monitoring of bioprocess -...

InstrumentationofBiotechnologicalProcesses

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Monitoring of Bioprocess

Process Monitoring Requirements

Cellular activities such as those enzymes,DNA,RNA and other components are the primary variables which determine the performance of microbial or cellular cultures. The development of specific analytical tools for measurement of these activities in vivo is therefore essential importance in order to achieve direct analytical access to these primary variables. The focus needs to be the minimization of relevant disturbances of cultures by measurements,i.e. rapid, non-invasive concepts should be promoted in bioprocess engineering science. What we can measure routinely today are the operating and secondary variables such as the concentrations of metabolites which fully depend on primary and operating variables.

In comparison to other disciplines such as physics or engineering,sensors useful for in situ monitoring of biotechnological processes are comparatively few; they measure physical and chemical variables rather than biological ones. The reason are manifold but, generally,biologically relevant variables are much more difficult and complex than others.

Standard Techniques ( State of Routine)

There are undoubtedly a few variables that are generally regarded as a must in bioprocess engineering. Among these are several physical, less chemical and even less biological variables.

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Biomass

Biomass concentration is of paramount importance to scientists as well as engineers. It is a simple measure of the available quantity of biocatalyst and is definitely an important key variable because it determines-simplifying-the rates of growth and / or product formation. Almost all mathematical models used to describe growth or product formation contain biomass as a most important state variable. Many control strategies involve the objective of maximizing biomass concentration; it remains to be decided whether this is always wise.

The measure of mass is important with respect to calculating mass balance. However, the elemental composition of biomass is normally ill defined. Another reason for determining biomass is the need for a reference when calculating specific rates. qi = ri/x. An ideal measure for the biocatalysts in a bioreaction system of interest would be their activity,physiological state,morphology or other classification rather than just their mass.


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Unfortunately, these are even more difficult to quantify objectively and this is obviously why the biomass concentration is still the greatest interest.

On-Line Sensing Devices

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On-line is synonymous for fully automatic. No manual interaction is necessary to obtain the desired results.

In Situ Instruments

Temperature

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Generally, the relationship between growth and temperature is strain-dependent and shows a distinct optimum. Hence, temperature should be maintained at this level by closed loop control. Industry seems to be satisfied with control precision +/- 0.4 K. Temperature can be the variable most often determined in bioprocess. In the range between o and 130 °C, this can be performed using thermoelements or by thermometers based on resistance changes, e.g. of a platinum wire ( then this sensor is called a Pt-100 or Pt-1000 sensor; the resistance is either 100 or 1000 Ohm at 0°C. This is, although not linear per se, one of the most reliable but not necessarily most accurate measures in bioprocesses. The necessary calibration references are usually not available. Temperature is most often controlled. ( Precision 1-10 mK)

pH

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pH is one of the often controlled in bioprocess operated in bioreactors because enzymatic acitivities and, therefore, metabolism is very sensitive to pH changes. The acidification derives in most cases predominantly from the ammonia uptake when ammonium ions are provided as the nitrogen source; NH3 is consumed and the proton left over from the NH4+ causes a drop in pH. In shake flask cultures, there is only one reasonable possibility to keep pH within a narrow range,namely the use of a very strong buffer,usually phosphate buffer. This is the major reason why culture media often contain a tremendous excess of phosphate. Insertion of multiple pH probes and titrant-addition tubes into shakers has, however, been proposed and marketed.


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Describe picture 7

Schematic design of a sterilizable pH electrode made of glass. The pH-sensitive glass which develops a gel layer with highest mobility for protons is actually only the tip (calotte) of the electrode.Electrloytes can contain gelling substances.Double ( or so called bridged) electrolyte electrodes are less sensitive to poisoning of the reference electrode.

Describe picture 10 Field effect Transistor

Schematic design of a usual metal oxide field effect transistor (MOSFET;top) and of an ion-sensitive field effect transistor (IsFET,bottom).The voltage applied to the gate- which is the controlling electrode-determines the current that flowes between source and drain. The substrate is p-Si,source and drain are n-Si, the metal contacts are made from Al, and the insulators are Si3N4.Instead of a metallic gate, a pH-FET hast a gate from nitrides or oxides, for instance Ta2O5. Depending on the pH of the measuring solution , the voltage at the interface solution/gate-oxide changes and controls the source drain current. Generally, in bio-FETs (which are also biosensors, of course) and additional layer of immobilized enzymes, whole cells, antibodies, antigens or receptor is mounted on top of the gate;the reaction must, of course, affect the pH producing or consuming protons to be detectable with this transducer.Note that the refernce electrode is still necessary; this means that all problems associated with the refernce pertain also to such a semiconductor-based electrode.

Oxygen Partial Pressure (pO2)

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Oxygen solubility is low in aqueous solutions, namely 36 mg l-1 bar -1 at 30 °C in pure water. Mass transfer is, therefore, derminant whether a culture suffers from oxygen limitaton or not. Several attempts to measure pO2 have been made in the past. Generally , oxygen is reduced by means of a cathode operated at a polarizing potential of 600-750 mV which is generated either externally (polagraphic method) or internally (galvanic method).A membrane separates the electrolyte from the medium to create some selectivity for diffusible substances rather than nondiffusible materials. The membrane is responsible for the dynamic sensor characteristics which are diffusion controlled. Less sensivity to membrane fouling and changes in flow conditions have been reported for transient measuring techniques, where the reducing voltage is applied in a pulsed mode, a deviation from common continous oxygen reduction. A control loop for low pO2 (<100ppb) based on a fast but non-sterilizable sensor.


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Describe picture

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Schematic design of a Clark-type oxygen partial pressure (pO2) electrode. A (sandwiched) membrane through which oxygen must diffuse separates the measuring solution from the electrolyte. Oxygen is reduced by electrons coming from the central platinum cathode which is surrounded by a glass insulator. The anode is a massive silver ring usually mounted around the insulator. This design , a so called polarographic electrode, needs an external power supply. For oxygen, the polarization voltage is the order of 700 mV and the typical current for atmospheric pO2 is in the order of 10-7 A. A built in thermistor allows automatic correction of the temperature dependet drift of approxiametely 3 % K-1 at around 30°C.

Oxygen in the Gas Phase

siehe Bild Seite 18 paramagnetic

Measurements of oxygen in the gas phase are based on its paramagenetic properties. Any change in the mass concentration of O2 affects the densitiy of a magnetic field and thus the forces on any (dia-or para) magnetic material in this field. These forces on , for example, an electroblance can be compensated electrically and the current can be converted into mass concentrations:further conversion into a molar ratio, e.g. % O2, requires the knowledge of total pressure. The effect of oxygen on metabolism is better known than the effects of other nutrients. Analysis of O2 as well CO2 in exhaust gas in becoming generally accepted and is likely to be applied as a standard measuring technique in bioprocessing. It is possible to multiplex the exhaust gas lines from several reactors in order to reduce costs.However, it should be taken into account that the time delay of depending on the efforts for gas transport (active,passive) and sample pretreatment (drying,filtering of the gas aliquot).

Describe picture with magnet

Schematic design of a paramagnetic oxygen analyzer. A diamagnetic electrobalance is placed in a permanent magnetic field. Whenever the paramagnetic oxygen enters this space, the field lines intensify and exert a force on the diamagnetic balance trying to move it out of the field. This force is compensated by powering the electric coils around the balance so much that it does not change is position in the field. The current is proportional to the mass of paramagnetic matter (i.e. oxygen) in the measuring cell, i.e. concentration and not a fraction or content.


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Carbon Dioxide Partial Pressure pCO2

siehe Seite 16 und 17

CO2 effects microbial growth in various ways according to its appearance in catabolism as well as in anabolism. Morphological changes and variations in growth and metabolic acid rates in response to pCO2 have been demonstrated. pCO2 can be measured indirectly: the pH value of a bicatbonate buffer, separated from the medium by a gas-permeable membrane, drops whenever CO2 diffuses into this compartiment and vice versa. The response of the pCO2 sensor is not exclusively CO2 dependet.

Describe picture

Schematic design of a carbon dioxide partial pressure electrode. CO2 diffuses trough the membrane into or out of the electrolyte where it equilibrates with HCO3- thus generating or consuming protons. The respective pH change of the electrolyte is sensed with a pH electrode and is logarithmically proportional to the pCO2 in the measuring solution. Since the electrolyte may become exhausted, one can replace it trough in /out lines. These can also be used to re-calibrate the pH electrode. Therefore, the electrode os retractable by means of a mechanical positioner.

Carbon Dioxide in the Gas Phase

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CO2 in the Gas phase can be determined by means of this significant infrared absorbance at wave lengths lambda < 15 µm, particularly at 4.3 µm, or by acoustic means. Integrated photoacoustic spectroscopy and magnetoacoustic PAS/MA technology for combined CO2 and O2 analysis rapid response time and a small sample volume is sufficient. The acoustic methods are accurate, stable over long periods and very simple to use.

Describe picture

Schematic design of a CO2 analyzer based on absorption a infrared IR radiation. An IR geerator illuminates both the measuring and the reference cuvette. The letter is used to adapt the measuring range is often filled with just a noble gas (zero). The remaining radiation then passes a filter cuvette which can be filled with interesting gad that absorbs all radiation energy at the respective wavelength in both light paths equally. A light chopper (electrically driven with a few 100 Hz) lets the light alternatively pass from the measuring and from the reference path. A thermoaneometric detector quantifies the arriving IR radiation which is inversely proportional to the CO2 present in the cuvettes


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State of the art

Thermodynamics

An elegant, completely non-invasive method is to exploit the heat generated during growth and other metabolic activities of organisms which is also proportional to the amount of active cells in a reaction system. Under welldefined conditions, calorimetry can be an excellent tool for the estimation of total biomass, even for such slow growing organisms as hybridoma cells or for anaerobic bacteria growing with an extremely low biomass yield. In flow calorimeters, samples of a culture grown in a bioreactor are continuously pumped through the measuring cell of a microcalorimeter. The sensivity of the differential signal between the reaction vessel and the refernce vessel is comparable to that obtained from microcalorimetry. From a practical point of view, they are quite flexible because they can connected to any reactor but, due to transfer times in the minutes range, gas and substrate limitations must be considered. Heat flux calorimeters are bioreactors equipped with special temperature control tools. They provide a sensivity which is approximately two orders of magnitude better than that of microcalorimeters. The evaluation and description of microbial heat release is based on a heat balance; heat yields an the heat of combustion of biological components are central paramters for quantification. Measurements obtained so far have been used to investigate growth,biomass yield maintenance energy the role of the reduction degree of substrates oxygen uptake and product formation.

Describe picture side 21

Schematic design of a heat flux calorimeter. Both the temperature in the reactor and in the circuit are measured as sensitively and reproducibly as possible. A welltuned temperature controller keeps the reactor temperature constant by feeding the circuit with warmer or colder water or oil. The circulating water or oil can be taken from either a chilled and a heated reservoir or, as shown, be heated or cooled via external heat exchangers. Calibration is made possible via an electric heater of known power.

Culture Fluorescence

Fluorescence measurements have been used for both characterization of technical properties of bioreactors, and for basic scientific investigations of physiology.Technically, either intra- or extracellular fluorophores are excited by visible or ultraviolet light generated by a low pressure mercury lamp and filtered according to the fluorophore of interest prior to emission into the reactor. Fluorescent light is emitted by the excited fluorophores at a longer characteristic wavelength. Only the backward fluorescence can be collected with appropriate (fiber)optics, is most likely filteredm and the residual light is detected by a sensitve photodetector (picture 22) Most investigators have measured NAD(P)H-dependent culture fluorescence but other fluorophores are also interesting.


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NAD(P)H-dependent culture fluorescence has mainly been exploited for metabolic investigations.The signal is sensitive to variables such as substrate concentration or oxygen supply.

Describe picture

Schematic design of a fluorescence sensor. A strong light source creates readiation with low wavelengths. Optics like lenses and filters extract and focus the desired excitation light which is sent through the window into the measuring solution. Only a small fraction of the fluorescent light arrives at the window, passes this, and is collected by appropriate optics and fed to a sensitive detector (usually a photomultipler). Variations in the light source intensity can be compensated by a comparative measurement.

Optical Density

siehe Seite 26 picture

Schematic design of the Aquasant probe. This is a sensor for optical densitiy measuring the reflected light. Precision optics focus and collect the incident and the reflected light. Left: cross section, right: front view

Interferences

Interferences from gas bubbles or particulate matter other than cells even report on a are common to almost all sensors but different methods are available to circumvent and minimize such problems.

The FundaLux system for instance aspirates a liquid aliquot with a Teflon piston into an external glass cell, allows a time to degas, measures transmission in comparison to an air blank, and realeses the aliquot back to the reactor;an interesting feature-specific to this instrument-in the repetitive cleaning of the optical window by the moving Teflon piston. Some problems with infections have been communicated with this device since the measuring cell is external to the bioreactor and the sensor is probably insufficiently sterilized in situ.


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Side 32

Cerex Foxbro multi purpose sensor

Describe picture

Schematic design of the Cerex Probe. This is a sensor for optical density and mounted vertically in situ. Suspension enters the side drain ports deliberately and can be trapped inside the sensor by powering the solenoid coils; the magnetic plunger closes the side ports. In the meantime , the trapped dispersion degasses and bubbles disappear through the upper vent hole. After some time the optical density reading os declared representative. The next cycle starts with opening the side drain ports.

Komatsugawa transmission sensor

Describe picture

Schematic design of the Komatsugawa probe. This is a sensor for measuring light transmission. It is powered with a laser that has enough energy to also measure highly dense cultures. Optical fibres send and collect light. Around the measuring zone, a stainless steel grid basket is mounted. Its function is to let cells pass and , at the same time exclude gas bubbles. This is why the mesh size of the gird must be selescted according to the type of cells being measured.

BTG Mettler and Wedgewood

siehe Seite 33 Bild

Schematic design of the MEX probe. (top: top and front view) This is a sensor measuring light transmission using four different light paths with two emitters and two detectors. The emitters are alternately switched on and off. The electronics determine the rations of received intensities , Q1 and Q2. The created signal is again a ratio of these values which is virtually independent of fouling of the window surfaces. Alternative constructions are shown below: a single-beam sensor and a variant allowing the comparison of the transmitted light with forward scattered light.


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Options

Chromatography such as GC HPLC

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A review of chromatographic methods is beyond the scope of this contribution. Both liquid chromatography (LC) and gas chromatography (GC) have been applied in numerous cases to off-line analyses of biotechnological samples but the online application has only recently been developed. The scope of chromatographic methods is the seperation of the individual constituents of mixtures as they pass trough colums filed with suitable stationary phases.

Describe picture

siehe Seite 40

Schematic design of linking a chromatograph on-line to a bioprocess. In principle the design is almost identical to an FIA system. This is why FIA is often characterized as chromatography without a column. However, degassing of the sample is essential , in particular, when no internal standard is added . In addition the technical designs of injection valves differ and the injector to a gas chromatograph is heated to 200 °C or 250 °C which means it needs therefore a special construction.

Mass spectrometry

Mass spectrometry MS has been applied mainly for the on-line detection and quantification of gases such as pO2,pCO2,pN2,pCH4 and even H2S or volatiles (alcohols acetoin butanediol).The detection principle allows simultaneous monitoring and, consequently control of import metabolites.

Describe picture

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Schematic design of a mass spectrometer connected on-line to bioprocess. Two alternative uses are sketched and two alternative separation principles. Top: Pressure of a gas is converted down to approximately 1 mbar on its way through a capillary through which is sucked using a mechanical pump. A fraction of this low-pressure gas can enter the high vacuum system of the mass spectrometer via frit or tiny hole. The alternative inlet is a direct membrane inlet. A thin tight membrane mechanically re-enforced to withstand the pressure gradient, is both a pressure and sterile barrier; the membrane is mounted in situ and interfaces cultivation liquid to high vacuum. Molecules entering the high vacuum system are ionized and electromagnetically focused into the mass separation space which can be either a magnetic sensor field (left) or a quadrupole system(right). After mass separation, the ions of interest are quantified usig either a highly sensitive secondary electron multiplier SEM or an an inexpensive pump TMP cascaded to a mechanical pump (not shown)


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Flow Injection Analysis (FIA)

Describe picture

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Schematic design of a flow injection analysis FIA system. A selection valve (top) allows a selection between sample stream and standard. The selected specimen is pumped through an injection loop. Repeatedly , the injection valve is switched for a short while so that the contents of the loop are transported by the carrier stream into the dispersion/reaction manifold. In this manifold, any type of chemical or physical reaction can be implemented . On its way through the manifold, the original plug undergoes axial dispersion which results in the typical shape of the finally detected signal peak

Biosensor

Describe picture

siehe Seite 45

Schematic design of a biosensor that can be mounted in situ. The biosensor itself sits in a housing and consits of a biocomponent such as one or more immobilized enzymes or cells on top of and in close contact with a suitable type of transducer. A buffer or diluent stream can help to extend the useful dynamic range of the biosensor. The analyte arrives at the biosensor by passing a suitable membrane which enhances selectivity and protects the biosensor. An additional mechanical shield in the form of a mesh, grid or frit may be necessary to assure mechanical stability in the highly turbulent zone.

Flow Cytometry

siehe Seite 47

Flow cytometry is a very versatile technique which allows the analysis of more than 10 5 cells per second. This high number results in statistically significant data and distributions of cell properties.Therefore, flow cytometry is a key technique to segregate biomass and to study microbial populations and their dynamics, specifically the cell cycle.Individual cells are aligned by means of controlled hydrodynamic flow patterns and pass the measuring cell one by one. One or more light sources, typically lasers are focused onto the stream of cells and a detection units measure the scattered and/or fluorescent light. Properties of whole cells such as size and shape can be estimated as well as distinct cellular components. The latter requires specific staining procedures which is normally do not allow this technique to be simply used on-line. So far, there has only been one report of an on line application.


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Among the items that have been measured are: vitality intracellular pH, DNA, and RNA content, and specific plasmids. Besides nucleic acids other intracellular components can also be analyzed, storage materials, enzymes and protein content or the cell size. Furthermore , this technique allows the separation of certain cells using a cell sorter.

Describe picture

siehe Seite 47

Schematic design of a flow cytometer. The exciting light is created by one or more laser sources which is focused by means of mirrors and lenses to a small measuring space in the flow channel.Whenever a particle passes this space, exctinction, forward or side scatter of light, or emission of fluorescent light occurs. These different light qualities are separated by lenses and mirrors and quantified by detectors mounted in appropriate positions. The particles are hydrodynamically aligned in the thin flow channel so that only individual single particles pass the measuring zone sequentially. If necessary, a cell sorter can be appended: the on-line executed data evaluation algorithms must classify every measured event in a short time so that an appropriate voltage can be applied to the deflection plates as soon as the respective particle arrives in this space. A tiny fraction of the liquid stream,most probably containg the particle of interest, can be deflected into one container. This allows particles to be sorted according to indvidual properties determined by the flow cytometer.

Field Flow Fractionation FFF

siehe Seite 46

FFF is an elution technique suitable for molecules with a molecularweight >1000 up to a particle size of some 100µm. Separating driving, external field forces are applied perpendicular to a liquid carrier flow, causing different species to be placed in different stream lines. Useful fields are gravity, temperature, cross flow electrical charge and others. The range of the molecular size of the analytes usually exceeds that which can be determined by classical laboratory analytical methods such as size exclusion chromatography.

Reports on investigated substances are widerspread and cover applications such as the separation and characterization of proteins and enzymes of viruses, the separation of human and animal cells, the isolation of plasmid DNA and the molecular weight and particle size distribution of polymers.


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Describe picture

siehe Seite 46

Schematic design of FFF analysis. A sample is transported along the flow channels by a carrier stream after injection and focusing into the injector zone. Depending on the type and strength of the perpendicular field, a separation of molecules or particles takes place: the field drives sample components towards the so-called accumulation wall.Diffusive forces counteract this field resulting in discrete layers of analyte components while parabolic flow profile in the flow channels elutes the various analyte components according to their mean distance from the accumulation wall. This is called normal mode. Particles larger than approximately 1 µm elute in inverse order: hydrodynamic lift forces induce steric effects: the larger particles cannot get sufficiently close to the accumulation wall and, therefore, elute quicker than smaller ones; this is calld steric mode. In asymmetrical flow FFF, the accumulation wall is a mechanically supported frit or filter which lets the solvent pass; the carrier system separates asymmetrically into the eluting flow and the permeate flow which creates the (asymmetrical) flow field.


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Zusatzmaterial aus Diplomarbeit von Res Odermatt

5.7. MONITORING Die routinemässige Überwachung eines Bioprozesses ist sehr wichtig in der Bioingenieurtechnik. Um einen Bioprozess kontrollieren und auf bestimmte Umstände sofort reagieren zu können, müssen wichtige Prozessvariablen möglichst in Echtzeit (real-time) überwacht werden können. Heutzutage werden die meisten Bioreaktoren standardmässig mit Kontroll- und Messorganen ausgestattet (siehe Abb.6).

Die in der Grafik dargestellten Prozessvariablen können in real-time und vollautomatisch (on- line) überwacht werden, was aus regeltechnischer Sicht essentiell ist. Ausserdem befinden sich einige Messorgane im Reaktor (in-situ). Mit einer in-situ Messanordnung kann der „momentane“ Rektorzustand sehr gut beschrieben werden. Es gilt zu beachten, dass die in- situ Messorgane sterilisiert werden müssen, da sie im direkten Kontakt mit der Zellsuspension stehen. Zurzeit können - nicht nur aus diesem Grund - bei weitem noch nicht alle wichtigen Parameter eines Bioprozesses in-situ erfasst werden. Vor allem Konzentrationsmessungen verschiedener Medium- oder Produktkomponenten sind hiervon betroffen. Diese Daten können aber im Bypass oder manuell (off-line) generiert werden. Die Nachteile solcher Messungen sind der zusätzliche Zeit- bzw. Arbeitsaufwand sowie die Zeitverzögerung zwischen der Probenahme und der Probenanalyse. Dies bedeutet, dass ohne Abstoppung des Zellmetabolismus bis zur Probenanalyse eine signifikante Änderung der Analytkonzentration auftreten kann. Es sollte also das Ziel eines Bioprozesses sein, möglichst wenige Prozessvariablen off-line erfassen zu müssen.


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Durch die Fortschritte in der Molekulargenetik konnten in diesem Bereich neue Türen geöffnet werden. Mit dem Einsatz fluoreszenter Reporterproteine und der Möglichkeit bereits gut funktionierende in-situ Trübungssonden (erfassen die optische Dichte der Zellsuspension) ohne grossen Aufwand in Fluoreszenzsonden umzurüsten, bietet sich die Möglichkeit, die heterologe Proteinexpression in real-time, on-line und in-situ erfassen zu können. Da diese Messtechnik aber noch nicht sehr ausgereift ist, gibt es noch einige Prozessparameter die eine Messung beeinflussen können (siehe Tabelle 2).

Bestimmt haben nicht alle erwähnten Faktoren einen grossen Einfluss auf die Messung, jedoch muss eine Messbeeinflussung in Betracht gezogen werden. Eine besondere Beachtung sollte der Begasung und der Rührerdrehzahl gelten, da diese aktiv beeinflusst werden können und während einer Kultivation möglicherweise verändert werden müssen. Da mit einer optischen Sonde gearbeitet wird, kann auch die Blasenkoaleszenz der Biosuspension einen Einfluss auf das Messsignal haben. Unter Blasenkoaleszenz versteht man, dass sich kleine Blasen zu grösseren zusammenschliessen. Sie ist abhängig von der Grenzflächenspannung der Biosuspension [16]. Dieser Effekt verändert die Lichtstreuung der Biosuspension und somit auch das Sondensignal. Folglich kann gesagt werden, dass durch eine Zugabe von Anti- Schaummittel (Polypropylenglykol) oder anderen Substanzen die die Grenzflächenspannung verändern, das Messsignal während einer Kultivation beeinflusst werden kann.


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