Biochemical Engineering Journal (v.7, #2)

Organisers’ preface by Markus Kühner (89).

Editor’s preface by Colin Webb; Shintaro Furusaki (89).

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.
Keywords: Shaking bioreactors; Baffle; Power consumption; Mixing; Fluid mechanics; Oxygen transfer;

The maximum gas–liquid mass transfer capacity of 250 ml 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 (OTRmax) 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).
Keywords: Shaking bioreactors; Oxygen transfer; Screening; Process development;

Mass transfer resistance of sterile plugs in shaking bioreactors by Christian Mrotzek; Tibor Anderlei; Hans-Jürgen Henzler; Jochen Büchs (107-112).
One of the mass transfer resistances for the gas exchange of shaking flasks is the sterile plug. The gas exchange through the sterile plug is described by an extended model of Henzler and Schedel [Bioprocess Eng. 7 (1991) 123]. Based on this model, a new method was developed to obtain the mass transfer resistance of various sterile closures. It consists of measuring the water evaporation rate of the shaking flask and is therefore very easily applied. Sterile plugs made of cotton, wrapped paper, urethane foam and fibreglass and caps made out of aluminium and silicone have been examined. Instead of the oxygen transfer coefficient (k O2 ), which is commonly found in the literature, the carbon dioxide diffusion coefficient (D CO2 ) is used to describe the mass transfer resistance of the sterile plug. The investigation revealed that this resistance is mainly dependent on the neck geometry and to a lesser extent on the plug material and density. The gas exchange of aluminium-caps was not reproducible.
Keywords: Sterile plug; Shaking bioreactor; Oxygen transfer; Fermentation; Diffusion; Modelling;

Growth of heterogeneous culture collections in microtiter plates is advantageous for logistic reasons and also in enabling significant savings in medium costs, labor input and use of equipment during large screening projects. The main hurdles to overcome for aerobic microbial strains are the prevention of cross-contamination and excessive evaporation while assuring sufficient aeration rates. For this purpose we developed a sandwich spongy silicone/cotton wool cover to close the wells of square-deepwell microtiter plates. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and were shown to be threefold higher during orbital shaking at a shaking diameter of 5 cm at 300 rpm (24 mmol O2  l−1  h−1 at a culture volume of 0.75 ml) in comparison to a shaking diameter of 2.5 cm. Photographic analysis showed a clear influence of the shaking diameter on the hydrodynamic behavior in the wells; during shaking at a 2.5 cm amplitude, out-of-phase conditions occurred resulting in poor vertical mixing, while a 5 cm shaking amplitude led to an optimal surface to volume ratio and a turbulent flow.
Keywords: Heterogeneous culture; Microtiter plate; Oxygen transfer rate;

Small-scale bioreactor system for process development and optimization by P. Girard; M. Jordan; M. Tsao; F.M. Wurm (117-119).
An agitated 12-well microtiter plate system with a working volume of 2 ml 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 NaHCO3-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.
Keywords: Bioreactors; Small-scale; Optimization; Screening tool; Animal cell culture; Transient transfection;

The feasibility of using shake flasks to culture animal cells was evaluated using various sizes of cylindrical shaped vessels as bioreactors. It was found that conditions can be optimized so that hybridoma, Chinese Hamster Ovary cells, and insect cells can be efficiently cultured in the shaking reactors to cell densities comparable to that obtained with stirred-jar bioreactors, and the system is scalable to larger volumes for the production of recombinant proteins or cell mass production in the laboratory.
Keywords: Animal cell culture; Bioreactors; Fed-batch culture; Fermentation; Insect cell culture; Hybridoma culture;

Scale-up of filamentous organisms from tubes and shake-flasks into stirred vessels by Werner Katzer; Mark Blackburn; Kevin Charman; Steven Martin; Julia Penn; Stephen Wrigley (127-134).
The choice of small-scale fermentation systems contributes significantly to a successful scale-up. Creasing of flasks and the chosen shaker parameters influence the production of secondary metabolites in a strain- and even compound-specific manner. Using actinomycetes and fungi as model organisms the influence of the small-scale fermentation system on the production of various secondary metabolites is described and the effects on screening success and scale-up are considered.
Keywords: Filamentous organism; Fungi; Antinomycetes; Tubes; Shake-flask; Secondary metabolite; Fermentation;

Out-of-phase operating conditions, a hitherto unknown phenomenon in shaking bioreactors by Jochen Büchs; Stefan Lotter; Claudia Milbradt (135-141).
One of the important parameters in characterising fermentations of aerobic microorganisms is the specific power consumption. A new method has been introduced which enables the accurate determination of the power consumption in shaking bioreactors. It is based on torque measurements in the drive and the appropriate compensation of the friction losses. Measurements of the power consumption revealed the phenomenon of the liquid being ‘out-of-phase’ for the first time for shaking bioreactors. This occurs at certain operating conditions and is characterised by an increasing amount of liquid not following the rotating movement of the shaker table, thus reducing the specific power consumption, mixing and the gas/liquid mass transfer. With respect to this, different hydrodynamic cases have to be distinguished. All these cases have in common, however, that the probability of ‘out-of-phase’ conditions increases with lower shaking diameters, lower filling volumes, larger number and sizes of baffles and higher viscosity. For unbaffled flasks with a nominal volume ≤1 l the ‘out-of-phase’ phenomenon is described in the form of a newly defined non-dimensional Phase number (Ph). To avoid the (unidentified) development of a screening project in unfavourable directions or even its complete failure, researchers must be aware of the ‘out-of-phase’ phenomenon. The experimental protocols have to be carefully selected so that the occurrence of such unwanted hydrodynamic conditions is not possible under all experimental circumstances.
Keywords: Shaking bioreactors; Agitation; Power consumption; Mixing; Screening; Process development;

Performance of a shaking vessel with current pole by Yoshihito Kato; Setsuro Hiraoka; Yutaka Tada; Toshio Nomura (143-151).
To improve solid particle suspensions in liquids in a shaking vessel, a pole was installed at the axis of the shaking vessel, which was referred to as the “current pole”. The performance of a shaking vessel with current pole at its central axis was examined experimentally with respect to particle dispersion, power consumption, mixing time and solid–liquid mass transfer coefficient. The current pole improved the particle suspension without an increase in power consumption and reduced the critical circulating frequency for complete suspension. The current pole was very effective in eliminating the stagnation point on the vessel bottom and to decrease the mixing time. The mass transfer coefficient with a current pole had the same value as that without a current pole above the critical circulating frequency for complete suspension. As the diameter of the current pole increased, the mixing time decreased. A pole diameter of 5% of the vessel diameter was effective for suspension.
Keywords: Solid particle suspension; Current pole; Shaking vessel;

A new mixing probe has been developed which measures the motions of the fluid during mixing as pressure fluctuations and converts the measurements into a mixing signal (MS). The MS is the root mean square (RMS) pressure fluctuation in the 1–64 Hz range as determined by a sensitive pressure sensor and a digital signal processor specifically designed for the purpose. The MS is a measure of the actual mixing flow of the fluid rather than a measurement of the input motions or energies into the reactor system (e.g. RPM, torque or power). In other studies, the MS has been measured as a function of mixing speed in numerous sized reactors from 10 to 1000 l, and provides consistent and reproducible measurements. The MS increases monotonically as a function of mixing speed, with a change of slope corresponding to the transition from laminar to turbulent mixing regimes. Maps of MS as a function of location in the reactor are useful in understanding stirred tank reactor design and performance. Quantitative measurements of mixing are especially useful during process development as a tool to increase the success of scale-up during the transition from process development to manufacturing. Measurements at a fixed location in a given reactor are useful in understanding changes in mixing that occur during the course of a given process, and are useful in manufacturing situations where validated documentation of lot-to-lot consistency of mixing is required (e.g. pharmaceutical manufacturing). In addition, the probe has been used to measure mixing in vessels with vibrational mixers with similar results. The probe has been successfully used in feedback loops to control either mixing speed or vibrational mixing amplitude in order to maintain constant mixing of the fluid during processing. With this system it is possible to maintain constant mixing over a wide range of fluid volumes in a given reactor, and, for instance, to compensate for changes in viscosity throughout the course of the process. Adaptations of this system for the measurement of mixing in shake-flasks is described in this paper.
Keywords: Mixing signal; Root mean square; Reactor system; Mixing; Shake-flask;

The oxygen transfer rate (OTR) is the most suitable measurable parameter to quantify the physiological state of a culture of aerobic microorganisms since most metabolic activities depend on oxygen consumption. Online measurement of the oxygen transfer rate in stirred bioreactors is state of the art although technically difficult. However, the online determination of the oxygen transfer rate in shaking bioreactors under sterile conditions has not been possible until recently. A newly developed measuring device eliminates this deficit. Extremely useful information about cultivating conditions and the physiological state of microorganisms can be gained in early stages of research and bioprocess development from many reactors operated in parallel.
Keywords: Shaking bioreactor; Oxygen transfer; Bioprocess monitoring; Respiration; Bioreactor; Fermentation;

Parallel substrate feeding and pH-control in shaking-flasks by Dirk Weuster-Botz; Jutta Altenbach-Rehm; Matthias Arnold (163-170).
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.
Keywords: Parallel substrate feeding; pH-control; Shaking-flask; Intermittent feeding; Fed-batch; E. coli;