioreactor systems require integration into liquid handling stations to perform a range of tasks stretching from substrate addition to automated sampling and sample analysis. To orchestrate these tasks, the authors propose a scheduling software to fully leverage the advantages of a state-of-the-art liquid handling station (LHS) and to enable improved process control and resource allocation. Fixed sequential order execution, the norm in LHS software, results in imperfect timing of essential operations like feeding or Ph control and execution intervals thereof, that are unknown a priori. However, the duration and control of, e.g., the feeding task and their frequency are of great importance for bioprocess control and the design of experiments. Hence, a software solution is presented that allows the orchestration of the respective operations through dynamic scheduling by external LHS control. With the proposed scheduling software, it is possible to define a dynamic process control strategy based on data-driven real-time prioritization and transparent, user-defined constraints. Drivers for a commercial 48 parallel bioreactor system and the related sensor equipment were developed using the SiLA 2 standard greatly simplifying the integration effort. Furthermore, this paper describes the experimental hardware and software setup required for the application use case presented in the second part.
... Over the last decades, various microbioreactor designs, including stirred [1][2][3][4], shaken [5][6][7], sparged [8][9][10] and pumped systems [11,12] have been developed to accelerate early up-stream bioprocess development [13,14]. To follow the Process Analytical Technology (PAT) guidance for process analytics and control [15], these microscale cultivation vessels are often equipped with a miniaturised periphery [16], enabling at-line and online process monitoring down to droplet-size cultivation volumes [17,18]. ...
Multi-wavelength (2D) fluorescence spectroscopy represents an important step towards exploiting the monitoring potential of microtiter plates (MTPs) during early-stage bioprocess development. In combination with multivariate data analysis (MVDA), important process information can be obtained, while repetitive, cost-intensive sample analytics can be reduced. This study provides a comprehensive experimental dataset of online and offline measurements for batch cultures of Hansenula polymorpha. In the first step, principal component analysis (PCA) was used to assess spectral data quality. Secondly, partial least-squares (PLS) regression models were generated, based on spectral data of two cultivation conditions and offline samples for glycerol, cell dry weight, and pH value. Thereby, the time-wise resolution increased 12-fold compared to the offline sampling interval of 6 h. The PLS models were validated using offline samples of a shorter sampling interval. Very good model transferability was shown during the PLS model application to the spectral data of cultures with six varying initial cultivation conditions. For all the predicted variables, a relative root-mean-square error (RMSE) below 6% was obtained. Based on the findings, the initial experimental strategy was re-evaluated and a more practical approach with minimised sampling effort and elevated experimental throughput was proposed. In conclusion, the study underlines the high potential of multi-wavelength (2D) fluorescence spectroscopy and provides an evaluation workflow for PLS modelling in microtiter plates.
... The ability to incorporate a broad range of additional devices into the workstation means that the Tecan has vast experimental potential. Puskeiler et al. (2005) was among the first to describe an E.coli fermentation performed within a Tecan platform. Automated capabilities were utilised for media additions in the 48 novel parallel reactors, and for at-line analytics, which were achieved using an integrated plate reader. ...
Perfusion culture is becoming an increasingly popular choice for the production of therapeutic proteins, however few scale-down devices capable of small scale and/or high throughput optimisation of high cell density perfusion cultures have been published. To address this technology gap, this thesis describes the development, implementation, and engineering characterisation of two scale-down technologies; (i) a quasi-perfusion method at mL-scale in microwell plates (MWPs) and (ii) a purposely designed novel bioreactor (BR) at 250mL scale. Quasi-perfusion in MWP was developed at mL-scale and is capable of achieving many of the specific characteristics of perfusion culture, namely elevated cell density, cell retention and good productivity. The quasi-perfusion methodology was implemented to screen a range of process conditions, exchanging at a constant vessel volume per day (VVD) between 0.5-1.8, or at constant cell specific perfusion rate (CSPR) with a range of media, with cell retention achieved via sedimentation or centrifugation. Viable cell densities (VCDs) of 42 × 10^{6} cells mL-1and volumetric productivities up to 2 fold greater than fed-batch were achieved. Design and engineering characterisation of the novel 250mL BR ensured favourable hydrodynamics, with a dual impeller system selected to maximise mass transfer. Cell retention was achieved via a tangential flow filter (TFF) at perfusion rates between 0.5- 1.8 VVD, maintaining maximum VCDs of 92 × 10^{6} cells mL-1 at >95% viability. Good scalability was demonstrated for a range of performance metrics, including µmax, qAb and biomass production, between each system when compared to a bench scale 5L BR, while scaling was based on constant volumetric power input. The combined use of quasi-perfusion methodologies, shown to be sensitive to changes in perfusion rate and media composition, for high throughput screening studies, followed by the 250mL BR for in-depth study of a small range of conditions, is shown to be a powerful tool for the development and optimisation of perfusion processes.
... Hence, a large amount of data is needed to obtain a mathematical model which is capable of describing the non-steady-state dynamics of highly nonlinear and complex systems such as cells . The recent development of high-throughput (HT) technology allows for performing a large number of laborious and time-consuming experiments by automatizing, parallelizing, and miniaturizing the experimental facilities (Puskeiler et al., 2005;Bunzel et al., 2018;Hemmerich et al., 2018). Liquid handling stations support the parallel cultivation on a large numbers of mini-bioreactors, based on the automated sampling, on-line and at-line analytics operation, real-time control, and data acquisition (Kusterer et al., 2008;Tai et al., 2015;Haby et al., 2019;Hans et al., 2020b). ...
We discuss the application of a nonlinear model predictive control (MPC) and a moving horizon estimation (MHE) to achieve an optimal operation of E. coli fed-batch cultivations with intermittent bolus feeding. 24 parallel experiments were considered in a high-throughput microbioreactor platform at 10 mL scale. The robotic island in question can run up to 48 fed-batch processes in parallel with an automated liquid handling and online and atline analytics. The implementation of the model-based monitoring and control framework reveals that there are mainly three challenges which need to be addressed; First, the inputs are given in an instantaneous pulsed form by bolus injections, second, online and atline measurement frequencies are severely imbalanced, and third, optimization for the distinctive multiple reactors can be either parallelized or integrated. We address these challenges by incorporating the concept of impulsive control systems, formulating multi-rate MHE with identifiability analysis, and suggesting criteria for deciding the reactor configuration. In this study, we present the key elements and background theory of the implementation with \textit{in silico} simulations for the bacterial fed-batch cultivation.
... Coupled with the increasing propagation of advanced microcultivation systems , a variety of technologies for laboratory automation have found their way into research and development laboratories over the past two decades (Baumann et al., 2015;Cruz Bournazou et al., 2017;Funke et al., 2010;Hemmerich, Tenhaef, et al., 2018;Heux et al., 2014;Puskeiler et al., 2005;Rohe et al., 2012). By integrating liquid handling robots and other connected devices such as centrifuges, microtiter plate (MTP) readers and others, automated microcultivation platforms have been created that fundamentally changed the way process developers work. ...
Given its geometric similarity to large‐scale production plants and the excellent possibilities for precise process control and monitoring, the classic stirred tank bioreactor (STR) still represents the gold standard for bioprocess development at a laboratory scale. However, compared to microbioreactor technologies, bioreactors often suffer from a low degree of process automation and deriving key performance indicators (KPIs) such as specific rates or yields often requires manual sampling and sample processing. A widely used parallelized STR setup was automated by connecting it to a liquid handling system and controlling it with a custom‐made process control system. This allowed for the setup of a flexible modular platform enabling autonomous operation of the bioreactors without any operator present. Multiple unit operations like automated inoculation, sampling, sample processing and analysis, and decision making, for example for automated induction of protein production were implemented to achieve such functionality. The data gained during application studies was used for fitting of bioprocess models to derive relevant KPIs being in good agreement with literature. By combining the capabilities of STRs with the flexibility of liquid handling systems, this platform technology can be applied to a multitude of different bioprocess development pipelines at laboratory scale.
... Meanwhile, microcultivation technology, such as minibioreactors [34] or microbioreactors [35], has been matured to mimic lab-scale bioreactor cultivations to a high extent [36,37]. Such devices are often integrated in laboratory robotic systems to increase versatility and performance, providing process samples and process information in a comparable manner to traditional laboratory-scale bioreactors, but with elevated throughput in the order of up to 2 orders of magnitude. ...
With the utilization of small-scale and highly parallelized cultivation platforms embedded in laboratory robotics, microbial phenotyping and bioprocess development have been substantially accelerated, thus generating a bottleneck in bioanalytical bioprocess sample analytics. While microscale cultivation platforms allow the monitoring of typical process parameters, only limited information about product and by-product formation is provided without comprehensive analytics. The use of liquid chromatography mass spectrometry can provide such a comprehensive and quantitative insight, but is often limited by analysis runtime and throughput. In this study, we developed and evaluated six methods for amino acid quantification based on two strong cation exchanger columns and a dilute and shoot approach in hyphenation with either a triple-quadrupole or a quadrupole time-of-flight mass spectrometer. Isotope dilution mass spectrometry with ¹³ C ¹⁵ N labeled amino acids was used to correct for matrix effects. The versatility of the methods for metabolite profiling studies of microbial cultivation supernatants is confirmed by a detailed method validation study. The methods using chromatography columns showed a linear range of approx. 4 orders of magnitude, sufficient response factors, and low quantification limits (7–443 nM) for single analytes. Overall, relative standard deviation was comparable for all analytes, with < 8% and < 11% for unbuffered and buffered media, respectively. The dilute and shoot methods with an analysis time of 1 min provided similar performance but showed a factor of up to 35 times higher throughput. The performance and applicability of the dilute and shoot method are demonstrated using a library of Corynebacterium glutamicum strains producing l -histidine, obtained from random mutagenesis, which were cultivated in a microscale cultivation platform.
Graphical abstract
... Agitation can also be achieved using a magnetic stirrer bar or gas-inducing impeller (Puskeiler, Kaufmann and Weuster-Botz, 2005;Klein, Schneider and Heinzle, 2013) MSBRs can achieve relatively high k L a values which are comparable to laboratory scale bioreactors; a k L a of 1600h -1 -was reported by , however this was for a novel MSBR with a gasinducing impeller. Aeration is achieved by direct sparging or surface aeration (Table1.2). ...
The rate limiting steps of biopharmaceutical process development are clone evaluation and process optimisation. To improve the efficiency of this step, miniature bioreactors are increasingly being used as a tool for high throughput experimentation. At industrial scale, microbial cultivations are usually performed in fed-batch mode to allow for high cell density cost-effective processes; however, many commercially available miniature bioreactors do not have an inbuilt feeding capacity. There are several challenges that need to be addressed to establish high cell density fed-batch cultivation at microscale: attaining high oxygen mass transfer rates, achieving good mixing for the duration of the culture and implementation of an industrially relevant feeding strategy requiring low volume additions. The overall aim of this project was to develop a scale-down fermentation platform suitable for the study and optimisation of high cell density cultures. The first objective of this work was to evaluate options for fed-batch cultures in a commercially available 24-well shaken microbioreactor. To achieve this, two feeding strategies were evaluated using an E. coli strain expressing a domain antibody: in situ feeding by the enzymatic release of glucose from polymeric starch, and direct feeding using a bespoke feed delivery system. In situ feeding was investigated as it is a simple option that does not require a physical method of feed delivery; cellular productivity was enhanced in comparison to batch cultures, however the glucose release was insufficient to sustain high cell density cultures representative of laboratory and pilot scale processes. To enable direct and continuous feed delivery to the microbioreactor a bespoke 3D-printed feeding system was developed that can operate at flow rates of 20μL h-1 and above, and enables up to twelve fed-batch cultures to be run in parallel. E. coli fermentations were performed on complex medium containing glycerol with direct feeding of a 23% w/v glycerol solution initiated at around 18 hours. The second objective of this project was to establish an industrially relevant feeding strategy in the microbioreactor, comparable to a laboratory scale fed-batch process. To this end, the direct feeding strategy was refined in terms of cell growth and product expression; the feed rate and concentration were modified, the DO set point was increased, and a pre-feeding hold period was implemented to allow for consumption of the inhibitory by-products generated in the batch phase. It was found that direct feeding enhanced biomass production by ~70% and product expression by ~2.4 fold in comparison to non-fed cultures. The third objective of this work was to demonstrate the applicability of the new feeding system as a tool for process optimisation experiments. The effect of IPTG concentration and post-induction temperature on product expression was performed using the both the microbioreactor feeding system and the 1L laboratory scale process. The data trends were consistent between scales; product expression was enhanced at a higher post-induction temperature, and IPTG concentration did not affect product expression over the concentration range tested. This demonstrates that the microbioreactor, is predicative of the 1L laboratory scale process terms of sensitivity to change in process conditions The fourth objective of this work was to characterise the microbioreactor in terms of oxygen transfer capability and fluid mixing. To achieve this aim, the volumetric oxygen mass transfer coefficient (kLa) and liquid phase mixing time (tm) of the microbioreactor were determined. The impact of shaking frequency, total gas flow rate and fill volume on oxygen transfer and fluid mixing were investigated and the optimum operating conditions were determined. Within the operating ranges of the miniature bioreactor system, it was found that oxygen transfer was dependant on both shaking frequency and gas flow rate, but was independent of fill volume. The oxygen mass transfer coefficient, kLa increased with both increasing shaking frequency (500-800rpm) and gas flow rate (0.1-20 mL min-1) over the range 3-101h-1; this is at the lower end of the range for conventional stirred tank reactors. It was demonstrated that the miniature bioreactor system is well mixed under the range of operating conditions evaluated. The liquid phase mixing time, tm under non-aerated conditions increased with shaking frequency and decreased with fill volume over the range 0.5-15s. The final objective this project was to demonstrate suitability of the microbioreactor as a scale-down model of an industrial fermentation process. 50L pilot scale, 1L laboratory scale, and 4mL microbioreactor fed-batch fermentations were performed under optimum conditions. The 4mL microbioreactor fed-batch process was shown to better predict the 50L pilot-scale process than the 1L laboratory-scale process based on cell growth, product expression and product quality. This could be explained by mixing and oxygen mass transfer phenomena. At 1L scale, oxygen mass transfer and fluid mixing are most efficient, meaning cell growth and productivity were the highest of the three processes. It appears that the limitations in oxygen mass transfer in the microbioreactor and fluid mixing in the 50L scale vessel, results in a comparable cellular environment, and therefore cell growth, productivity and product quality. In summary, this work has demonstrated the ability to conduct high cell density, fed-batch microbial cultures in parallel, using a shaken miniature bioreactor system. A bespoke, 3D-printed feed delivery system was developed allowing for twelve industrially-relevant microbial fed-batch cultures to be run in parallel. The microbioreactor fed-batch cultures were shown to be predictive of, a 50L pilot scale process in terms of cell growth, productivity and product quality.
... Parallel whole-cell biotransformations of geraniol on mL-scale Whole-cell biotransformations of geraniol on mLscale were performed as batch processes in a parallel stirred-tank bioreactor system (bioREACTOR, 2mag AG) equipped with gas-inducing stirrers (Puskeiler et al. 2005) using sterile single-use unbaffled bioreactors (2mag AG) with a reaction volume of 10 mL. Reactions were executed for 24 h at an impeller speed of 2200 or 4000 rpm, an aeration rate of 0.1 L min -1 in the headspace, a temperature of 30 or 37°C and activated headspace cooling (20°C) to prevent losses in liquid volume by evaporation. ...
Objective
Geraniol, a fragrance of great importance in the consumer goods industry, can be glucosylated by the UDP-glucose-dependent glucosyltransferase VvGT14a from Vitis vinifera, yielding more stable geranyl glucoside. Escherichia coli expressing VvGT14a is a convenient whole-cell biocatalyst for this biotransformation due to its intrinsic capability for UDP-glucose regeneration. The low water solubility and high cytotoxicity of geraniol can be overcome in a biphasic system where the non-aqueous phase functions as an in situ substrate reservoir. However, the effect of different process variables on the biphasic whole-cell biotransformation is unknown. Thus, the goal of this study was to identify potential bottlenecks during biotransformation with in situ geraniol supply via isopropyl myristate as second non-aqueous phase.ResultsFirst, insufficient UDP-glucose supply could be ruled out by measurement of intracellular UDP-glucose concentrations. Instead, oxygen supply was determined as a bottleneck. Moreover, the formation of the byproduct geranyl acetate by chloramphenicol acetyltransferase (CAT) was identified as a constraint for high product yields. The use of a CAT-deficient whole-cell biocatalyst prevented the formation of geranyl acetate, and geranyl glucoside could be obtained with 100% selectivity during a biotransformation on L-scale.Conclusion
This study is the first to closely analyze the whole-cell biotransformation of geraniol with Escherichia coli expressing an UDP-glucose-dependent glucosyltransferase and can be used as an optimal starting point for the design of other glycosylation processes.
... Further findings suggest that viscosity up to 35 mPa did not show any major influence on the oxygen transfer rate and the mixing process. The details on microscale cultivation technology and machines have been reviewed recently (Puskeiler et al. 2005). ...
Microorganisms are indispensable in the food industry, but wild-type strains hardly meet the current industrial demands due to several undesirable traits. Therefore, microbial strain improvement offers a critical solution to enhance the food industry. Traditional techniques for food microbial improvement, such as the use of chemical mutagens and manual isolation/purification, are inefficient, time-consuming, and laborious, restricting further progress in the area of food fermentation. In this review, the applications of novel mutagenesis and screening technologies used for the improvement of food microbes were summarized, including random mutagenesis based on physical irradiation, microbial screening facilitated by a microtiter plate, fluorescence-activated cell or droplet sorting, and microscaled fermentation in a microtiter plate or microbioreactor. In comparison with conventional methods, these new tools have the potential in accelerating microbial strain improvement and their combined applications could create a new trend for strain development. However, several problems that could affect its potential application may include the following: the lack of specific mutagenesis devices and biosensing systems, the insufficient improvement of the mixed culture system, the low efficiency when using filamentous fungi and flocculating bacteria, and the insufficient safety assessment on harnessing genome-editing technology. Therefore, future works on strain improvement remain challenging for the food industry.
... A vast number of HT cultivation platforms have been subject of research studies, while several systems (miniature shaken vessel/well, 0.2-4.0 mL; bubble column-or microplate-based mini-bioreactors, 1.0-10 mL; stirred mini-tank bioreactors, 10 mL) have been successfully commercialized in the past decade [50][51][52]. HT cultivation platform allows strains to be screened under conditions comparable to those in the manufacturing process, which can make timeline remarkably shorter for establishing optimal processes in many bio-industrial sectors [53]. ...
Le métabolisme définit l’ensemble des réactions biochimiques au sein d’un organisme, lui permettant de survivre et de s’adapter dans différents environnements. La régulation de ces réactions requiert un processus complexe impliquant de nombreux effecteurs interagissant ensemble à différentes échelles.Développer des modèles de ces réseaux de régulation est ainsi une étape indispensable pour mieux comprendre les mécanismes précis régissant les systèmes vivants et permettre, à terme, la conception de systèmes synthétiques, autorégulés et adaptatifs, à l'échelle du génome. Dans le cadre de ces travaux interdisciplinaires, nous proposons d’utiliser une approche itérative d’inférence de réseau et d’interrogation afin de guider l’ingénierie du métabolisme de la levure d’intérêt industriel Yarrowia lipolytica.À partir de données transcriptomiques, le premier réseau de régulation de l’adaptation à la limitation en azote et de la production de lipides a été inféré pour cette levure. L’interrogation de ce réseau a ensuite permis de mettre en avant et valider expérimentalement l’impact de régulateurs sur l'accumulation lipidique.Afin d’explorer davantage les liens entre régulation et métabolisme, une nouvelle méthode, CoRegFlux, a été proposée pour la prédiction de phénotype métabolique à partir des profils d’activités des régulateurs dans les conditions étudiées.Ce package R, disponible sur la plateforme Bioconductor, a ensuite été utilisé pour mieux comprendre l’adaptation à la limitation en azote et identifier des phénotypes d’intérêts en vue de l’ingénierie de cette levure, notamment pour la production de lipides et de violacéine.Ainsi, par une approche itérative, ces travaux apportent de nouvelles connaissances sur les interactions entre la régulation et le métabolisme chez Y. lipolytica, l’identification de motifs de régulation chez cette levure et contribue au développement de méthodes intégratives pour la conception de souches assistée par ordinateur.
... The bioreactor was connected to a Tecan ® pipetting robot (Tecan Group Ltd, Männedorf, Switzerland) and to a central database where all online and at-line data were directly saved. The aeration of each mini-bioreactor was achieved by an induced draft mechanism, where the air was drawn into the broth through a hollow shaft on which the impeller was mounted (Puskeiler, Kaufmann, & Weuster-Botz, 2005). By this mechanism, the whole bioreactor block was aerated to 10 L air ·min −1 during cultivation. ...
Concentration gradients that occur in large industrial‐scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the response of strains to such heterogeneities to reduce the risk of failure during process scale‐up. Although there are various scale‐down techniques to study these effects, scale‐down strategies are rarely applied in the early developmental phases of a bioprocess, as they have not yet been implemented on small‐scale parallel cultivation devices.
In this study, we combine mechanistic growth models with a parallel mini‐bioreactor system to create a high‐throughput platform for studying the response of Escherichia coli strains to concentration gradients. As a scaled‐down approach, a model‐based glucose pulse feeding scheme is applied and compared with a continuous feed profile to study the influence of glucose and dissolved oxygen gradients on both cell physiology and incorporation of noncanonical amino acids into recombinant proinsulin. The results show a significant increase in the incorporation of the noncanonical amino acid norvaline in the soluble intracellular extract and in the recombinant product in cultures with glucose/oxygen oscillations. Interestingly, the amount of norvaline depends on the pulse frequency and is negligible with continuous feeding, confirming observations from large‐scale cultivations. Most importantly, the results also show that a larger number of the model parameters are significantly affected by the scale‐down scheme, compared with the reference cultivations.
In this example, it was possible to describe the effects of oscillations in a single parallel experiment. The platform offers the opportunity to combine strain screening with scale‐down studies to select the most robust strains for bioprocess scale‐up.
... The system can reach a volumetric mass transfer coefficient (k L a) of up to 0.4 s -1 . 39 The power input and local energy dissipation are comparable to standard stirredtank bioreactors and computational fluid dynamics simulations showed similar hydrodynamics to bench-top stirred bioreactors on a liter scale. 40 In the lid of the system, the supplied air is distributed to the headspace of each single bioreactor. ...
During process development, the experimental search space is defined by the number of experiments that can be performed in specific time frames but also by its sophistication (e.g., inputs, sensors, sampling frequency, analytics). High-throughput liquid-handling stations can perform a large number of automated experiments in parallel. Nevertheless, the experimental data sets that are obtained are not always relevant for development of industrial bioprocesses, leading to a high rate of failure during scale-up. We present an automated mini bioreactor platform that enables parallel cultivations in the milliliter scale with online monitoring and control, well-controlled conditions, and advanced feeding strategies similar to industrial processes. The combination of two liquid handlers allows both automated mini bioreactor operation and at-line analysis in parallel. A central database enables end-to-end data exchange and fully integrated device and process control. A model-based operation algorithm allows for the accurate performance of complex cultivations for scale-down studies and strain characterization via optimal experimental redesign, significantly increasing the reliability and transferability of data throughout process development. The platform meets the tradeoff between experimental throughput and process control and monitoring comparable to laboratory-scale bioreactors.
... Mixing and mass transfer in miniature bioreactors commonly rely on shaking or stirring with magnetic rings, which can provide a mixing time less than 2 s and an oxygen transfer rate higher than 1000 h −1 under certain conditions. 9,19,20,8,14,15,[21][22][23] Puskeiler et al. 24 developed a gas-inducing impeller to improve the oxygen transfer at milliliter scale. Another challenge for miniature bioreactors is process control, particularly for pH and DO in minimal space. ...
BACKGROUND
Sufficient oxygen supply and stable process control are crucial for the successful screening and process optimization of aerobic microorganisms in small‐scale bioreactors. In this work, a miniature bioreactor (volume 80 mL) equipped with non‐invasive biosensors was developed and characterized.
RESULTS
To enable proper mixing and high oxygen transfer rate, a mechanical agitator with large diameter (0.56 tank diameter) and submerged aeration were applied. The flow fields formed in the miniature bioreactor under a wide range of conditions (filling volume 30–70 mL, agitation speed 300–1100 rpm and aeration rate 0–4 vvm) were numerically analyzed by computational fluid dynamics (CFD). The miniature bioreactor performed with a suitable mixing time (<4 s) and a high oxygen transfer coefficient (kLa > 1000 h⁻¹). It showed that engineering parameters including mass transfer, mixing and shear were more sensitive to agitation than either filling volume or aeration. Correlations for quantitative evaluation of the engineering parameters were established. The predicted oxygen transfer coefficient by CFD showed good agreement with both experimental and reported data. Furthermore, cultivations of Escherichia coli and Pichia pastoris in the mini bioreactor verified the excellent performance.
CONCLUSION
The developed miniature bioreactor will provide a reliable tool for screening and process optimization, and the presented correlations that predict the engineering parameters will make it convenient to achieve scale‐up. © 2019 Society of Chemical Industry
... The combination of autoclavable gas distributors and stirrers with disposable reactor vessels provides the practical benefits of disposables while keeping the material costs low. [6,7] Lacking any robotic augmentation or liquid pumps, custom integration into a laboratory robot system is inevitable for the bioREACTOR as well. However, the successful integration in Tecan and Hamilton systems or combinations of both for automated cultivation with integrated sample preparation has already been reported. ...
Advances in molecular biotechnology have resulted in the generation of numerous potential production strains. Because every strain can be screened under various process conditions, the number of potential cultivations is multiplied. To exploit this potential without increasing the associated timelines requires a cultivation platform that offers increased throughput and flexibility to perform various bioprocess screening protocols.
Currently, there is no commercially available fully automated cultivation platform that can operate multiple microbial fed‐batch processes, including at‐line sampling, deep freezer off‐line sample storage and complete data handling.
To enable scalable high‐throughput early‐stage microbial bioprocess development, a commercially available microbioreactor system and a laboratory robot were combined to develop a fully automated cultivation platform. By making numerous modifications, as well as supplementation with custom‐built hardware and software, fully automated milliliter‐scale microbial fed‐batch cultivation, sample handling and data storage were realized.
The initial results of cultivations with two different expression systems and three different process conditions were compared using 5‐L scale benchmark cultivations, which provided identical rankings of expression systems and process conditions.
Thus, fully automated high‐throughput cultivation, including automated centralized data storage to significantly accelerate the identification of the optimal expression systems and process conditions, offers the potential for automated early‐stage bioprocess development.
This article is protected by copyright. All rights reserved.
... In any commercial bioprocess, the strain, medium culture and fermentation conditions should be developed and optimized, which are costly and time-consuming processes [1,4]. The miniaturized parallel bioreactors can overcome these obstacles by simultaneously providing a large number of experiments at low volume, and thus, reducing the bioprocess development time and cost [1][2][3]5]. Miniaturized bioreactors are classified into three different categories according to their agitation: shaken flasks, bubble columns and stirred tanks [2,6]. ...
Miniaturized bubble columns (MBCs) have different hydrodynamics in comparison with the larger ones, but there is a lack of scientific data on MBCs. Hence, in this study, the effect of gas hold-up, flow regimes, bubble size distribution on volumetric oxygen mass transfer coefficient at different pore size spargers and gas flow rates in MBCs in the presence and absence of microorganisms were investigated. It was found that flow regime transition occurred around low gas flow rates of 1.18 and 0.85 cm/s for small (16–40 µm) and large (40–100 µm) pore size spargers, respectively. Gas hold-up and KLa in MBC with small size sparger were higher than those with larger one, with an increasing effect in the presence of microorganisms. A comparison revealed that the wall effect on the flow regime and gas hold-up in MBCs was greater than bench-scale bubble columns. The KLa values significantly increased up to tenfold using small pore size sparger. In the MBC and stirred tank bioreactors, the maximum obtained cell concentrations were OD600 of 41.5 and 43.0, respectively. Furthermore, it was shown that in MBCs, higher KLa and lower turbulency could be achieved at the end of bubbly flow regime.
... The bioreactor was connected to a Tecan ® pipetting robot (Tecan Group Ltd, Männedorf, Switzerland) and to a central database where all online and at-line data were directly saved. The aeration of each mini-bioreactor was achieved by an induced draft mechanism, where the air was drawn into the broth through a hollow shaft on which the impeller was mounted (Puskeiler, Kaufmann, & Weuster-Botz, 2005). By this mechanism, the whole bioreactor block was aerated to 10 L air ·min −1 during cultivation. ...
... Microwell bioreactors allow for speedier process development times and are commonly used for process optimization and screening with shaking platforms instead of mechanically stirred bioreactors which have comparatively reduced scope for parallelization typically being capable of running between only 4 and 48 experiments together (Puskeiler et al. 2005;Gill et al. 2008). Microwell plates can also be readily incorporated into automation systems to deliver important process design considerations at the lab-scale making them a useful choice for developing new and optimising existing bioprocesses (Lye et al. 2003;Micheletti & Lye 2006). ...
Human induced pluripotent stem cells (hiPSC) have the potential to provide patient and disease specific cells for research and act as therapeutic agents in unlimited supply. To translate lab-scale research toward clinical applications we need to reduce variability from complex differentiation protocols which often include xenogeneic components. A move toward more defined culture systems will improve predictability and process control as well as reduce risks of exposure to animal pathogens. Cell therapy development, also requires flexibility in scalability as cell numbers for therapies vary greatly between disease indications. Improved control over the microenvironment at the lab scale would offer more defined parameters amenable to scale up with better flexibility, reduced waste and more precision. This thesis describes the use of forced aggregation to improve the initiation of differentiation combined with culture in pre-validated and scalable, 24 microwell plates on shaking platforms, for stem cell differentiation toward retinal lineages. Using an established hiPSC line (MSU001) we show using forced aggregation to form embryoid bodies (EBs) promotes efficient initiation of differentiation. We next determined 2 EB sizes (5K and 10K cells/EB) which improved initiation of retinal differentiation compared to scraped EBs as demonstrated by >3fold increase in expression of early eye field transcription factor Rax at day 3 of culture. The 5K and 10K EBs also facilitated the adaption of an adherent protocol for retinal differentiation to an orbital shaken suspension culture system. Size controlled EBs also enabled the selection of a permissive shaking speed (120rpm) suitable for orbital shaken culture for initiating retinal differentiation with 5K and 10K EBs. Furthermore orbital shaken culture enabled elimination of the undefined xenogeneic ingredients of Matrigel, from the culture system. This thesis demonstrates that combining size control for hiPSC derived EBs and orbital shaken culture is a feasible method for the initiation of retinal differentiation. By facilitating removal of xenogeneic materials from the culture system; the combination of orbital shaken culture with size controlled EBs may be of value to other complex stem cell differentiation systems to improve the initiation of differentiation whilst providing the potential for scalability.
... A vast number of HT cultivation platforms have been subject of research studies, while several systems (miniature shaken vessel/well, 0.2-4.0 mL; bubble column-or microplate-based mini-bioreactors, 1.0-10 mL; stirred mini-tank bioreactors, 10 mL) have been successfully commercialized in the past decade [50][51][52]. HT cultivation platform allows strains to be screened under conditions comparable to those in the manufacturing process, which can make timeline remarkably shorter for establishing optimal processes in many bio-industrial sectors [53]. ...
Synthetic biology is an emergent field of research whose aim is to make biology an engineering discipline, thus permitting to design, control, and standardize biological processes. Synthetic biology is therefore expected to boost the development of biotechnological processes such as protein production and enzyme engineering, which can be significantly relevant for lipases and esterases.
... To make the wide range of soft sensors routinely available for MBR systems, intense research is needed to enable the miniaturization of sensing probes providing rich analytical data [91] and increased flexibility to act on the individual cultures by integrated microfluidics. [25,[92][93][94] In combination with liquid handling systems, [41,42,95] software modules connecting multiple input data streams with multiple hardware actors will be used to control the individual cultures and, finally, enable the robust interplay of all hardware and software components. ...
In recent years, microbioreactor (MBR) systems have evolved towards versatile bioprocess engineering tools. They provide a unique solution to combine higher experimental throughput with extensive bioprocess monitoring and control, which is indispensable to develop economically and ecologically competitive bioproduction processes. MBR systems are based either on down-scaled stirred tank reactors or on advanced shaken microtiter plate cultivation devices. Importantly, MBR systems make use of optical measurements for non-invasive, online monitoring of important process variables like biomass concentration, dissolved oxygen, pH and fluorescence. The application range of MBR systems can be further increased by integration into liquid handling robots, enabling automatization and, thus standardization, of various handling and operation procedures. Finally, the tight integration of quantitative strain phenotyping with bioprocess development under industrially relevant conditions greatly increases the probability of finding the right combination of producer strain and bioprocess control strategy. This review will discuss the current state of the art in the field of MBR systems and we can readily conclude that their importance for industrial biotechnology will further increase in the near future.
... This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. culture), Biolector V R from m2p labs 16,17 (32 3 800 mL to 2,000 mL mammalian and microbial), 2mag bioreactor from 2mag AG (8 or 48 3 8 to 15 mL, aerobic and anaerobic microorganism) 18 and ambr V R 15 cell culture 5,15,[19][20][21] (24 3 10 to 15 mL, mammalian). ...
High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-up. This article is protected by copyright. All rights reserved.
We discuss the application of a nonlinear model predictive control (MPC) and moving horizon estimation (MHE) framework to achieve optimal operation of E. coli fed-batch cultivations with intermittent bolus feeding. 24 parallel experiments were considered in a high-throughput mini-bioreactor platform at a 10 mL scale. The robotic facility can run up to 48 fed-batch processes in parallel with automated liquid handling, online and at-line analytics. Three main challenges emerge in implementing the model-based monitoring and control framework: First, the inputs are given in an instantaneous pulsed form by bolus injections; second, online and at-line measurement frequencies are severely imbalanced; and third, optimization for the distinctive multiple reactors can be either parallelized or integrated. We address these challenges by incorporating the concept of impulsive control systems, formulating multi-rate MHE with identifiability analysis, and suggesting criteria for deciding the reactor configuration. In this study, we present the key elements and background theory of the implementation with in silico simulations for bacterial fed-batch cultivations.
Metabolic flux analysis (MFA) quantitatively describes cellular fluxes to understand metabolic phenotypes and functional behaviour after environmental and/or genetic perturbations. In the last decade, the application of stable isotopes became extremely important to determine and integrate in vivo measurements of metabolic reactions in systems biology. 13C-MFA is one of the most informative methods used to study central metabolism of biological systems. This review aims to outline the current experimental procedure adopted in 13C-MFA, starting from the preparation of cell cultures and labelled tracers to the quenching and extraction of metabolites and their subsequent analysis performed with very powerful software. Here, the limitations and advantages of nuclear magnetic resonance spectroscopy and mass spectrometry techniques used in carbon labelled experiments are elucidated by reviewing the most recent published papers. Furthermore, we summarise the most successful approaches used for computational modelling in flux analysis and the main application areas with a particular focus in metabolic engineering.
Modern biotechnological laboratories are equipped with advanced parallel mini-bioreactor facilities that can perform sophisticated cultivation strategies (e.g. fed-batch or continuous) and generate significant amounts of measurement data. These systems require not only optimal experimental designs that find the best conditions in very large design spaces, but also algorithms that manage to operate a large number of different cultivations in parallel within a well-defined and tightly constrained operating regime. Existing advanced process control algorithms have to be tailored to tackle the specific issues of such facilities such as: a very complex biological system, constant changes in the metabolic activity and phenotypes, shifts of pH and/or temperature, and metabolic switches, e.g. by product induction, to name a few. In this work we implement a model-predictive control (MPC) approach based framework to demonstrate: 1) the challenges in terms of mathematical model structure, state and parameter estimation, and optimization under highly nonlinear and stiff constraints in biological systems, 2) the adaptations required to enable its application in High Throughput Bioprocess Development (HTBD), and 3) the added value of MPC implementations when operating parallel mini-bioreactors aiming to maximize the biomass concentration while coping with hard constrains on the Dissolved Oxygen Tension profile.
Modern biotechnological laboratories are equipped with advanced parallel mini-bioreactor facilities that can perform sophisticated cultivation strategies (e.g. fed-batch or continuous) and generate significant amounts of measurement data. These systems require not only optimal experimental designs that find the best conditions in very large design spaces, but also algorithms that manage to operate a large number of different cultivations in parallel within a well-defined and tightly constrained operating regime. Existing advanced process control algorithms have to be tailored to tackle the specific issues of such facilities such as: a very complex biological system, constant changes in the metabolic activity and phenotypes, shifts of pH and/or temperature, and metabolic switches, e.g. by product induction, to name a few. In this work we implement a model-predictive control (MPC) approach based framework to demonstrate: 1) the challenges in terms of mathematical model structure, state and parameter estimation, and optimization under highly nonlinear and stiff constraints in biological systems, 2) the adaptations required to enable its application in High Throughput Bioprocess Development (HTBD), and 3) the added value of MPC implementations when operating parallel mini-bioreactors aiming to maximize the biomass concentration while coping with hard constrains on the Dissolved Oxygen Tension profile.
Automation, parallelization and autonomous operation of standard lab equipment, usually applied for manual bioprocess development, is considered as the key for reduction of bioprocess development time and costs. An automated bioreactor system with 4 stirred-tank bioreactors on a L-scale was combined with a custom-made biomass transfer system to distribute the cell suspensions produced on the L-scale into 48 parallel stirred-tank bioreactors on a mL-scale. Afterwards parallel protein expression studies automated by a liquid handling system with integrated fluorescence reader were performed. Isopropyl β-D-1-thiogalactopyranoside-induced (IPTG) expression of the red fluorescence protein mCherry was studied as an example of using fed-batch processes with recombinant Escherichia coli. In a first automated study, IPTG concentrations were varied in 48 parallel fed-batch processes with E. coli cells produced at a growth rate of 0.1 h-1 on an L-scale and transferred automatically to the mL-scale. The mCherry expression rate increased with increasing inducer concentration until the highest protein expression rate was observed at > 9 µM IPTG. In a second automated study, the growth rate of E. coli was varied between 0.1 - 0.2 h-1 in parallelly-operated stirred-tank bioreactors on a L-scale. The cells were automatically transferred and distributed into the stirred-tank bioreactors on a mL-scale and the concentration of the inducer IPTG was varied as before in parallel fed-batch processes. An increased growth rate during the production of the recombinant E. coli cells and/or higher cell densities during protein expression resulted in the increased IPTG concentrations necessary to achieve identical expression rates compared to a growth rate of 0.1 h-1 with the exception of very low inducer concentrations and inducer concentrations in excess. The new automated multi-scale cascade of parallel stirred-tank bioreactors should easily be applicable for performing fast optimisation studies with other microbial production systems and will have the potential to reduce bioprocess development time and staff assignment considerably.
The production capability of a fermentation process is predominately determined by individual strains, which are ultimately affected ultimately by interactions between the scale-dependent flow field developed within bioreactors and the physiological response of these strains. Interpreting these complicated interactions is key for better understanding the scale-up of the fermentation process. We review these two aspects and address progress in strategies for scaling up fermentation processes. A perspective on how to incorporate the multiomics big data into the scale-up strategy is presented to improve the design and operation of industrial fermentation processes.
This is the first comprehensive review on methods and materials for use in optical sensing of pH values, and on applications of such sensors. The Review starts with an introduction that contains subsections on the definition of the pH value, a brief look back on optical methods for sensing of pH, on the effects of ionic strength on pH values and pKa values, on the selectivity, sensitivity, precision, dynamic ranges and temperature dependence of such sensors. Commonly used optical sensing schemes are covered in a next main chapter, with subsections on methods based on absorptiometry, reflectometry, luminescence, refractive index, surface plasmon resonance, photonic crystals, turbidity, mechanical displacement, interferometry and solvatochromism. This is followed by sections on absorptiometric and luminescent molecular probes for use pH in sensors. Further large sections cover polymeric hosts and supports, and methods for immobilization of indicator dyes. Further and more specific sections summarize the state of the art in materials with dual functionality (indicator and host), nanomaterials, sensors based on upconversion and 2-photon absorption, multiparameter sensors, imaging, and sensors for extreme pH values. A chapter on the many sensing formats has subsections on planar, fiber optic, evanescent wave, refractive index, surface plasmon resonance and holography based sensor designs, and on distributed sensing. Another section summarizes selected applications in areas such as medicine, biology, oceanography, bioprocess monitoring, corrosion studies, on the use of pH sensors as transducers in biosensors and chemical sensors, and their integration into flow-injection analyzers, microfluidic devices and lab-on-a-chip systems. An extra section is devoted to current challenges, with subsections on challenges of general nature and those of specific nature. A concluding chapter gives an outlook on potential future trends and perspectives.
Miniaturized stirred-tank bioreactor systems provide a scalable platform for high-throughput bioprocess development. Online measurement of process variables is a major demand to enable efficient process monitoring and control in parallel operated bioreactors. One miniaturized laser light source and two photodiodes were placed around a cylindrical disposable bioreactor made of polystyrene for individual and contact-free measurement of optical density (OD). One photodiode was positioned at an angle of 28° to the laser for measuring the scattered light, a second photodiode was positioned face to face with the laser for measuring the transmitted light. Miniaturized lasers with wavelengths of 650 nm (orange), 780 nm (NIR) and 850 nm (IR) were evaluated. The best results were achieved with a laser emitting at 850 nm. Both signals (scattered and transmitted light) were influenced by the stirrer speed (usually constant) and the microorganisms under study. After individual calibration, online OD monitoring of pH-controlled fed-batch processes was successfully shown with Escherichia coli, Corynebacterium glutamicum, and Trichosporon oleaginosus. Online OD measurements based on the transmitted light signals showed low standard deviation at low cell densities, whereas scattered light signals were more accurate at higher cell densities. Correlations were reliable up to cell dry weight concentrations of 46 g L⁻¹ in stirred-tank bioreactors on a milliliter scale.
Yeast fields of application are extensive, ranging from food, brewing to green energy. The yeast Saccharomyces cerevisiae is the worldwide dominating species. In addition, S. cerevisiae is also an important model organism in modern cell biology research and is one of the most thoroughly studied eukaryotic microorganisms.This work focuses on the behaviour of yeast culture exposing to pressure induced by CO2 and O2. The pressure is set up to 9 bar (A) due to the highest pressure can be reached in industrial scale bioreactors is 8 bar (A). In order to expose yeast culture to pressure conditions, new bioreactors were built and characterised. Two experiments are designed: an experiment to investigate the yeast growth and the metabolites under pressure, as well as the molecular biology experiments to better understand yeast cells behaviour under various O₂ pressure.The first experiment has offered a better understanding of the influence of CO₂ and O₂ pressures on S. cerevisiae culture behaviour. Regarding the impact of CO₂,the study has shown that the yeast culture has consistent behaviours under different pressures. While, in terms of O₂ pressure, under 2 to 5 bar (A) air pressure, yeast cells show higher growth rates compared with atmospheric pressure. Furthermore, the antioxidant molecular glutathione kept a redox balance. Under 6 to 9 bar (A), the cells growth is inhibited and 9 bar (A) leads to the excessive oxidised glutathione accumulation.On the other hand, the molecular experiment has derived further insights on the culture behaviour under O2 pressures. The investigation of several oxidative stress induced genes has highlighted the cellular effects of oxidative stress induced by oxygen pressure and molecular mechanisms of oxidative stress response in yeast cell. It was shown that several oxidative stress induced genes were upregulated: transcription factor gene Msn2/4 and Yap 1, glutathione metabolism genes GSH2 and GLR, as well as a superoxide dismutase synthesis gene SOD2.
Microbioreactors (MBRs) have emerged as potent cultivation devices enabling automated small-scale experiments in parallel while enhancing their cost efficiency. The widespread use of MBRs has contributed to recent advances in industrial and pharmaceutical biotechnology, and they have proved to be indispensable tools in the development of many modern bioprocesses. Being predominantly applied in early stage process development, they open up new fields of research and enhance the efficacy of biotechnological product development. Their reduced reaction volume is associated with numerous inherent advantages - particularly the possibility for enabling parallel screening operations that facilitate high-throughput cultivations with reduced sample consumption (or the use of rare and expensive educts). As a result, multiple variables can be examined in a shorter time and with a lower expense. This leads to a simultaneous acceleration of research and process development along with decreased costs.MBRs range from simple miniaturized cultivations vessels (i.e., in the milliliter scale with limited possibilities for process control) to highly complex and automated small-scale microreactors with integrated sensors that allow for comprehensive screenings in very short time or a precise reflection of large-scale cultivation conditions. Progressive developments and improvements in manufacturing and automation techniques are already helping researchers to make use of the advantages that MBRs offer. This overview of current MBR systems surveys the diverse application for microbial and mammalian cell cultivations that have been developed in recent years.
Limited throughput represents a substantial drawback during bioprocess development. In recent years, several commercial microbioreactor systems have emerged featuring parallelized experimentation with optical monitoring. However, many devices remain limited to batch mode and do not represent the fed-batch strategy typically applied on an industrial scale. A workflow for 32-fold parallelized microscale cultivation of protein secreting Corynebacterium glutamicum in microtiter plates incorporating online monitoring, pH control and feeding was developed and validated. Critical interference of the essential media component protocatechuic acid with pH measurement was revealed, but was effectively resolved by 80% concentration reduction without affecting biological performance. Microfluidic pH control and feeding (pulsed, constant and exponential) were successfully implemented: Whereas pH control improved performance only slightly, feeding revealed a much higher optimization potential. Exponential feeding with µ = 0.1 h−1 resulted in the highest product titers. In contrast, other performance indicators such as biomass-specific or volumetric productivity resulted in different optimal feeding regimes.
Use of a membrane within a bioreactor (MBR), either microbial or enzymatic, is a technology that has existed for 30 years to increase process productivity and/or facilitate the recovery and the purification of biomolecules. Currently, this technology is attracting increasing interest in speeding up the process and in better sustainability. In this work, we present the current status of MBR technologies. Fundamental aspects and process design are outlined and emerging applications are identified in both aspects of engineering, i.e., enzymatic and microorganism (bacteria, animal cells, and microalgae), including microscale aspects and wastewater treatment. Comparison of this integrated technology with classical batch or continuous bioreactors is made to highlight the performance of MBRs and identify factors limiting their performance and the different possibilities for their optimization.
Unter einem Bioreaktor wird ein Apparat verstanden, in dem unter Mitwirkung von Biokatalysatoren Stoffumwandlungen mit Enzymen, Mikroorganismen oder Zellen stattfinden. Zum Homogenisieren, Suspendieren und Dispergieren müssen fluide Phasen im Bioreaktor transportiert und intensiv miteinander in Kontakt gebracht werden. Hierzu ist Energie erforderlich. Daher ist es konsequent, die vielfältigen Bauformen von Bioreaktoren nach Art des Energieeintrags zu klassifizieren. In diesem Kapitel werden die entsprechenden Bioreaktorklassen sowie geschüttelte Bioreaktoren jeweils getrennt voneinander behandelt, die wesentlichen Eigenschaften im Hinblick auf die Grundaufgaben beschrieben und die Vorgehensweisen zur Maßstabsvergrößerung von Bioprozessen mit den jeweiligen Bioreaktoren erläutert.
Geranyl glucoside, the glucosylated, high-value derivative of the monoterpenoid geraniol, has various applications in the flavor and fragrance industry and can be produced through whole-cell biotransformation of geraniol with Escherichia coli whole-cell biocatalysts expressing the glucosyltransferase VvGT14a. However, the low water solubility and high cytotoxicity of geraniol require the design of a proper biphasic system where the second, non-aqueous phase functions as an in-situ substrate reservoir. In this work, a rational selection strategy was applied for choosing suitable sequestering phases for geranyl glucoside production by whole-cell biotransformation of geraniol. Hansen solubility parameters and octanol/water distribution coefficients were used as first principle methods in combination with extensive database research to preselect 12 liquid and 6 solid sequestering phases. Subsequently, experimental approaches were applied to determine physicochemical characteristics and the distribution of geraniol and geranyl glucoside between the phases. Moreover, the effects of the sequestering phases on the whole-cell biocatalysts and on the produced geranyl glucoside concentration were measured during parallel biotransformations in milliliter-scale stirred-tank bioreactors. The fatty acid ester isopropyl myristate emerged as the best choice due to its low viscosity, very poor water solubility, low price and compatibility with the whole-cell biocatalyst. The biphasic system containing 20. % (v/v) of this solvent boosted geranyl glucoside production (4.2-fold increase of geranyl glucoside concentration in comparison to aqueous system) and exhibits advantageous partitioning of geraniol into the organic phase (logD of 2.42. ±. 0.03) and of geranyl glucoside into the water phase (logD of -2.08. ±. 0.05). The systematic selection of a suitable biphasic system constitutes basic groundwork for the development of new bioprocesses involving geraniol. Moreover, this study can serve as a guideline for selecting sequestering phases for other whole-cell biotransformation processes.
A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.
Steady state studies in a chemostat enable the control of microbial growth rate at defined reaction conditions. The effects of bacteriophage M13 infection on maximum growth rate of Escherichia coli JM109 were studied in parallel operated chemostats on a milliliter-scale to analyze the steady state kinetics of phage production. The bacteriophage infection led to a decrease in maximum specific growth rate of 15 % from 0.74 h⁻¹ to 0.63 h⁻¹. Under steady state conditions, a constant cell specific ssDNA formation rate of 0.15 ± 0.004 mgssDNA gCDW⁻¹ h⁻¹ was observed, which was independent of the growth rate. Using the estimated kinetic parameters for E. coli infected with bacteriophage M13, the ssDNA concentration in the steady state could be predicted as function of the dilution rate and the glucose concentration in the substrate. Scalability of milliliter-scale data was approved by steady state studies on a liter-scale at a selected dilution rate. An ssDNA space-time yield of 5.7 mg L⁻¹ h⁻¹ was achieved with increased glucose concentration in the feed at a dilution rate of 0.3 h⁻¹, which is comparable to established fed-batch fermentation with bacteriophage M13 for ssDNA production.
The microbial expression of intracellular, recombinant proteins in continuous bioprocesses suffers from low product concentrations. Hence, a process for the intracellular production of photoactivatable mCherry with Escherichia coli in a continuously operated cascade of two stirred-tank reactors was established to separate biomass formation (first reactor) and protein expression (second reactor) spatially. Cascades of miniaturized stirred-tank reactors were implemented, which enable the 24-fold parallel characterization of cascade processes and the direct scale-up of results to the liter scale. With PAmCherry concentrations of 1.15 g L−1 cascades of stirred-tank reactors improved the process performance significantly compared to production processes in chemostats. In addition, an optimized fed-batch process was outperformed regarding space–time yield (149 mg L−1 h−1). This study implicates continuous cascade processes to be a promising alternative to fed-batch processes for microbial protein production and demonstrates that miniaturized stirred-tank reactors can reduce the timeline and costs for cascade process characterization.
Transport limitation is regarded as one of the major phenomena leading to process yield reduction in large-scale fermentations. Knowledge of both the fluid dynamics and the microbial kinetics is needed for understanding and describing situations in large-scale production bioreactors. Microbial kinetics of Escherichia coli including flow metabolism was determined in lab-scale batch and fed-batch experiments. The effect of high substrate fluctuations on metabolism was quantified in scale-down experiments. This knowledge was incorporated into a flow model based on the compartment model approach (CMA). The flow model was verified by mixing time experiments on aerated reactors mixed with multiple impellers at different regimes with liquid volumes 8–22 m3. The integral model, predicting local glucose, acetate and biomass concentrations in different parts of the reactor, was compared to three large-scale fermentations performed in two different reactors. If lab-scale kinetics was used, the biomass prediction overestimated the biomass concentration. Lab-scale kinetics modified by the results of scale-down experiments incorporating the effect of substrate fluctuations gave a rather satisfying description of biomass concentration. Glucose gradients in different parts of the reactor and acetate produced as a result of overflow metabolism were predicted on a qualitative level. The simulations show that at present the decisive factor for a successful integration of fluid dynamics and microbial kinetics is the kinetics.
The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type). The amounts of each of these enzymes present in the E. coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration. Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force. The same is true for the two ubiquinol oxidases. Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized. In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated. The results are compatible with the proton translocation values of the various components reported from in vitro measurements. The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.
Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation
space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on
the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing
the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains
frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38
mmol liter−1 h−1, corresponding to a mass transfer coefficient of 188 h−1) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking
diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter−1 during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated
for a culture collection consisting of aerobic strains, mainly from the generaPseudomonas, Rhodococcus, andAlcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately
prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.
Mycelium-forming Streptomyces strains were grown in one milliliter liquid micro-cultures in square deep-well microtiter plates. Growth was evaluated with respect to biomass formation and production of secondary metabolites which were found to be very similar in the micro-cultures, bioreactor, and shake flask cultivations, respectively. Despite repetitive sampling and extensive growth on the walls of the wells, no cross contamination occurred. Furthermore, we successfully employed cold storage at -20 degrees C of spore suspensions (in the 96-well format), directly prepared from cultures grown on agar in the microtitre plate. Cultures were retrieved by replicating aliquots from the frozen spore suspensions.
In this work, the correct placement of a Rushton disk turbine in reactors for improving gas-liquid mass transfer has been studied experimentally in the case of surface aeration, and in particular, the power input through surface aeration is compared with that through sparger aeration. It is found that in the case of surface aeration the power input at a fixed value of the mass-transfer parameter kLa decreases as the turbine moves towards the static liquid surface. Moreover, it is interesting to find out that, for the standard sparger aeration to get the same kLa value at low or intermediate gassing rates, the power input can be greater than that for the surface aeration with the turbine placed very near the static liquid surface.
A gas-inducing impeller enables efficient recycling of gas from the headspace into the liquid. Historically, these impellers were used for the first time in froth flotation machines. The various designs of gas-inducing impellers (including those used in froth flotation) could be classified into three categories, depending on the flow pattern coming into and leaving the impeller zone. These are denoted as type 11, type 12, and type 22 systems. The critical impeller speed for the onset of gas induction (NCG) is governed by a balance between the velocity head generated by the impeller and the hydrostatic head above the impeller. A number of correlations (for types 11 and 22) are based on this balance (Bernoulli's equation). The rate of gas induction (QG) for the type 11 system can be accurately determined by equating the pressure difference (between the impeller zone and the headspace) generated by the impeller and the pressure drop required for the flow of gas. For type 22 systems, the correlations for QG are mainly empirical in nature. Correlations for the power consumption, fractional gas holdup, mass-transfer coefficient, and so forth are also available in the literature, although these studies on are not comprehensive. A process design algorithm has been presented for the design of gas-inducing impellers. The algorithm consists of the determination of the rate-controlling step, selection of geometry and the operating conditions, and an economic analysis to choose the optimum design. Guidelines have been given about the desired geometry of gas-inducing impellers for achieving different design objectives such as heat transfer, mass transfer, mixing, solid suspension, froth flotation, and so forth. It has been shown that the use of a gas-inducing impeller in a conventional stirred vessel can lead to a substantial increase in the productivity. It has been shown that the optimum geometry may not correspond to the maintenance of equal power consumption per unit volume, or equal tip speed on scale-up. Suggestions have been made for future work in this area.
The rate of gas induction was measured in gas-inducing-type mechanically agitated contactors (GIMAC) provided with two impellers. Three vessels of 0.57, 1.0, and 1.5 m i.d. were used. Tap water was used as the liquid phase, and air was used as the gas phase. Six different impeller designs (pitched-blade downflow and upflow turbines straight-blade turbine, disk turbine, and upflow and downflow propellers) were employed. From these designs, six different impeller combinations were made and an optimum combination has been proposed. The impeller speed was varied in the range of 0.30 to 15.45 rev/s. The ratio of impeller diameter to tank diameter (D/T) and the submergence (S) of upper impeller from the top were varied. The effects of clearance of lower impeller from the tank bottom (C-1) and the impeller spacing (C-3, distance between the two impellers) were also varied over a wide range. The design of the lower impeller was optimized in terms of diameter (D), blade width (W), blade angle (B-phi), number of blades (n(b)), and the blade thickness (t(b)). Rational correlations have been developed for the critical impeller speed for gas induction and the rate of gas induction.
The purpose of this work was to determine the effect of surface aeration on scale-up procedures based on maintaining a constant volumetric oxygen transfer coefficient, (K La). Oxygen mass transfer rates were measured both with and without air-sparging in fermentors of various sizes (1 to 51,000 liters). The K La values were determined from the data of transient dissolved oxygen concentration obtained during degassing and aeration periods with the aid of a fast response oxygen probe and by correcting for the probe response. Results showed that: The contribution of surface aeration to the overall K La value increases as the scale of fermentors decreases; for scale-up of aerobic fermentation process that is based on maintaining a constant K La, especially at high volumetric power input, fermentors of 200 liters or smaller are not satisfactory, unless proper consideration is given to surface aeration; and, for geometrically similar fermentors between 200 and 51,000 liters, we can propose that the K La values owing to surface aeration be determined as (K La) sj ∝ (P/v) jσj and (σ j/σ) = (v j/v) -0.3.
A new method to estimate the oxygen transfer coefficient (KLa) from the experimental dynamic response data is presented. Employing a linear model which allows for gas phase, diffusion film, and oxygen electrode dynamics, the first moment of the response curve is simply related to the sum of the model parameters. Two separate experiments are used to characterize the measurement dynamics and to measure the unknown KLa parameter. The simple calculation procedure involves only measuring the area above the response curves.
The placement of two Rushton disk turbines (DTs) in reactors for gas–liquid mass transfer has been studied in this work, and the correct position for each DT has been proposed. It is found that the volumetric mass transfer parameter kLa obtained with the correct placement of the two DTs can be substantially greater than that with the commonly adopted placement. The influence of the agitator positions on the correlation for kLa is also investigated, and it is shown that the dependence of kLa on power input is determined mainly by the upper DT position, while positions of both lower and upper DTs are important for the dependence of kLa on the superficial velocity.
The high information demand in biotechnical process development makes shaken bioreactors an essential tool for process development. By far most of all biotechnological experiments are carried out in shaken bioreactors. Nevertheless, the reaction conditions in these systems often are not representative of the following conditions of the technical scale. Therefore, results obtained in the screening system frequently can not be transferred to the production scale. This article describes a way to pragmatically scale down the technical scale to the screening system, to validate the screening system and finally to improve the validity of the generated data.
Magnetically driven stirred-columns designed for parallel operation in an incubator chamber operated like shaking flasks were characterized with respect to oxygen mass transfer and volume related power input. Power inputs of 770±60 W m−3 were measured at a stirrer speed of 900 rpm, which is in the same order of magnitude as in stirred-tank reactors and shaking flasks. Oxygen mass transfer coefficients of up to 0.34±0.05 s−1 were achieved in stirred-columns (E. coli medium). Due to their similarity to stirred-tank reactors with respect to oxygen mass transfer and power input, parallel-operated stirred-columns may be a useful new tool for microbial process development.
Rates of gas induction and fractional gas hold-up were measured in a 0.57 m i.d. gas inducing type mechanically agitated contactor. Several designs of the impeller were employed for optimizing the rate of gas induction with respect to power consumption. The effect of blade width blade, angle, blade thickness, blade curvature, number of blades, etc. on gas induction, has been investigated in detail. An optimum design has been proposed. Hysteresis in the gas induction rate and power consumption was observed for a wider impeller projecting out of stator.
On a mesuré les vitesses d'induction de gaz et de rétention fractionnelle de gaz dans un contacteur à induction de gaz agité mécaniquement, de 0,57 m de diamètre intérieur. Plusieurs types de turbines ont été utilisés pour l'optimisation de la vitesse d'induction de gaz par rapport à la consommation d'énergie. L'effet des caractéristiques des pales (largeur, angle, épaisseur, courbure, etc. ainsi que le nombre) a été étudié en détail. Une conception optimale est proposée. Une hystérésis dans l'induction de gaz et la consommation d'énergie a été observéé pour les turbines les plus larges déchargeant sur un stator fixe.
Volumetric oxygen transfer rates and power inputs were estimated by a model of the formation of primary gas bubbles at the static sparger (sinter plate) of small-scale bubble columns and a common mass-transfer correlation for bubbles rising in a non-coalescent Newtonian electrolyte solution of low viscosity. Estimations were used to assess the dimensioning and possibilities of small-scale bubble column application with an height/diameter ratio of about 1. Estimations of volumetric oxygen transfer rates (<0.16 s-1) and power inputs (<100 W m-3) with a mean pore diameter of the static sparger of 13 m were confirmed as function of the superficial air velocity (<0.6 cm s-1) by measurements using an Escherichia coli fermentation medium. Small-scale bubble columns are thus to be classified between shaking flasks and stirred-tank reactors with respect to the oxygen transfer rate, but the maximum volumetric power input is more than one magnitude below the power input in shaking flasks, which is of the same order of magnitude as in stirred-tank reactors. A small-scale bubble columns system was developed for microbial process development, which is characterized by handling in analogy to shaking flasks, high oxygen transfer rates and simultaneous operation of up to 16 small-scale reactors with individual gas supply in an incubation chamber.
Online measurement of respiration activity (including oxygen transfer rate (OTR), carbon dioxide transfer rate (CTR), respiratory quotient (RQ)) of microbial cultures in stirred bioreactors with exhaust gas analysis has been state of the art for years. As much more experiments are conducted in shaking bioreactors compared to stirred bioreactors, Anderlei and Buchs [Biochem. Eng. J. 7 (2001) 157] developed a measuring device (OTR-Device) for online determination of the oxygen transfer rate in shake flasks under sterile conditions. In this paper, an extension of the OTR-Device, termed respiration activity monitoring system (RAMOS) is described, which allows additional measurement of the carbon dioxide transfer rate and the respiratory quotient in shaking bioreactors. Fermentations of the yeasts Saccharomyces cerevisiae and Pichia stipitis carried out with RAMOS are presented. These measurements show very clearly the differences in respiration activities between the Crabtree-positive yeast S. cerevisiae and the Crabtree-negative yeast P stipitis. Furthermore, a fermentation of the bacterium Corynebacterium glutamicum is presented, showing the influence of an oxygen limitation on the metabolic activities of the culture. Also, a fermentation of a hybridoma cell line was carried out with RAMOS to elucidate the measuring sensitivity of the system. The new device provides the most important and characteristic parameters (OTR, CTR, RQ) representing biological cultures online, enabling users to draw conclusions on metabolisms of microorganisms already in shaking bioreactors. (C) 2003 Elsevier B.V. All rights reserved.
Shaking bioreactors are the most frequently used reaction vessels in biotechnology and have been so for many decades. In spite of their large practical importance, very little is known about the characteristic properties of shaken cultures from an engineering point of view. The few publications available contain to some extent contradicting statements and conflicting advice concerning the correct operating conditions of shaking bioreactors. Depending on the investigated microbial system, the engineering parameters may more or less significantly influence the experimental results in a quantitative as well as in a qualitative manner. Unfortunately, these kind of interactions are often overlooked or ignored by scientists. Precise knowledge about the controlling hydrodynamic phenomena in shaking bioreactors and quantitative information about the physical parameters influencing the cultures are needed to assure reproducible and meaningful operating conditions. In this introduction, the state of the art of culturing microorganisms in shaking bioreactors is reviewed and some issues of their practical application in screening and process development projects are addressed.
The effects of dissolved salts on the solubilities of gases were analysed based on a comprehensive set of literature data for the temperature of 298.2 K. A new empirical model was suggested which, at no increase in the number of adjustable parameters, described the data with a lower standard deviation than previously suggested models. The parameter values evaluated for the new model allow to estimate the effects of 20 cations and 19 anions on the solubilities of 15 gases.
An intermittent feeding system for shaking-flasks was developed to close the gap between batch operated shaking-flasks and fed-batch operated as well as pH-controlled stirred tank reactors. A precise syringe pump was connected via a substrate distribution system to individual 2/2-way miniature valves, one for each of up to 16 shaking-flask. The shaking-flasks were equipped with pH-probes. A process computer controls the intermittent feeding of substrates by tracking predefined individual feeding profiles as well as the base (or acid) addition for individual pH-control of the shaking-flasks. Higher concentrations of aerobic cells with higher cellular activities were achieved in fed-batch operated and pH-controlled shaking-flasks as compared to the conventional batch operation. Physiological effects of an intermittent feeding were studied in a stirred tank reactor with a recombinant E. coli strain, which expressed the GDP-mannose-pyrophosphorylase enzyme under the control of the lac-promoter.
Inconsistencies are frequently observed in data for the volumetric mass transfer coefficient for oxygen kℓa obtained by various dynamic methods. The reasons for this phenomenon are critically reviewed. On the basis of our own experimental data for water, solutions of electrolytes, glycerol and carboxy-methylcellulose, the various dynamic methods are compared with the result that only one variant gives correct results over a wide range of power dissipated per unit volume. In this variant, pure oxygen is introduced as a step input to a batch freed from any dissolved gas by e.g. vacuum desorption. All the data obtained by this method agree with those measured by the steady state sulphite method and conform to a simple relation of the form kℓa ∼ en1. The decrease in the slope n1 of a log(kℓa) vs. log e plot, or even a decrease in kℓa with increasing e at higher e values, as reported by a number of researchers, is merely a result of incorrect techniques used for kℓa measurement.
A new miniature bioreactor with a diameter equal to that of a single well of a 24-well plate is described and its engineering performance as a fermenter assessed. Mixing in the miniature bioreactor is provided by a set of three impellers mechanically driven via a microfabricated electric motor and aeration is achieved with a single tube sparger. Parameter sensitive fluorophors are used with fibre optic probes for continuous monitoring of dissolved oxygen tension and an optical based method is employed to monitor cell biomass concentration during fermentation. Experimental measurements are provided on volumetric mass transfer coefficient for air–water and bacterial fermentation data are presented for Escherichia coli.The local and average power input, energy dissipation rate and bubble size are derived from an analysis of the multiphase flow in the miniature bioreactor using computational fluid dynamics (CFD). Volumetric mass transfer coefficients are predicted using Higbie's penetration model with the contact time obtained from the CFD simulations of the turbulent flow in the bioreactor. Comparative data are provided from parallel experiments carried out in a ( working volume) conventional fermenter. Predicted and measured volumetric mass transfer coefficients in the miniature bioreactor are in the range 100–, typical of those reported for large-scale fermentation.
The gas–liquid mass transfer in 250 ml shake flasks has previously been sucessfully modelled on basis of Higbie’s penetration theory. The current contribution presents advances in understanding and modelling the gas–liquid mass transfer in shake flasks at waterlike liquid viscosity in flask sizes between 50 and 1000 ml. An experimental investigation of the maximum gas–liquid mass transfer capacity OTRmax using the sodium sulphite system was extended to relative filling volumes of 4–16%, shaking diameters of 1.25, 2.5, 5, 7, 10 cm and shaking frequencies of 50–500 rpm for the above flask sizes. Simultaneously, the previous model of the gas–liquid mass transfer was extended to a “two sub-reactor model” to account for different mechanisms of mass transfer in the liquid film on the flask wall and the bulk of the liquid rotating within the flask. The shake flask is for the first time considered to be a two-reactor system consisting of a stirred tank reactor (bulk liquid) and a film reactor (film on flask wall and base). The mass transfer into the film on the flask wall and base at “in-phase” operating conditions is described by Higbie’s penetration theory. Two different mass transfer theories were applied to successfully describe the mass transfer into the bulk liquid: a model by Kawase and Moo-Young and a model by Gnielinski. The agreement between the new modelling approach, which requires absolutely no fitting parameters and the experimental is within ±30%. The applicability of the models to a biological system was shown using a Pichia pastoris culture. This is particularly notable since geometrically non-similar liquid distributions in very different sizes of shaking flasks are covered. A comparable description of the gas–liquid mass transfer in bubble aerated reactors like stirred tanks is absolutely out of reach. A spatially- and time-resolved consideration of the mass transfer in the liquid film on the flask wall and base has shown that the validity of Higbie’s theory sensitively depends on the film thickness and contact time.
The creation of microscale fermentation procedures could have significant benefits at all stages of fermentation process development from discovery through to process optimisation. For both microbial and mammalian fermentations, pH is a vital process parameter as it has a marked affect on cell growth rate, viability and product synthesis. In this work, we describe the influence of various pH control strategies on growth and erythromycin synthesis by Saccharopolyspora erythraea CA340 at the 7 l scale and show that the effects can be reproduced in pH-controlled microscale fermentations (thousandfold scale translation). At the 7 l scale the implementation of base only or full pH control (NaOH and H3PO4 additions) significantly increased both the maximum growth rate and biomass concentrations attained compared to fermentations without pH control. There was over a twofold increase in erythromycin biosynthesis and the ratio of erythromycin A (EA) to erythromycin C (EC) increased from 2:1 to 6:1 (base only pH control) to 11:1 (full pH control). In order to measure pH during microscale fermentations, a specially designed microtitre plate was built that allowed the insertion of a micro-pH probe into each well (total well volume 7 ml). This, allowed manual base only pH control to be implemented in microwell fermentations which enhanced both the maximum specific growth rate and the maximum biomass concentration. Total erythromycin synthesis and the ratio of EA:EC were also significantly enhanced. This work has demonstrated the benefits of implementing pH control in microscale fermentations and now allows the specification of an automated pH control system.
High-cell-density cultivations of Escherichia coli in glucose-mineral-salt media produce more than 100 g dry cells litre-1 in special fed-batch modes with feeding of glucose and ammonia only. The specific growth rate can be adjusted to allow optimum recombinant protein generation.
The growth characteristics of Escherichia coli K-12 in the continuous culture with a smooth increase in the dilution rate (A-stat) of various carbon sources (glucose, acetate, succinate, glycerol, lactate, acetate + succinate, casamino, acids + glucose) were studied. For all substrates studied the maximum value of specific respiration rate, Q
O2, remained between 14–18 mmol O2 h-1 g dwt-1 and the maximum growth rate varied from 0.22 h-1 on acetate to 0.77 h-1 on glucose + casamino acids. After the respiratory capacity of the cells was exhausted at growth rates µ < µcrit, the growth yield YXO2, increased slightly when the dilution rate increased. The maximum growth rate of Escherichia coli K12 was dependent on growth yield, respiratory capacity and glycolytic capacity of the strain. Analysis of the cultivation data using a stoichiometric flux model indicated that ATP synthesis in E. coli exceeds by two-fold that (theoretically) required to build up biomass. The experimental value of mATP < 4 mmol ATP h-1 g dwt-1 determined from A-stat cultivation data was low compared with the calculated ‘unproductive hydrolysis’ of ATP (64–103 mmole ATP g dwt-1).
The design of a microbioreactor is described. An optical sensing system was used for continuous measurements of pH, dissolved oxygen, and optical density in a 2 mL working volume. The K(L)a of the microbioreactor was evaluated under different conditions. An Escherichia coli fermentation in both the microbioreactor and a standard 1 L bioreactor showed similar pH, dissolved oxygen, and optical density profiles.%The low cost of the microbioreactor, detection system, and the small volume of the fermentation broth provide a basis for development of a multiple-bioreactor system for high-throughput bioprocess optimization.
The maximum gas-liquid mass transfer capacity of 250ml shaking flasks on orbital shaking machines has been experimentally investigated using the sulphite oxidation method under variation of the shaking frequency, shaking diameter, filling volume and viscosity of the medium. The distribution of the liquid within the flask has been modelled by the intersection between the rotational hyperboloid of the liquid and the inner wall of the shaking flask. This model allows for the calculation of the specific exchange area (a), the mass transfer coefficient (k(L)) and the maximum oxygen transfer capacity (OTR(max)) for given operating conditions and requires no fitting parameters. The model agrees well with the experimental results. It was furthermore shown that the liquid film on the flask wall contributes significantly to the specific mass transfer area (a) and to the oxygen transfer rate (OTR).
Shake flasks and pH-controlled small-scale bubble columns were compared with respect to their usefulness as a basic tool for process development for human calcitonin precursor fusion-protein production with Staphylococcus carnosus. Parallel control of the pH (and making use of the base addition data) is necessary to study the effects of medium composition, to identify pH-optima and to develop a medium, which minimizes the acid excretion of S. carnosus. This medium with glycerol as energy source and yeast extract as carbon and nitrogen source resulted in cell dry weight concentration in shake flasks of 5 g l(-1), which were thus improved by a factor of 10. Cell dry weight concentrations of up to 12.5 g l(-1) were measured in the batch process with pH-controlled small-scale bubble columns due to their higher oxygen transfer capability. In contrast to shake flasks it was demonstrated, that the batch process performance of recombinant S. carnosus secreting the human calcitonin precursor fusion-protein was identical within the estimation error in pH-controlled small-scale bubble columns compared to the stirred-tank reactor.
Mixing in 96-well microplates was studied using soluble pH indicators and a fluorescence pH sensor. Small amounts of alkali were added with the aid of a multichannel pipet, a piston pump, and a piezoelectric actuator. Mixing patterns were observed visually using a video camera. Addition of drops each of about 1 nL with the piezoelectric actuator resulted in umbrella and double-disklike shapes. Convective mixing was mainly observed in the upper part of the well, whereas the lower part was only mixed quickly when using the multichannel pipet and the piston pump with an addition volume of 5 microL or larger. Estimated mixing times were between a few seconds and several minutes. Mixing by liquid dispensing was much more effective than by shaking. A mixing model consisting of 21 elements could describe mixing dynamics observed by the dissolved fluorescence dye and by the optical immobilized pH sensor. This model can be applied for designing pH control in microplates or for design of kinetic experiments with liquid addition.
Microscale processing techniques would be a useful tool for the rapid and efficient collection of biotransformation kinetic data as a basis for bioprocess design. Automated liquid handling systems can reduce labor intensity while the small scale reduces the demand for scarce materials such as substrate, product, and biocatalyst. Here we illustrate this concept by establishing the use of several microwell formats (96-round, 96-deep square and 24-round well microtiter plates) for quantification of the kinetics of the E. coli TOP10 [pQR239] resting cell catalyzed Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2en-6-one using glycerol as a source of reducing power. By increasing the biocatalyst concentration until the biotransformation rate was oxygen mass-transfer limited we can ensure that kinetic data collected are in the region away from oxygen limitation. Using a 96-round well plate the effect of substrate (bicyclo[3.2.0]hept-2en-6-one) concentration on the volumetric CHMO activity was examined and compared to data collected from 1.5-L stirred-tank experiments. The phenomenon and magnitude of substrate inhibition, observed at the larger scale, was accurately reproduced in the microwell format. We have used this as an illustrative example to demonstrate that under adequately defined conditions, automated microscale processing technologies can be used for the collection of quantitative kinetic data. Additionally, by using the experimentally determined stoichiometry for product formation and glycerol oxidation, we have estimated the maximum oxygen transfer rates as a function of well geometry and agitation rate. Oxygen-transfer rates with an upper limit of between 33 mmol. L(-1). h(-1) (based solely on product formation) and 390 mmol. L(-1). h(-1) (based on product formation and glycerol oxidation) were achieved using a 96-square well format plate shaken at 1300 rpm operated with a static surface area to volume ratio of 320 m(2). m(-3).
Gas-liquid mass transfer properties of shaken 96-well microtiter plates were characterized using a recently described method. The maximum oxygen transfer capacity (OTR(max)), the specific mass transfer area (a), and the mass transfer coefficient (k(L)) in a single well were determined at different shaking intensities (different shaking frequencies and shaking diameters at constant filling volume) and different filling volumes by means of sulfite oxidation as a chemical model system. The shape (round and square cross-sections) and the size (up to 2 mL maximum filling volume) of a microtiter plate well were also considered as influencing parameters. To get an indication of the hydrodynamic behavior of the liquid phase in a well, images were taken during shaking and the liquid height derived as a characteristic parameter. The investigations revealed that the OTR(max) is predominantly dependent on the specific mass transfer area (a) for the considered conditions in round-shaped wells. The mass transfer coefficient (k(L)) in round-shaped wells remains at a nearly constant value of about 0.2 m/h for all shaking intensities, thus within the range reported in the literature for surface-aerated bioreactors. The OTR(max) in round-shaped wells is strongly influenced by the interfacial tension, determined by the surface tension of the medium used and the surface properties of the well material. Up to a specific shaking intensity the liquid surface in the wells remains horizontal and no liquid movement can be observed. This critical shaking intensity must be exceeded to overcome the surface tension and, thus, to increase the liquid height and enlarge the specific mass transfer area. This behavior is solely specific to microtiter plates and has not yet been observed for larger shaking bioreactors such as shaking flasks. In square-shaped microtiter plate wells the corners act as baffles and cause a significant increase of OTR(max), a, and k(L). An OTR(max) of up to 0.15 mol/L/h can be reached in square-shaped wells.
Microscale processing techniques are rapidly emerging as a means to increase the speed of bioprocess design and reduce material requirements. Automation of these techniques can reduce labour intensity and enable a wider range of process variables to be examined. This article examines recent research on various individual microscale unit operations including microbial fermentation, bioconversion and product recovery techniques. It also explores the potential of automated whole process sequences operated in microwell formats. The power of the whole process approach is illustrated by reference to a particular bioconversion, namely the Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one for the production of optically pure lactones.
Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.
The reliability of dynamic measurement methods of k(l)a in fermentors using a step oxygen concentration change in the feed gas was tested. The tests were performed both for the original variant using the nitrogen right harpoon over left harpoon air exchange and the newly presented variant using the oxygen-enriched air (27 vol % O(2)) --> air exchange. The testing consisted in comparing k(l)a values determined from these methods with values determined from the steady-state Na(2)SO(3) feeding method and the dynamic pressure method, the reliability of which was proven earlier. The measurements were done in water (coalescent batch) and in 0.5M Na(2)SO(4) solution with and without the addition of 1 wt % carboxymethylcellulose (noncoalescent batches). It was found that in noncoalescent liquids the methods tested give extremely low k(l)a values (as low as 15% of the correct value). The methods are defective in principle irrespective of the gases used for exchange.
- Kostov