C - DTU Nanotech - Danmarks Tekniske Universitet

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14 2 Overview of Small–Scale Cultivation Systems cultivation is very simple and cheap, but the oxygen transfer is usually low and there is no online–monitoring or control of pH and oxygen available [23]. Microtiter plates have been used for decades in medical diagnostics and later by the pharmaceutic industry. They offer the possibility of providing a large number of parallel and small reactors with identical shape. The handling can be automated using robotics with modern pipetting and dispensing systems, etc., so that a large number of samples can be handled at the same time. In the past microtiter plates have been often used for enzyme linked immunosorbent assays and high–throughput screening [23]. But recently microtiter plates are as well used for small–scale cultivation [17, 50]. The typical culture volume varies from 0.025 − 5 ml and standard sizes are 6, 12, 24, 48, 96, 384 and 1536 wells. Microtiter plates with integrated sensors for pH [40] or dissolved oxygen [17] measurements are available. 2.2 Stirred small-scale cultivation systems Stirred small-scale cultivation systems are superior to most static or perfusion systems in terms of sampling, data collection, online–monitoring and control, and they provide a ho- mogeneous environment. Stirred minibioreactors are in their performance and monitoring very similar to conven- tional stirred bioreactors, except for their size. Temperature, pH and dissolved oxygen can be controlled. Additionally the oxygen transfer capacity is high [44]. The volume of minibioreactors ranges between 50 − 300 ml. They are ideal for dedicated research projects with costly substrates. The primary limitation, compared to other small–scale devices, is the significant effort and time to operate these reactors. Due to this reason it is difficult to apply them for high–throughput screening. 2.3 Special small–scale cultivation systems Cuvette–based minibioreactors have been developed, which employ optical sensors to suc- cessfully measure pH, dissolved oxygen and optical density [21]. The polystyrene cuvette with a total volume of 4 ml is equipped with a silicone rubber cap, which has openings for fresh air and exhaust air. The fermentation medium is agitated by small magnetic beads. The resulting pH, dissolved oxygen and optical density profiles are very similar to a parallel fermentation in a 1 l bioreactor [21]. Miniature bioreactors with integrated membrane inlet mass spectrometer probe are used for biological processes with advanced online analysis of volatile compounds like H2, CH4, O2, N2, CO2, ethanol or methanol [18]. These reactors, which are useful in analysis of respiratory dynamics, consist of a small, stirred reaction vessel with thermocouple, a pH
2.3 Special small–scale cultivation systems 15 probe, agitation, temperature control and an option for aeration. The mass spectrometer is linked to the reactor by silicon rubber or fluorohydrogcarbon membranes, which separate the reactor from the high vacuum in the mass spectrometer. Selectively only volatile compounds are introduced into the mass spectrometer, where they are ionized and separated according to their mass to charge ratio. A big advantage is the continuous and sensitive detection of small changes in the concentration of dissolved gases, which allows fast kinetic measurements and in–depth metabolic studies [28, 46]. But these reactors are expensive and the measurement of organic volatile compounds is more difficult. Microbioreactors with a working volume of 5 µl and with integrated optical sensors for the measurement of pH, optical density and dissolved oxygen have been developed recently [36]. The main part of the microbioreactor is a round chamber with a diameter of 5 mm, which is fabricated from poly (dimethyl siloxane) (PDMS), which has a high biocompatibil- ity, transparency and a high gas permeability, so that it can be used as aeration membrane. This microbioreactor has been used in batch mode with E. coli, optical density has been monitored via transmittance measurements through the well of the microbioreactor, while dissolved oxygen and pH have been detected by using fluorescence lifetime–based sensors incorporated into the body of the microbioreactor [49]. Observed dissolved oxygen, optical density and pH profiles are similar to those of a 500 ml bench–scale bioreactor [36]. This small-scale cultivation system seems to be very useful for future screening applications with extended monitoring of cultivation profiles, however the sampling has to be optimized. There are a couple of other small–scale cultivation systems, like a bubble–column minibiore- actor [43], which is useful if the required oxygen supply is moderate and if a pH controlled fed–batch cultivation is needed. Or minibioreactors for online measurements of organic compounds using NMR spectroscopy [24]. Furthermore, there are specific small-scale cultivation systems for growing mammalian cells, for example spinner–flasks [39], tissue culture flasks [39], roller bottles [14] or hollow fiber based bioreactors [13, 20, 23].
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64 7 Concluding Remarks Reactor cha
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Appendix Freezing experiments with
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Experiments with 20 µl PBS-buffer
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72 List of Figures 6.8 Heat wire de
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Bibliography [1] Arisawa, H.; Brill
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Bibliography 77 [26] Mark, H. F.: E
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Bibliography 79 [50] Zimmermann, H.
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