Process performance of parallel bioreactors for batch cultivation of Streptomyces tendae

Batch cultivations of the nikkomycin Z producer Streptomyces tendae were performed in three different parallel bioreactor systems (milliliter-scale stirred-tank reactors, shake flasks and shaken microtiter plate) in comparison to a standard liter-scale stirred-tank reactor as reference. Similar dry cell weight concentrations were measured as function of process time in stirred-tank reactors and shake flasks, whereas only poor growth was observed in the shaken microtiter plate. In contrast, the nikkomycin Z production differed significantly between the stirred and shaken bioreactors. The measured product concentrations and product formation kinetics were almost the same in the stirred-tank bioreactors of different scale. Much less nikkomycin Z was formed in the shake flasks and MTP cultivations, most probably due to oxygen limitations. To investigate the non-Newtonian shear-thinning behavior of the culture broth in small-scale bioreactors, a new and simple method was applied to estimate the rheological behavior. The apparent viscosities were found to be very similar in the stirred-tank bioreactors, whereas the apparent viscosity was up to two times increased in the shake flask cultivations due to a lower average shear rate of this reactor system. These data illustrate that different engineering characteristics of parallel bioreactors applied for process development can have major implications for scale-up of bioprocesses with non-Newtonian viscous culture broths.     

Reaction engineering studies of acetone-butanol-ethanol fermentation with Clostridium acetobutylicum

Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum has been extensively studied in recent years because the organism is recognized as an excellent butanol producer. A parallel bioreactor system with 48 stirred-tank bioreactors on a 12 mL scale was evaluated for batch cultivations of the strictly anaerobic, butanol-producing C. acetobutylicum ATCC 824. Continuous gassing with nitrogen gas was applied to control anaerobic conditions. Process performances of ABE batch fermentations on a milliliter scale were identical to the liter-scale stirred-tank reactor if reaction conditions were identical on the different scales (e.g., initial medium, pH, temperature, specific evaporation rates, specific power input by the stirrers). The effects of varying initial ammonia concentrations (0.1-4.4 g L(-1) ) were studied in parallel with respect to glucose consumption and butanol production of C. acetobutylicum ATCC 824 as a first application example. The highest butanol yield of 33% (mol mol(-1) ) was observed at initial ammonia concentrations of 0.5 and 1.1 g L(-1) . This is the first report on the successful application of a 48 parallel stirred-tank bioreactor system for reaction engineering studies of strictly anaerobic microorganisms at the milliliter scale.     

Economic comparison of diagnostic antibody production in perfusion stirred tank and in hollow fiber bioreactor processes

The total operating costs of small-scale monoclonal antibody production were calculated for two different upstream options and general downstream procedure based on protein A chromatography. The upstream options were a spin-filter equipped stirred-tank bioreactor (STR) and a hollow fiber bioreactor (HFB). Both the bioreactors were operated in perfusion mode. The total operating costs of the processes were 6,900 €/g for STR option and 6,400 €/g for the HFB option. In the both systems, the costs were dominated by expenses derived from the downstream section (almost 80%) that was almost identical in the both systems. In the upstream section, the investment depreciation was the largest cost item. The lower total costs of the HFB option were a result of lower investment costs and more concentrated product that led into savings also in downstream section. This study brings out the HFB as on viable alternative for stirred-tank bioreactor, especially in small-scale diagnostic monoclonal antibody production.     

New milliliter‐scale stirred tank bioreactors for the cultivation of mycelium forming microorganisms

A novel milliliter‐scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter‐scale. A newly designed one‐sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface‐to‐volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k_La) > 0.15 s^?1 were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter‐scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory‐scale stirred tank bioreactor with six‐bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter‐scale stirred tank bioreactor was reduced compared to the laboratory‐scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale‐up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter‐scale (standard deviation < 8%) with up to 48 stirred tank bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear‐thinning non‐Newtonian behavior. The newly developed one‐sided paddle impellers operated in unbaffled reactors on a 10 milliliter‐scale with a magnetic inductive drive for up to 48 parallel bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically. Biotechnol. Bioeng. 2010; 106: 443–451. © 2010 Wiley Periodicals, Inc.     

Process-engineering characterization of small-scale bubble columns for microbial process development

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 µm 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.     

Miniature bioreactors: current practices and future opportunities

This review focuses on the emerging field of miniature bioreactors (MBRs), and examines the way in which they are used to speed up many areas of bioprocessing. MBRs aim to achieve this acceleration as a result of their inherent high-throughput capability, which results from their ability to perform many cell cultivations in parallel. There are several applications for MBRs, ranging from media development and strain improvement to process optimisation. The potential of MBRs for use in these applications will be explained in detail in this review. MBRs are currently based on several existing bioreactor platforms such as shaken devices, stirred-tank reactors and bubble columns. This review will present the advantages and disadvantages of each design together with an appraisal of prototype and commercialised devices developed for parallel operation. Finally we will discuss how MBRs can be used in conjunction with automated robotic systems and other miniature process units to deliver a fully-integrated, high-throughput (HT) solution for cell cultivation process development.     

Biocatalytic transformations in ionic liquids

Room temperature ionic liquids are non-volatile, thermally stable and highly polar; they are also moderately hydrophilic solvents. Here, we discuss their use as reaction media for biocatalysis. Enzymes of widely diverging types are catalytically active in ionic liquids or aqueous biphasic ionic liquid systems. Lipases, in particular, maintain their activity in anhydrous ionic liquid media; the (enantio)selectivity and operational stability are often better than in traditional media. The unconventional solvent properties of ionic liquids have been exploited in biocatalyst recycling and product recovery schemes that are not feasible with traditional solvent systems.     

High-performance recombinant protein production with <Emphasis Type="Italic">Escherichia coli</Emphasis> in continuously operated cascades of stirred-tank reactors

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.     

Design and development of a new ambr250® bioreactor vessel for improved cell and gene therapy applications

The emergence of cell and gene therapies has generated significant interest in their clinical and commercial potential. However, these therapies are prohibitively expensive to manufacture and can require extensive time for development due to our limited process knowledge and understanding. The automated ambr250® stirred-tank bioreactor platform provides an effective platform for high-throughput process development. However, the original dual pitched-blade 20 mm impeller and baffles proved sub-optimal for cell therapy candidates that require suspension of microcarriers (e.g. for the culture of adherent human mesenchymal stem cells) or other particles such as activating Dynabeads® (e.g. for the culture of human T-cells). We demonstrate the development of a new ambr250® stirred-tank bioreactor vessel which has been designed specifically to improve the suspension of microcarriers/beads and thereby improve the culture of such cellular systems. The new design is unbaffled and has a single, larger elephant ear impeller. We undertook a range of engineering and physical characterizations to determine which vessel and impeller configuration would be most suitable for suspension based on the minimum agitation speed (N_JS) and associated specific power input (P/V)_JS. A vessel (diameter, T, = 60 mm) without baffles and incorporating a single elephant ear impeller (diameter 30 mm and 45° pitch-blade angle) was selected as it had the lowest (P/V)_JS and therefore potentially, based on Kolmogorov concepts, was the most flexible system. These experimentally-based conclusions were further validated firstly with computational fluid dynamic (CFD) simulations and secondly experimental studies involving the culture of both T-cells with Dynabeads® and hMSCs on microcarriers. The new ambr250® stirred-tank bioreactor successfully supported the culture of both cell types, with the T-cell culture demonstrating significant improvements compared to the original ambr250® and the hMSC-microcarrier culture gave significantly higher yields compared with spinner flask cultures. The new ambr250® bioreactor vessel design is an effective process development tool for cell and gene therapy candidates and potentially for autologous manufacture too.

     

Water immiscible ionic liquids as solvents for whole cell biocatalysis

Whole cell biocatalysis can effectively be used for the production of enantiomerically pure compounds, but efficiency is often low. Toxicity and poor solubility of substrates and products are the main obstacles. In this study, water immiscible ionic liquids are shown to have no damaging effects on the cell membranes of Escherichia coli and Saccharomyces cerevisiae. Thus, they can be used as biocompatible solvents for microbial biotransformations exemplified by an increase in yield of chiral alcohol synthesis. As key point to the success of these processes, the distribution ratio of the reactants between the ionic liquid and the aqueous phase was identified. The use of ionic liquids as substrate reservoir and in situ extracting agent for the asymmetric reduction of various ketones resulted in an increase of chemical yield from <50% to 80-90% in simple batch processes. (R)-1-(4-chlorophenyl)ethanol was produced at a higher initial reaction rate in the biphasic system (>50 microM s(-1) L(-1)) compared to the aqueous system. This result demonstrates that good mass transfer rates can be obtained despite the relatively high viscosity of ionic liquids.     

Small-scale bioreactor system for process development and optimization

An agitated 12-well microtiter plate system with a working volume of 2ml was investigated for cell culture process development. Agitation assures homogeneity in wells and enhances mass transfer between the gas and the liquid phase, thus improving maximum cell density and pH stability. The pH of the NaHCO(3)-buffered system can be adjusted by altering the carbon dioxide content of the gas phase. The non-toxic, visual pH indicator phenol red was used in combination with a spectrophotometric plate reader for rapid and precise pH measurements. For high throughputs, cell growth was assessed non-invasively using stable green fluorescent protein (GFP) expressing cells and a fluorescence plate reader. The setup is simple and inexpensive. The system can be automated and allows several hundred small-scale bioreactor experiments to be run in parallel.     

Pervaporation-aided enzymatic esterifications in non-conventional media

This paper focuses on enzymatic esterifications in non-conventional media (organic solvents, ionic liquids, and solvent-free systems) with reference to the water removal. Different types of water removal techniques are reviewed with a special emphasis on pervaporation. Pervaporation is a separation process in which liquid is transported through a selective membrane with simultaneous evaporation of permeates. In an integrated process where pervaporation is coupled with a bioreactor where esterification is performed, selective removal of water or other esterification products can be achieved. In this manner benefit can be doubled, due to the equilibrium shift and possible pure product recovery. Available literature on esterifications coupled with pervaporation is presented in detail. Reviewed examples are divided according to the type of reaction media.     

Enzyme catalysis in ionic liquids

Ionic liquids offer new possibilities for the application of solvent engineering to biocatalytic reactions. Although in many cases ionic liquids have simply been used to replace organic solvents, they have often led to improved process performance. Unlike conventional organic solvents, ionic liquids possess no vapor pressure, are able to dissolve many compounds, and can be used to form two-phase systems with many solvents. To date, reactions involving lipases have benefited most from the use of ionic liquids, but the use of ionic liquids with other enzymes and in whole-cell processes has also been described. In some cases, remarkable results with respect to yield, (enantio)selectivity or enzyme stability were observed.     

Scale-up of Escherichia coli growth and recombinant protein expression conditions from microwell to laboratory and pilot scale based on matched k(L)a

Fermentation optimization experiments are ideally performed at small scale to reduce time, cost and resource requirements. Currently microwell plates (MWPs) are under investigation for this purpose as the format is ideally suited to automated high-throughput experimentation. In order to translate an optimized small-scale fermentation process to laboratory and pilot scale stirred-tank reactors (STRs) it is necessary to characterize key engineering parameters at both scales given the differences in geometry and the mechanisms of aeration and agitation. In this study oxygen mass transfer coefficients are determined in three MWP formats and in 7.5 L and 75 L STRs. k(L)a values were determined in cell-free media using the dynamic gassing-out technique over a range of agitation conditions. Previously optimized culture conditions at the MWP scale were then scaled up to the larger STR scales on the basis of matched k(L)a values. The accurate reproduction of MWP (3 mL) E. coli BL21 (DE3) culture kinetics at the two larger scales was shown in terms of cell growth, protein expression, and substrate utilization for k(L)a values that provided effective mixing and gas-liquid distribution at each scale. This work suggests that k(L)a provides a useful initial scale-up criterion for MWP culture conditions which enabled a 15,000-fold scale translation in this particular case. This work complements our earlier studies on the application of DoE techniques to MWP fermentation optimization and in so doing provides a generic framework for the generation of large quantities of soluble protein in a rapid and cost-effective manner.     

Production of biodiesel fuel from soybean oil catalyzed by fungus whole-cell biocatalysts in ionic liquids

The methanolysis of soybean oil to produce a fatty acid methyl ester (ME, i.e., biodiesel fuel) was catalyzed by lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst in the presence of ionic liquids. We used four types of whole-cell biocatalysts: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL), recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL), Candida antarctica lipase B (r-CALB), and mono- and diacylglycerol lipase from A. oryzae (r-mdlB). w-ROL gave the high yield of fatty acid methyl ester (ME) in ionic liquid [Emim][BF₄] or [Bmim][BF₄] biphasic systems following a 24 h reaction. While lipases are known to be severely deactivated by an excess amount of methanol (e.g. 1.5 Mequiv. of methanol against oil) in a conventional system, methanolysis successfully proceeded even with a methanol/oil ratio of 4 in the ionic liquid biphasic system, where the ionic liquids would work as a reservoir of methanol to suppress the enzyme deactivation. When only w-ROL was used as a biocatalyst for methanolysis, unreacted mono-glyceride remained due to the 1,3-positional specificity of R. oryzae lipase. High ME conversion was attained by the combined use of two types of whole-cell biocatalysts, w-ROL and r-mdlB. In a stability test, the activity of w-ROL was reduced to one-third of its original value after incubation in [Bmim][BF₄] for 72 h. The stability of w-ROL in [Bmim][BF₄] was greatly enhanced by cross-linking the biocatalyst with glutaraldehyde. The present study demonstrated that ionic liquids are promising candidates for use as the second solvent in biodiesel fuel production by whole-cell biocatalysts.     

A review of advanced small-scale parallel bioreactor technology for accelerated process development: current state and future need

The pharmaceutical and biotech industries face continued pressure to reduce development costs and accelerate process development. This challenge occurs alongside the need for increased upstream experimentation to support quality by design initiatives and the pursuit of predictive models from systems biology. A small scale system enabling multiple reactions in parallel (n ≥ 20), with automated sampling and integrated to purification, would provide significant improvement (four to fivefold) to development timelines. State of the art attempts to pursue high throughput process development include shake flasks, microfluidic reactors, microtiter plates and small-scale stirred reactors. The limitations of these systems are compared to desired criteria to mimic large scale commercial processes. The comparison shows that significant technological improvement is still required to provide automated solutions that can speed upstream process development.     

Regime analysis of a Baeyer–Villiger bioconversion in a three-phase (air–water–ionic liquid) stirred tank bioreactor

The aim of this work was to conduct a regime analysis on a three-phase (air–water–ionic liquid) stirred tank bioreactor of the Baeyer–Villiger bioconversion process, using [MeBuPyrr][BTA] ionic liquid as the dispersed phase. The regime analysis based on characteristic times of the different mechanisms involved (mixing, mass transfer, reaction) can yield a quantitative estimate of bioreactor performance. The characteristic time obtained for oxygen uptake rate (54 s⁻¹) was among the characteristic times determined for oxygen transfer (13–129 s⁻¹) under different operating conditions, suggesting that the oxygen transfer rate under certain operating conditions could be a limiting step in the bioconversion process. Further enhancement of oxygen transfer rates requires proper selection of the bioreactor operational conditions, and improved design of the ionic liquid used as oxygen transfer vector.     

Methods and milliliter scale devices for high-throughput bioprocess design

Based on electromagnetic simulations as well as on computational fluid dynamics simulations gas-inducing impellers and their magnetic inductive drive were optimized for stirred-tank reactors on a 10 ml-scale arranged in a bioreaction block with 48 bioreactors. High impeller speeds of up to 4,000 rpm were achieved at very small electrical power inputs (63 W with 48 bioreactors). The maxima of local energy dissipation in the reaction medium were estimated to be up to 50 W L⁻¹ at 2,800 rpm. Total power input and local energy dissipation are thus well comparable to standard stirred-tank bioreactors. A prototype fluorescence reader for 8 bioreactors with immobilized fluorometric sensor spots was applied for online measurement of dissolved oxygen concentration making use of the phase detection method. A self-optimizing scheduling software was developed for parallel control of 48 bioreactors with a liquid-handling system for automation of titration and sampling. It was shown on the examples of simple parallel batch cultivations of Escherichia coli with different media compositions that high cell densities of up to 16.5 g L⁻¹ dry cell mass can be achieved without pH-control within 5 h with a high parallel reproducibility (standard deviation<3.5%, n=48) due to the high oxygen transfer capability of the gas-inducing stirred-tank bioreactors.     

Mathematical modeling of the integrated process of mercury bioremediation in the industrial bioreactor

The mathematical model of the integrated process of mercury contaminated wastewater bioremediation in a fixed-bed industrial bioreactor is presented. An activated carbon packing in the bioreactor plays the role of an adsorbent for ionic mercury and at the same time of a carrier material for immobilization of mercury-reducing bacteria. The model includes three basic stages of the bioremediation process: mass transfer in the liquid phase, adsorption of mercury onto activated carbon and ionic mercury bioreduction to Hg(0) by immobilized microorganisms. Model calculations were verified using experimental data obtained during the process of industrial wastewater bioremediation in the bioreactor of 1 m³ volume. It was found that the presented model reflects the properties of the real system quite well. Numerical simulation of the bioremediation process confirmed the experimentally observed positive effect of the integration of ionic mercury adsorption and bioreduction in one apparatus.     

A recyclable enzymatic biodiesel production process in ionic liquids

Immobilized Candida antarctica lipase B suspended in ionic liquids containing long alkyl-chain cations showed excellent synthetic activity and operational stability for biodiesel production. The interest of this process lies in the possibility of recycling the biocatalyst and the easy separation of the biodiesel from the reaction mixture. The ionic liquids used, 1-hexadecyl-3-methylimidazolium triflimide ([C(16)MIM][NTf(2)]) and 1-octadecyl-3-methylimidazolium triflimide ([C(18)MIM][NTf(2)]), produced homogeneous systems at the start of the reaction and, at the end of the same, formed a three-phase system, allowing the selective extraction of the products using straightforward separation techniques, and the recycling of both the ionic liquid and the enzyme. These are very important advantages which may be found useful in environmentally friendly production conditions.