Journal of Biotechnology, 25 (1992) 23-53

23

© 1992 ElsevierScience Publishers B.V. All rightsreserved 0168-1656/92/$05.00

BIOTEC 00781 Minireview

On-line measurement in biotechnology: Techniques Georg Locher, B e r n h a r d Sonnleitner and Armin Fiechter Institute of Biotechnology, ETH Ziirich H6nggerberg, Ziirich; Switzerland

(Received 12 May 1991;revisionaccepted 16 June 1991)

Summary Bioprocesses are generally ill controlled. This is due to the fact that the measurement of relevant variables is difficult. Therefore, fundamental knowledge of metabolic interrelations is, at least in vivo, limited. In this article, some of the most important measurement techniques are reviewed in order to provide an evaluation of their current state. Emphasis is given to the underlying principles and on-line capability which allow to judge their importance and potential for exploitation resulting in well (maybe entirely) controlled bioprocesses in the future. Sensors; Measurement and control; On-line techniques; Sampling; pH; Temperature; Pressure; Oxygen; Carbon dioxide; Turbidity; Redox potential; Fluorometry; Mass spectrometry (MS); Flow injection analysis (FIA); Viscosity; Calorimetry; High performance liquid chromatography (HPLC); Gaschromatography (GC); Biosensors; Flow cytometry; Nuclear magnetic resonance spectroscopy (NMR); Acoustic resonance densitometry (ARD); Electrochemistry; Ultrasound (US); Hydrogen; Field flow fractionation (FFF); Filtration; ATP

Introduction Measurement is fundamental to all scientific and technical disciplines. It quantifies an unknown variable by comparing it with a generally accepted standard. Typical classifications in biotechnology are based on the nature of a variable, e.g. Correspondence to: Georg Locher, Institute of Biotechnology,ETH Ziirich H6nggerberg, CH 8093

Zfirich, Switzerland.

24 physical, chemical, biological, or on the principle of measurement, e.g. optical, electrochemical, thermoelectrical, on the location of the sensor, e.g. in situ, in line, on the state of automation, i.e. off-line or on-line, or on the technique, e.g. FIA, MS, GC. The measurement is normally displayed in order to inform the user about the actual state of the investigated object (monitoring) and can be used for either manual or automatic control. In comparison to other disciplines such as physics or engineering, biotechnology is substantially underdeveloped with respect to both measurement and control. Sensors to be used in situ for biotechnological processes are rare; they measure physical and chemical variables rather than biological ones. The reasons are manifold but, generally, variables biologically relevant (e.g. energy charge, respiratory capacity) are much more complex than others (e.g. temperature, pressure). A second important reason derives from restricting requirements, namely to withstand sterilization procedures - to be stable and reliable over extended periods - to be applicable over an extended dynamic range not to interfere with the sterile barrier to be insensitive towards protein adsorption and surface growth - to endure enzymatic breakdown. Finally, material problems arise from the requirements of aseptic culture conditions and the necessity to measure over extended dynamic ranges (e.g. glucose: from < 1 mg 1-1 in physiological studies (Filippini et al., 1991) to >> 10 g I-1 in technical media for industrial production) and often make the construction of sensors rather difficult. With this article, we intend to review the contribution of selected measuring techniques to bioprocess perception. A brief description of the measuring principle is given, followed by a survey of actual applications. In order to complement other reviews (e.g. Schiigerl, 1990a; Schiigerl et al., 1987; Clarke et al., 1985; Harris and Kell, 1985; Clarke et al., 1986; Fleischaker et al., 1981) special attention is devoted to evaluation and interpretation of on-line techniques 1. In combination with the second article in this special issue (Locher et al., 1992) some important steps on the way from raw data generation inside the reactor until sophisticated information exploitation on the level of bioprocess supervision will be reviewed. -

-

-

Application of sensors Measurement of relevant variables in submerged cultures requires the analyte being in contact or interrelation with the measuring device. Gaseous, dissolved and solid compounds are of interest, either in the medium (extracellular) or in the cells. Sampling methods have been discussed by Schiigerl (1990b) and Spohn and Vo13 (1989). Meiners and Schaller (1986) discussed the application of autoanalyzers. i An excellent, comprehensivereview of measuring principles has recently been published in German by Schfigerl(1991).

25 The terms on-line and off-line code for the automation degree of measurements. In situ and ex situ describe the location of the sensor. Real time applies to the delay of a measured signal. Continuous and discontinuous or discrete refer to the temporal characteristics of data generation. However, various other terms are in use but their meaning is often not clear. In-situ sensors are optimal with respect to fast and representative measurements but further criteria have to be envisaged, too. This can result in the choice of a complex instrumentation instead, because such a sensor - if available in the analytical laboratory - might not be capable to withstand cultivation a n d / o r sterilization conditions. Others must be protected (biosensors) or the detection principle might require sample preparation steps prior .to measurement (e.g. degassing, controlled flow patterns, cell disruption to access intracellular components, dilution). In such cases, a representative sample(flow) is needed which can be either recirculated into the reactor (after non-invasive measurement; closed loop) or discarded (after modification by chemical reactions; open loop). Generally, careful investigation of the representativeness of the sample withdrawn from the reactor is necessary and, further, all sampling operations must warrant monoseptic conditions (exactly one single and distinct - intentionally inoculated - species of organisms present), i.e. with a sterilizable apparatus, not corrupting the sterile barrier. Samples can be: gaseous compounds (soluble gases or volatiles), aliquots of the cell suspension or cell free supernatants. Volatile compOunds are usually determined after pervaporation through suited membranes (silicon, teflon); however, major problems are encountered with clogging, and fouling of the membrane as well as with unknown permeability characteristics which depend on the varying composition of the medium. Cell-free supernatant is produced by micro- or ultrafiltration, often as crossflow in a closed bypass loop or by in-situ filters. Aliquots of the cell suspension are usually needed for cellular measurements; here, the challenging task is to interface the sterile barrier efficiently and safely.

Standard measurements

Measuring techniques for temperature, pH and pressure are well known from chemical engineering. Being essential parameters to reaction conditions, frequent measurements and an appropriate control are indispensable in bioreactors. Further, oxygen and carbon dioxide are most important substances in the metabolism of many microorganisms and several approaches to bioprocess control are based on these data. Reliable equipment for the determination is available; the mentioned variables should be measured routinely in bioreactors.

Temperature Generally, there exists a strain-dependent relation between growth and temperature (approximated by the Arrhenius equation at suboptimal temperatures) with a distinct optimum. Hence, temperature should be maintained on this level by

26

removing the heat produced by the organism. Temperature might be the variable most often determined in bioprocesses. In the range between 0°C and 130°C, this can be performed using thermoelements or, better, by thermometers based on resistance changes of a platinum wire (e.g. Pt-100) which is one of the most reliable measures in biotechnology. Temperature is most often also controlled, i.e., held constant or made a cultivation parameter. With a sound control system it is possible to obtain a precision of some 1 mK in laboratory-scale bioreactors. Control of metabolic activities can be achieved by object-oriented temperature changes.

Acidity (pH) Acidity is one of the variables most often controlled in biotechnology because enzymatic activities and, therefore, metabolism is very sensitive to pH changes. pH of process suspensions is measured potentiometrically using electrodes filled with liquid or gel electrolytes. A brief comparison of properties is given by Gary et al. (1988). Glass electrodes develop a gel layer with mobile hydrogen ions when dipped into an aqueous solution, p H changes cause ion diffusion processes generating an electrode potential. This is measured in comparison to a reference electrode. The electric circuit is closed by a diaphragm separating reference electrolyte from solution. Spoiling of the reference electrolyte is one of the major problems during long cultivations. Monzambe et al. (1988) reported on discrepancies of one pH unit between in situ on-line and off-line measurements in an anaerobic wastewater digestion process which were caused by black clogging of the porous diaphragm. Either acidification or pressurization of the electrolyte was suitable to restrain this. p H can be maintained within a few hundredths of a pH unit, provided mixing time is sufficiently small. Interestingly, many scientists 'control' the pH by exclusively adding alkali. Addition of acid is not foreseen assuming (not approving!) that acids are not necessary. But if pH is well controlled it is rewarding to monitor the pH controller output signal as well because it reveals the activities of the culture with respect to production and consumption of pH active substances, i.e. (de)protonized molecules such as organic acids or ammonium ion. This is valuable information which usually remains unused.

Pressure The direct dependence of microorganisms on pressure changes is negligible provided they do not exceed a few bars. But indirectly, the partial pressure of dissolved gases and their solubility is affected and must, therefore, be considered. In addition, the reduction of infection risks by a controlled overpressure is advantageous. During sterilization, pressure is of paramount interest for safety reasons. A variety of sterilizable detectors exist e.g. piezoresistive, capacitive or resistance strain gauge sensors, but not all of them are sufficiently temperature compensated. A data sampling frequency in the range of a few 100 ms is appropriate for digital pressure control in laboratory-scale bioreactors.

27

Oxygen Oxygen solubility is low in aqueous solutions. Mass transfer is, therefore, of considerable importance to avoid or control oxygen limitation. Several attempts to m e a s u r e p O 2 have been made in the past (e.g. Fatt, 1976; Brookman, 1969; Van Hemert et al., 1969; Ohashi et al., 1979). Generally, oxygen is reduced by means of a cathode applying a polarizing potential of 600-750 mV which is generated either externally (polarographic method) or internally (galvanic method). A membrane separates the electrolyte from the medium to improve selectivity. It is responsible for the sensor characteristics which are diffusion controlled. Less sensitivity to membrane fouling and changes in flow conditions is reported for transient measuring techniques, where the reducing voltage is applied in a pulsed mode, a deviation from common continuous oxygen reduction (Wang and Li, 1989). Measurements of oxygen in the gas phase are based on its paramagnetic properties. Changes of the mass concentration of O 2 affect the density of a magnetic field and thus the forces on (dia- or para)magnetic matter in this field. These forces can be compensated electrically and the current be converted into mass concentrations: further conversion into a molar ratio, e.g. % O z, requires the knowledge of total pressure. Merchuk et al. (1990) investigated the dynamics of oxygen electrode measurements when analyzing mass transfer and reported whether and when an instantaneous response occurs. A semi-empirical description of diffusion coefficients was found by Ju and Ho (1988). Bacillus subtilis cultures change the product concentration ratio between acetoin and butanediol rapidly in the 80-90 ppb range (Moes et al., 1985). This fact could be used for the characterization of bioreactor oxygen transport capabilities. A control loop for low oxygen concentrations ( < 100 ppb) based on a fast but non-sterilizable sensor (Marubishi DY-2) was devised by Heinzle et al. (1986). Lorenz et al. (1987) improved penicillin production by aeration control based o n pO 2 measurements. For biological systems, often the effect of oxygen on metabolism is by far better investigated than effects of other nutrients. For instance, Furukawa et al. (1983) reported on a long-term adaption of Saccharomyces cereuisiae to low oxygen levels and Pih et al. (1988) observed a clear relationship between p O 2 and catabolite repression, catabolite inhibition, and inducer repression for/3-galactosidase during growth of Escherichia coli. Wilson (1987) based on-line biomass estimation on dynamic oxygen balancing, i.e. the evaluation of culture responses to non-aerated phases. Analysis of O 2 as well as CO 2 in the exhaust gas is being generally accepted and applied as a standard measuring technique in biotechnology. It is possible to multiplex several streams in order to reduce costs but it should be taken into account that the time delay for measurements is in the range of several minutes, depending on the efforts for gas transport (active, passive) and sample preparation (drying, filtering).

28

Carbon dioxide C O 2 affects microbial growth in various ways according to its appearance in catabolism as well as in anabolism. Due to this complexity the lack of understanding is intelligible. Morphological changes (e.g. Edwards and Ho, 1988) and variations of growth and metabolic rates in response to p C O 2 have been investigated by several authors. pCO2 can be measured indirectly as p H of a bicarbonate buffer separated from the medium by a gas-permeable m e m b r a n e (Puhar et al., 1980): gases in the exterior sample equilibrate the bicarbonate solution in the electrode. CO 2 reacts with water and further into H + and H C O y . The resulting p H is measured with a p H electrode. The response of the p C O 2 sensor is not exclusively CO2-dependent (Donaldson and Palmer, 1979). Yegneswaran et al. (1990) modelled the effect of changes in physical conditions on the p C O 2 signal. A step up in external p H resulted in a p C O 2 downward spike and vice versa. Pressure shifts in the range of 1 - 2 bar caused p C O 2 fluctuations to an extent of 14%. Mass transfer is assumed to control the dynamics of CO 2 equilibration. The bicarbonate buffer solution must be replaced regularly due to the limited capacity. Otherwise, the equilibration will be prolonged and base line drifts occur. CO 2 in the gas phase can be determined by means of its significant infrared absorbance (,~ < 15/zm, particularly at 4.3/zm). Molin (1983) grouped certain types of food related bacteria in accordance to their CO 2 resistance and Jones and Greenfield (1982) reviewed inhibition of yeasts, distinguished by metabolic and m e m b r a n e effects. Supercritical CO 2 - an interesting extraction fluid - was found to be tolerated by yeasts (L'Italien et al., 1989). According to these investigations it is likely that an optimum CO 2 level exists which is also reported for the growth rate of bacteria like Escherichia coli (Lacoursiere et al., 1986) and for biomass yield, glucose uptake and ethanol production of Zymomonas mobilis (Nipkow et al., 1985). Hirose (1986) considered biochemical effects of O 2 supply and CO 2 removal and concluded that further physiological studies are needed to promote better understanding of the mechanisms involved. Park et al. (1983) assumed a linear correlation between biomass and carbon dioxide evolution rate and exploited this model for the estimation of cell concentration, an elegant tool for processes with technical media like highly colored molasses-mineral salts medium with large amounts of particles. Similarly, the cell concentrations of Streptococcus thermophilus in coculture with Lactobacillus thermophilus could be determined due to its property to metabolize urea in milk to CO 2 and ammonia (Spinnler et al., 1987). CO 2 was reported to serve as control variable in cultures of Candida brassicae and allowed to maintain automatically 0 2 and, thus, ethanol at a constant level (Suzuki et al., 1986). Further, CO 2 measurements could be used successfully for assays of enzyme activities (e.g. Salmon, 1987; Botr~ and Botr~, 1990).

29 Measurements providing a high range of information

Often, biologically relevant variables are simultaneously dependent on a variety of influences. Unless detailed (physiological, physical, chemical) knowledge about the respective causes and effects as well as a comprehensive set of data is available, a sound interpretation is not possible. Typical representatives are e.g. fluorometry, turbidity and redox potential.

Fluorometry Fluorescence measurements have been used for both technical characterization of bioreactors (e.g. Li et al., 1990; Scheper and Schfigerl, 1986; Gschwend et al., 1983) and basic scientific investigations of bioprocesses (physiology). The combination of fluorescence and fibre-optics is described in combination with biosensors in this article and the application for biomass estimation is discussed elsewhere in this special issue (Sonnleitner et al., 1992). Either intra- or extracellular fluorophores are excited by visible or ultraviolet light generated by a low pressure mercury lamp and filtered according to the fluorophor under investigation prior to emission into the reactor. Absorbed energy is emitted as radiation of a longer characteristic wavelength. The backward fluorescence can be collected with fibre optics, filtered and the residual light is detected by a photodetector, preamplified and then processed further. Descriptions of typical sensors are given by Beyeler et al. (1981) and Scheper et al. (1987b). Most of the investigators measured NADH-dependent culture fluorescence but other fluorophores are also interesting. Humphrey (1988) gives an (incomplete) survey of the historical evolution of fluorescence measurements for cultivation monitoring. All these data have to be interpreted carefully. Quantification appears difficult even if attempts for theoretical analysis of involved effects have been made (Wang and Simmons, 1987; Srinivas and Mutharasan, 1987b). Calibrations are difficult since quenching behavior of cell material and chemical composition of the medium contribute substantially to the measured signal (Samson et al., 1987). Further, production of interfering fluorophores has to be considered (Meyer et al., 1984; Horvath et aI., 1989). Turbidity must be low and the bubble spectrum should remain constant (Beyeler et al., 1981). NADH-dependent culture fluorescence has mainly been exploited for metabolic investigations (e.g. Rao and Mutharasan, 1989; Reardon et al., 1987; Srinivas and Mutharasan, 1987a). The signal is sensitive to variables such as substrate concentration or oxygen supply. Thus, all attempts to (mis)use this signal as a biomass sensor (Zabriskie and Humphrey, 1978) are limited to conditions where no metabolic alterations occur (Luong and Carrier, 1986; Sonnleitner et al., 1992). The outstandingly rapid principle fluorescence measurements served excellently for the suppression of ethanol formation during continuous baker's yeast production (Meyer and Beyeler, 1984). Rapidity, robustness and the capability to investigate intracellular components

30 are the main advantageous features of fluorescence sensors; however, critical interpretation (condensed e.g. in models) is needed prior to exploitation.

Turbidity Measurement of turbidity is very often applied to on-line as well as off-line biomass estimation and is a central topic of the review of Sonnleitner et al. (1992) in this special issue. Scatter of light which passes through a turbid medium depends on size, surface and number of particles. Analysis of the distribution allows to draw conclusions thereon. If only the absorption is measured the logarithmic ratio of emitted and transmitted light intensities (optical density: OD = log Io/I) is in a first approximation described by the L a m b e r t - B e e r equation (empirically valid for OD < 0.5). In biotechnology, turbidity has been determined in three different ways: light transmission or forward scatter (e.g. Lee, 1981), 90 ° scatter (nephelometry, e.g. Geppert et al., 1989), and retroreflective scatter (e.g. Hancher et al., 1974; Geppert and Thielemann, 1984). Dilution of the sample or appropriate techniques to change the length of the light path are necessary to achieve a useful dynamic range (Lee and Lim, 1980; Fujita and Nunomura, 1968). Main problems encountered with in situ optical measurements are linearity over an extended range, cleaning of the involved optical instruments and interferences with particles and gas bubbles. Optical sensors have been used to estimate biomass, e.g., in order to control the biomass-dependent feed of olive oil and Fe-ions to a semi-batch culture of Pseudomonas fluorescens maximizing lipase activity (Ishihara et al., 1989), to control glucose concentration in fed-batch baker's yeast cultivations (Iijima et al., 1987) or even in semi-solid state cultivations (Hong et al., t987), oD sensors were employed also for assaying enzyme activities (e.g. Virolle et al., 1990; Dean and Rollings, 1989). According to our experimental experience this measure does not justify - as far as accuracy and precision are concerned - the sometimes euphoric acceptance as indicator for biomass concentration found in literature.

Redox potential Biotechnological media and culture liquids contain many different components which are subject to chemical and biological redox reactions, i.e. as redox couples. The resulting redox potential, as measured by a redox electrode, is related to an 'overall availability of electrons' rather than to a specific compound. The fluorescence sensor signal (specific for N A D H / N A D P H ) refers to the intracellular redox state. The relation with the extracellular redox measurement is very instructive, specifically under microaerobic conditions where the pO 2 sensor signal becomes inaccurate (Winter et al., 1988). Redox potential is measured potentiometrically with electrodes made of noble metals (Pt, Au). The mechanical construction is similar to those of p H electrodes. Accordingly, the reference electrode must meet the same requirements.

31 The use and control of redox potential has been reviewed by Kjaergaard (1977). Considerations of redox couples, e.g. in yeast metabolism (Bruinenberg, 1986), are often restricted to theoretical investigations because the measurement is too unspecific and experimental proof cannot be given. Reports on the successful application of redox sensors (e.g. Jee et al., 1987) are confined to detailed description of observed phenomena rather than their interpretation. Flow cytometric methods (Severin et al., 1987) seem to be very promising but are presently not an on-line tool. The application of a redox sensor in a control loop has been reported by Memmert and Wandrey (1987) who controlled xylanase production of Bacillus amyloliquefaciens by defined oxygen limitation: redox electrodes refer essentially to dissolved oxygen concentration below 10/xM 0 2. This property was also promoted to determine the quality of anaerobic processes (Sonnleitner et al., 1984). The state of knowledge on redox data might be high, however, other data must be considered in addition. Electrochemical measurements Measurements of electrochemical properties of a biotechnological medium are affected by both the ceils and the chemical composition. Its linear, passive electrical properties are completely characterized by the electrical permittivity and conductivity (Harris et al., 1987). The biological cell consists of a poorly conducting lipid membrane separating a conducting cytoplasmic volume from the conducting extracellular medium. Thus, the capacitance of a cell suspension will be greater than that of a medium without cells, since only cells possess a membrane whose capacitance can be charged up. The impedance of a culture can be described by a capacitator and a resistor in series. The capacitance component of, e.g., an Escherichia coli culture is far greater than the resistance component. The conductivity of a cell suspension increases with frequency because the cytoplasmic conductivity is increasingly involved with higher frequencies. The chemical composition of the medium depends upon uptake, production and excretion of charged and uncharged metabolites. For instance, electrically inert carbohydrates are metabolized into electrically active products such as organic acids, changing the apparent conductivity. Both influences have to be taken into account for the interpretation of measured data. Equipment for electrochemical measurements is available (Kell et al., 1990; Ur and Brown, 1974) but the effect of medium components may be of dominant and badly explored importance, especially in complex media. Effects of yeast extract (added as a nutrient) were investigated by Ebina et al. (1989) who also found an important influence of acidic substances on impedance. In the work of Gencer and Mutharasan (1979), the determination of cells was limited to low salt concentrations. Owens and Wacher-Viveros (1986) performed research on pH buffer compounds and gave criteria for selection. The uptake of mineral salts was made responsible for the lowering of medium conductivity in plant cell cultures and served indirectly for biomass estimation (Taya et al., 1989). Owens et al. (1989)

32 calculated biomass after absorption of the metabolic CO 2 in an alkaline solution whose conductance was monitored. At present, the technique provides data which are only slightly understood (Connolly et al., 1988; Henschke and Thomas, 1988). Especially in undefined media we cannot expect a useful application as biomass sensors but rather for characterizing the medium composition.

Hydrogen The importance of hydrogen in biological wastewater treatment has been reviewed by Harper and Pohland (1986). Hydrogen detection was initially based on the very fast equilibrium of ions (H +) and molecular hydrogen (H 2) with noble metals. Sensors consist of a platinum (Wilkins, 1978) or platinum black covered gold electrode and a calomel reference. The relationship with redox sensors is obvious but calomel decomposes during sterilization. Generally, platinum refers to redox and pH 2 whereas gold is mainly sensitive to redox measurements (R. Bucher, Ingold AG, Urdorf, Switzerland, personal communication). A linear correlation was observed between inoculum size and the length of time during which H 2 could be detected in a batch (Wilkins et al., 1978). Several modern detection methods are reported by Pauss et al. (1990) who reported on a h y d r o g e n / a i r fuel cell detector. Cleland et al. (1990) used H 2 detection for bioreactor characterization in an Escherichia coli culture which increased its H 2 production under continued oxygen limitation. Due to the low solubility of H 2 in aqueous solution most of the investigators applied measurement techniques for the gas phase, usually gas chromatographs.

Measurements based on 'intelligent analytical subsystems' Several measurements, especially by wet chemical analysis, are based on a sequence of steps. Flow injection analysis, for example, is based on the highly synchronized operation of pumps and valves which must be controlled appropriately. For on-line operation, this must be automated; a 'classical indication' for the application of microprocessors. Hence, such equipment is designed as an ('intelligent') independent tool, capable to operate on its own.

Flow injection analysis (FIA) According to Ruzicka and Hansen (1981) FIA is: "Information gathering from a concentration gradient formed from an injected, well-defined zone of a fluid, dispersed into a continuous unsegmented stream of a carrier." Accordingly, basic components of FIA equipment are a transport system consisting of tubing, pumps, valves and a carrier stream in which an insertion system injects a sample. A reaction - typical for the substance to be measured - usually occurs during the flow and products or residual (co)substrates are measured by the sensing system. The latter, for instance, can be based on enzymatic or immunological reactions

33 (Schmid and Kiinneke, 1990; St6cklein and Schmid, 1990), may be either optical (e.g. Olsson et al., 1983), or thermal (e.g. Decristoforo, 1988) or even on other analytical devices such as mass spectrometers (e.g. Hayward et al., 1990) or biosensors (e.g. Liidi et al., 1990). Although FIA does not generate results continuously there are several important advantages: a high sampling frequency (up to > 100 h-l), small sample volumes, low reagent consumption, high reproducibility and versatility of sensing methods, no interference with the sterile barrier since the entire apparatus works outside the sterile range. But special emphasis must be given to the sampling device interfacing the sterile barrier (Ogbomo et al., 1990; Stamm et al., 1989). Even separation of compounds by HPLC prior to FIA analysis has been reported, a quite great expense (Yao et al., 1990). Kroner (1988) reports on good automation properties of FIA used for enzyme analysis. The stability of enzymes in the sensing systems is still limiting the usage of biosensors substantially even though improvements are reported (Dullau et al., 1989). FIA has been used for on-line determination of glucose (e.g. G a r n e t al., 1989) or to estimate biomass directly (Nielsen et al., 1990) or indirectly by means of an extended Kalman filter (Valero et al., 1990). Filippini et al. (1991) compared FIA with an in-situ enzyme electrode during continuous cultivation of Saccharomyces cerevisiae. FIA has been applied to detect microorganism indirectly by measuring the concentration of a mediator which is reduced by the organisms (Ding and Schmid, 1990). Amino acids, such as L-lysine were measured (Pohlmann et al., 1990) and even intracellular enzymes can be determined on-line (Ahlmann et al., 1986). Metabolic studies of a lactic acid production based on glucose, lactose, galactose, lactate, and protein determinations after nutrient pulses were reported by Nielsen et al. (1990). An outstanding property of FIA is its range of application. Rather a general solution-handling technique than a distinct sensor, this causes high flexibility with respect to analytical methods applicable and, advantageously, no need for sterilizability. A high degree of automation is necessary and desirable but this is not in opposition to the general trend of bioprocess control design. It must be expected to become one of the most powerful tools for quantifying bioprocesses in the near future.

Mass spectrometry (MS) MS has been applied mainly for the on-line detection and quantification of dissolved gases (pO 2, pCO 2, pN 2, pH 2, pCH 4) and volatiles (alcohols, acetoin, butanediol). The detection principle allows simultaneous monitoring - and consequently control - of important metabolites. The principles, sampling systems, control of the measuring device and application of MS for bioprocesses has been summarized by Heinzle (1987; and 1992, this issue). Samples are introduced into vacuum (< 10 -5 bar) via a capillary (heated, stainless steel or fused silica, 0.3 × 1000 mm) or a direct membrane inlet (e.g. teflon; Cox, 1987). Electron impact ionization with high energy (approx. 70 eV) causes (undesired) extensive fragmentation but is commonly applied. Mass separa-

34 tion can be obtained either by quadrupole or magnetic instruments and the detection should be performed by (fast and sensitive) secondary electron multipliers instead of (slower and less sensitive) Faraday cups. Capillary inlet MS are the exception (Camelbeeck et al., 1988), most scientific publications report on membrane inlet applications. An important problem in these cases is the quantification because the membrane behavior is more or less unpredictable (Griot et al., 1987; Rohner et al, 1988). There were approaches proposed which use whole mass spectra of complete supernatants containing even unknown compounds as typical fingerprints characterizing the process state (Heinzle et aI., 1985). This comprehensive view of process data is also applicable to pyrolysed samples (Sandmeier et al., 1987). The analysis of data makes computation power indispensable (e.g. Pungor et al., 1983). A complete interpretation of the peroxidase-catalyzed oxidation of uric acid was possible on-line with a thermospray tandem mass spectrometer coupled to an enzyme reactor (Volk et al., 1990). Coppella and Wang (1990) determined flow rates by spiking and balancing the gas stream with Ar gas. A control loop based on H 2 measurements has been set up by Lloyd and Whitmore (1988; Whitmore et al., 1987) in order to prevent inhibition of methanogenesis: they controlled the addition of the carbon source to a thermophilic anaerobic digestion process. Of course, mass spectrometry requires expensive equipment. But it should be taken into account that automatic multiplexing of different sample streams is possible and, in addition, a variety of different substances can be determined simultaneously.

Calorimetry Bioreactions are exothermic. The net heat released during growth represents the sum of the many enzymic reactions involved. Reasonably, this measure depends on both the biomass concentration and the metabolic state of the cells. Its use in biotechnology has recently been reviewed by von Stockar and Marison (1989). Mainly three different approaches are applied: micro-, flow- and bench-scale calorimetry. Microcalorimeters typically work with small (1-100 ml) reaction volumes and have a sensitivity of the order of 1 /zW (e.g. Ishikawa and Shoda, 1981; Dermoun et al., 1985). In addition to serious limitations when setting up the environmental conditions for the cells (pH or homogeneity), the impracticability to apply any further sensors is an important obstacle for their use. In flow calorimeters, samples of a culture grown in a bioreactor are continuously pumped through the measuring cell of a microcalorimeter. The sensitivity of the differential signal between the reaction and the reference vessel is comparable to that obtained from the mentioned microcalorimetry (e.g. Jolicoeur et al., 1988). From a practical point of view, they are quite flexible because they can be connected to any reactor but, due to transfer times in the minute(s) range, gas and substrate limitations must be considered. Finally, bench scale calorimeters are bioreactors equipped with special temperature control tools. They provide a sensitivity which is approximately two

35 orders lower, although the surface/volume ratio favors them in comparison to microcalorimeters. This approach to heat measurements generally refers to construction concessions rather than to questions of sensor application (e.g. Birou and von Stockar, 1989; Luong and Volesky, 1982). The evaluation and description of microbial heat release bases on a heat balance; heat yields and the heat of combustion of biological components are central parameters for quantification (Cordier et al., 1987). Measurements obtained so far were used to investigate growth, biomass yield, maintenance energy, the role of the reduction degree of substrates, oxygen uptake (von Stockar and Birou, 1989) and product formation. Parallels to other measurements are increasingly drawn (e.g. Lid6n et al., 1989; Roy and Samson, 1988). Analysis of enzyme kinetics (Owusu and Finch, 1986; Lovrien et al., 1987) and characterization of macromolecules and cellular systems is being performed by differential scanning calorimetry (Chowdhry and Cole, 1989). Approaches for practical exploitation have also been made. Fardeau et al. (1980) proposed integrated thermograms as a measure for biodegradability and Lovrien et al. (1987) used microcalorimetric analysis of Klebsiella sp. growth for sugar determination. The exploitation of calorimetry for biomass estimation has been compared to other methods (Boe and Lovrien, 1990). Although often proposed, there are only a few reports on control of processes based on calorimetric data (e.g. Silman, 1988). Entropy is closely related to heat (enthalpy) and energy. If all the ATP available from catabolic processes were used for anabolism (chemical synthesis), up to ten times more cellular material could be produced. First investigations of this large outflow of entropy from growing cells have been made (Bormann, 1988); however, classical thermodynamics are hardly applicable to complex, non-equilibrium metabolic systems and must be extended (Wilson and von Westerhoff, 1982).

Chromatographic methods A review on chromatographic methods and capabilities is beyond the scope of this article. Both, liquid chromatography (LC) and gas chromatography (GC) have been applied in many cases to off-line analyses of biotechnological samples but the on-line application of apparatus is only recently developing. The scope of chromatographic methods is the separation of constituents of mixtures on their passage through columns filled with suitable stationary phases. The retention in the column is determined by the interaction between the individual constituents of the mixture and the stationary phase. Comberbach and Bu'lock (1983) measured ethanol in the bioreactor headspace every 6 min using an electropneumatic sampling system connected to a GC. McLaughlin et al. (1985) provided cell-free samples of a butanol-acetone bioreaction by means of a tangential flow ultrafiltration membrane; they determined the components also in the headspace by a GC. Grobo'illot et al. (1989) monitored the kinetics of acetaldehyde, ethanol, fusel alcohols and CO 2 after gas chromatographic separation. DaPra et al. (1989) used GC data to control the influx rate of highly polluted wastewater to an anaerobic filter. HPLC systems were helpful in

36 monitoring cephalosporin production (Holzhauer-Rieger et al., 1990) and p-cresol degradation (Smolenski and Suflita, 1987). Other HPLC systems have been reported to serve for the control of penicillin production (precursor feed; Miiller et al., 1986), 3-chlorobenzoate conversion (Schmidt, 1988) or naphthalenesulfonic acid reduction (Meschke et al., 1988). It is possible to link such apparatuses - after some adaptation and modification to bioreactors ('intelligent analytical subsystems'). This trend towards increased automation is not restricted to chromatographic methods.

-

Viscosity The viscosity of biotechnological fluids is caused by a variety of inner frictions. Mainly, the effects of biomass concentration (e.g. Perley et al., 1979), morphology (e.g. Bongenaar et al., 1973) and individual metabolic compounds (e.g. Endo et al., 1990) on viscosity have been investigated. Multiple approaches for measurement cover classical rotational viscometers (e.g. Shimmons et al., 1976), capillary flow viscometers (e.g. Allen and Robinson, 1990) and impeller-type rheometers (e.g. Baker et al, 1988); even new apparatuses are reported such as vibrating rod sensors (Picque and Corrieu, 1988) or detectors for resonance frequency changes of oscillating crystals in contact with liquids (e.g. Kanazawa and Gordon, 1985). Data obtained from different techniques are sometimes not consistent due to physical measuring properties (e.g. Allen and Robinson, 1990; Blakebrough et al., 1978) and interpretation of data is said to be misleading caused by insufficiently examined theoretical assumptions (Chisti and Moo-Young, 1989). In most cases the reports concentrate on pros and cons of measuring principles and rheological models rather than on biotechnological interpretation or control. Adenosine triphosphate (A TP) ATP is central to the energy household of cells. Its quantification allows therefore, to investigate the relation between metabolism and energy charge. Measurements are based on the emission of light produced during the oxidation of luciferin by molecular oxygen in the presence of ATP, luciferase and magnesium ions. Chemical or physical disintegration of the cell envelope is difficult, especially for yeasts, but could successfully be performed by trichloro-acetic acid or benzalkonium (Siro et al., 1982). Both have an inhibitory but acceptable effect on the ATP assay. Due to the multifactorial effects on the energy charge of cells (Prior et al., 1988) ATP should not be regarded exclusively as an estimate for biomass (e.g. Kang et al., 1983) but as an indicator for the metabolic state. For instance, Farkas et al. (1986) monitored the correlation between ATP level and cellulase production of Trichoderma viride under repeated additions of lactose. Siro et al. (1982) applied the method on-line and found a constant level of ATP concentration in Saccharomyces cerevisiae during exponential growth preceded by a spike in the initial lag phase. Important information for the evaluation of the cell state would be made available with sound data on ATP concentration.

37

Filtration methods Nestaas and Wang (1983) estimated biomass by analysis of the filtration behavior of the culture. Gravimetrically determined filtrate volume, optically measured filter cake volume and the filtration t i m e s e r v e d for the correlation to dry weight data. Thomas et al. (1985) measured biomass in the range of 2-40 g 1-1 in a comparable setup. Contamination risks and clogging of the filter could be reduced by the approach of Reuss et al. (1987) who applied a band filter avoiding repeated use of the same filter. The method is limited to organisms generating a significant filter cake (compare Sonnleitner et al., 1992). Acoustic resonance densitometry (ARD) The relative density of aqueous solutions changes with biomass contents. Acoustic resonance densitometry is sufficiently precise to detect differences between culture and cell-free culture filtrate (Blake-Coleman et al., 1986). Basis of the measurement is the relationship between density and resonance frequency of a sample enclosed in a test chamber and electromagnetically excited to vibrate. Measurements with mammalian cell cultures (Kilburn et al., 1989) indicated a linear relationship between biomass concentration of a culture and the frequency shift compared to culture supernatant (valid above a minimum cell concentration). Although its robustness is advantageous, major drawbacks are: necessity to prepare a cell-free reference for comparison for each single measurement, interferences with gas bubbles and significant sensitivity to pH changes (CO 2 solubility). Ultrasound (US) Measurements are based on the extinction of ultrasound due to suspended particles. This technique has been applied for analysis of bubble size distribution in bioreactors (Bugmann and von Stockar, 1989; Stravs et al., 1986).

Promising techniques for the future Flow cytometry, nuclear magnetic resonance, biosensors and, to a minor extent, field flow fractionation have proven their potential for bioprocess evaluation and will be introduced in the following.

Flow cytometry Flow cytometry is a very versatile technique (Kruth, 1982) which allows to analyze more than 10 4 cells per s (Scheper et al., 1987a). This high number results in statistically significant data and distributions of cell properties. Therefore, flow cytometry is a key technique to segregate biomass (into distinct cell classes) and to study microbial populations and their dynamics. Cells are aligned by means of controlled hydrodynamical flow patterns and pass the measuring cell. One or more light sources, typically laser(s), are focussed onto the stream of single cells and a detection unit measures the scattered or fluores-

38 cence light. Data on whole cells, such as size and shape can be measured as well as distinct cellular components. The latter requires specific (tedious) staining procedures which actually do not facilitate to use this technique on-line. Due to the large amount and segregation of the measured data they are usually evaluated statistically by suitable computer programs (Kachel et al., 1983). Among the items that have been measured are: vitality, intracellular pH, DNA and R N A content, specific plasmids (Srienc et al., 1986), enzymes and protein content (Ranzi et al., 1986). Further, this technique allows to separate certain ceils using a cell sorter, e.g. for strain improvement (Betz et al., 1984). Unfortunately, the equipment is expensive and most of the measurements are tedious and laborious, consequently badly suited for on-line applications. Nuclear magnetic resonance spectroscopy (NMR) Responses of atomic nuclei with net magnetic moments, that are exposed to a magnetic field and irradiated by electromagnetic energy, reveal a variety of informations. Absorption of energy, i.e. resonance of exciting and nuclear frequency, is typical for a nucleus in a certain molecular-electronic environment. The resonance frequency is shifted according to shielding effects of the physical-chemical environment (other nuclei in the molecular neighborhood) onto the local magnetic field (chemical shift). NMR spectroscopy has recently been reviewed by Gillies et al. (1989) and by Fernandez and Clark (1987). It is suited for non-invasive investigation of biochemical structures, pathways and enzymatic mechanism rather than for quantitative determination of small molecules. Biochemical applications involved NMR mainly for structure determination of complex molecules (e.g. Huang et al., 1988; Berrada et al., 1987). In biotechnology, the potential of determining intracellular components without cell disruption is increasingly used for in-vivo studies of metabolism (e.g. Lohmeier-Vogel et al., 1989; Dijkema et al., 1988). Scanning times lie at least in the range of minutes. NMR can help to monitor energization (e.g. Galazzo et al., 1990), especially the levels of 31p-containing metabolites (e.g. Briasco et al., 1990; Sattur et al., 1988), enzyme kinetics, compartmentalized intracellular ion activities, the fate of 3H-, 2H-, 13C-, 15N-, or 19F-labeled tracers (e.g. Fernandez et al., 1988), 0 2 tension, compartmentalized redox potential, membrane potential, cell number, cell volume (see Gillies et al., 1989) and even pH. Major drawbacks are the costs for the equipment, low intrinsic sensitivity and the interpretation of spectra which - currently - scarcely allows automation or even control (Tellier et al., 1989). Biosensors The rationale for using biosensors is to combine the high specificity of biological components with the capabilities of electronical tools. Biosensors consist of a sensing biological module of either catalytical (e.g. enzymes, organism) or affinity reaction type (e.g. antibodies, cell receptors) in intimate contact with a physical transducer. The latter converts the chemical into an electric signal.

39 According to the diversity of possible applications many review-type articles on biosensors have been written during the last years (e.g. Hall, 1986; Owen and Turner, 1987; Wolfbeis, 1987). Often they concentrate especially on a certain group of biosensors but high redundancy is obvious. In the following, different sensor types, ordered by the transduction principle, are introduced: Electrochemical biosensors. Electrochemical transducers can work on an amperometric, potentiometric, or conductometric principle. Further, chemically sensitive semiconductors are recently under development. Commercially available are today: sensors for carbohydrates, such as glucose, sucrose, lactose, maltose, galactose, and the artificial sweetener (Nutra Sweet), for urea, creatinine, uric acid, lactate, ascorbate, aspirin, alcohol, amino acids and aspartate. The determinations are mainly based on the detection of simple cosubstrates and products such as 02, H 2 0 2 , N H 3 or CO 2 (Guilbault and Luong, 1989). Amperometric transducers measure the current (flux of electrons) caused by oxidation or reduction of the species of interest when a voltage is applied between working and reference electrode. Often, oxygen serves as electron acceptor but interferences encouraged development of new methods to avoid this, e.g. controlled oxygen supply and the application of mediators, such as ferrocene (e.g. Higgins et al., 1988). Other determinations base on the detection of H 2 0 2 or N A D H (Arnold and Meyerhoff, 1988). Potentiometric transducers measure a potential between the sensing element and a reference element. Thus, contrary to amperometric transducers, practically no mass transport occurs; the response depends on the development of the thermodynamic equilibrium, pH changes often correlate with the measured substance because many enzymatic reactions consume or produce protons. Conductometric transducers consist of two pairs of identical electrodes, one of which contains an immobilized enzyme. As the enzyme-catalyzed reaction causes concentration changes of the electrolyte the conductivity alters and can be detected. The conductivity of certain semiconductors such as field effect transistors (FETs) can be affected by specific chemicals. Ion selective FETs (ISFETs) are metal oxide semiconductor FETs (MOSFETs) with special properties (Bergveld, 1989). A great advantage is the small size (miniaturization) resulting in the possibility to combine several (identical or alternative) sensing units in multi-functionalized devices (Karube et al., 1989). They have already been used for the detection of urea, ATP, alcohol, glucose and glutamate (Guilbault and Luong, 1989). Fibre-optic sensors. Optical biosensors typically consist of an optical fibre which is coated with the indicator chemistry for the material of interest at the distil tip. The quantity or concentration is derived from the intensity of absorbed, reflected, scattered, or re-emitted electromagnetic radiation (e.g. fluorescence, bio- and chemiluminescence).

40 Advantageous are potentiality for miniaturization, low cost and the fact that fibre-optics are sterilizable (even if the analyte is not!). The most limiting disadvantages are actually interferences from ambient light and the comparatively small dynamic ranges. Applications so far reported in the literature appeared for pH (e.g. Attridge et al., 1987), CO 2 (e.g. Munkholm et al., 1988), NH 3 (e.g. Arnold and Ostler, 1986), CH 4 (see Guilbault and Luong, 1989), metal ions (see Guilbault and Luong, 1989), O 2 (e.g. Peterson et al., 1984), and even for biomass (e.g. Junker et al., 1988). Calorimetric sensors. This type of biosensors exploits the fact that enzymatic reactions are exothermic (5-100 kJ mol-1). The biogenic heat can be detected by thermistors or temperature sensitive semiconductor devices. A technical realization can be performed either by immobilizing enzymes on particles in a column around the heat sensing device or by direct attachment of the immobilized enzyme to the temperature transducer. Applications to measured substances biotechnologically relevant have been: ATP, glucose, lactate, triglycerides, cellobiose, ethanol, galactose, lactose, sucrose and penicillin (Guilbault and Luong, 1989). Acoustic/mechanical sensors. The piezo-electrical effect of deformations of quartz under alternating current (at frequency in the order of 10 MHz) is used by coating the crystal with a selectively binding substance, e.g. an antibody. When exposed to the antigen, the antibody-antigen complex will be formed on the surface and shift the resonance frequency of the crystal in correlation with the mass increment which is proportional to the antigen concentration. A similar approach is used with surface acoustic wave detectors. Currently, the technique is under investigation for gaseous substances, such as HC1, isocyanates, CO, NO x, and SO 2 (Guilbault and Luong, 1989). Generally, biosensors are difficult to handle. Due to the vulnerable biological element, e.g. a living microorganism (Karube et al., 1988), they cannot be sterilized. They must therefore be used in a suited environment, preferably after sample preparation like in FIA systems or auto-analyzers. The long-term stability under working conditions is generally poor. But research has been concerned with the construction (e.g. enzyme immobilization, housing) of sensors for (off-line) monitoring and only very little experience is made under technical process conditions. Examples of recent industrial applications of biosensors are given by Arnold and Meyerhoff (1988). Merten et al. (1986) gave a comparison of methods for glucose determination with respect to bioprocessing.

Field flow fractionation (FFF) FFF is an elution technique suitable for molecules with a molecular weight > 1000 up to a particle size of some 100 ~m. The separating, driving, external field forces are applied perpendicular to a liquid carrier flow, causing different species to be placed in different stream lines of open channels or hollow fibres. Useful fields are gravity, temperature, cross flow, electrical charge, or others (Giddings, 1989).

Dependent on energy charge of ceils and on biomass concentration, disintegration of the cell envelope necessary, provides a very important measure of the physiological state, on-line capability, but little experience is available Generally poor long term stability, non-sterilizable, multiple interferences, actually only suited as sensing element together with an automatic sampling system or off-line Microcalorimeters hardly provide realistic culture conditions, flow calorimeters require for careful investigation on representativity in the measuring cell, bench scale calorimeters provide least but sufficient sensitivity and sound culture conditions Established measuring technique, interferences with pH and pressure changes, can be recalibrated in situ (Ingold), minor dependencies on membrane characteristics, aging and flow patterns, temperature dependency should be compensated Robust standard measuring technique, gas preparation needed, overlapping absorption bands of contaminant gases HPLC and GC have proven useful as powerful on-line tools, however practical experience is rare Cells, but mainly medium components play the dominant role during signal generation, capacitance measurements detect all polarizable fluid elements that are enclosed by a membrane, low sensitivity, biotechnological experiences are rare

Oxidation of luciferin by 0 2 in the presence of ATP, luciferase and Mg-ions

Biological 'recognition' by catalytical or affinity reactions, transduced into an electronical signal via electrochemical, optical, calorimetrical or acoustical means or semiconductors

Thermopile (microcalorimeter), comparison of reference and reaction vessels (flow calorimeter), or measuring of the heat exchange (bench scale calorimeter)

pH measurement of a bicarbonate solution behind a gaspermeable membrane where CO 2 reacts into H + and HCO~

Infrared absorption

Separation methods based on elementary forces on molecules, performed in a column of suited material

Electrical permittivity and conductivity

ATP

Biosensor

Calorimetry

Carbon dioxide (partial pressure)

Carbon dioxide (exhaust gas)

Chromatography

Electrochemistry

Interferences from bubbles, pH dependency, robust, sterilizable, but relatively insensitive technique, needs cell-free reference

Change of resonance frequency caused by density changes expected through microorganism

ARD

Problems, solutions, comments

Measuring principle

Name

Summary of methods and techniques for bioprocess analytics

TABLE 1

4~

Measuring principle

Separation technique, performed in open channels or hollow fibers, in a perpendicular external field of e.g. either gravity, temperature, cross flow, electrical charge

Sample injection into a carrier stream, detection based on e.g. enzymatic, immunological, optical or thermal reactions

Analysis of filtration behavior: cake volume, filtration time, filtrate volume apply to the correlation with dry weight of biomass

Resolution of (stained) single cells or even cell compartments prior to distinct optical measures: transmitted, scattered and excited light

Excitation of fluorophores by visible or ultraviolet light, m e a s u r e m e n t of the characteristic emitted wavelength

Potentiometric m e a s u r e m e n t s of the (fast) equilibrium of molecular hydrogen with noble metals, such as platinum or gold

Mass separation after ionization in an electromagnetic field

Detection of the response of nuclei which are exposed to a magnetic field and irradiated by electromagnetic energy

Name

FFF

FIA

Filtration

Flow cytometry

Fluorometry

Dissolved hydrogen

MS

NMR

T A B L E 1 Continued Problems, solutions, comments

Theoretical background of signal generation and thus analysis of data is complex, expensive off-line tool, useful for research not mature for automatic control

Membrane inlets obstacle quantification, m e a s u r e m e n t of several components possible, spectra allow for fingerprinting of processes

Close relationship and thus interference with redox measurements

Valuable tool for N A D ( P ) H and thus for metabolic research, often misused as biomass sensor, interferences with fluorophores, sensitive to bubbles, chemical composition, should be related to other data

O n e of the rare techniques which allows for segregation of cell populations, expenditure is high, especially if staining procedures are involved, off-line technique

Low sensitivity, restricted to certain organism

Rather a highly flexible methodology than a sensor, thus presently, the most versatile on-line measuring technique, requiring a high degree of automation, long term stability of many enzymatic reaction systems is poor

High resolution capabilities, biotechnological experience is rare, on-line capability so far not investigated

4~ t~

Several (unknown) affectors such as oxygen, platinum measures also pH2, must be evaluated in conjunction with other data Optical density and biomass are correlated by a logarithmic dependency, applicable only for singly growing cells, imprecisions and problems do not justify the high acceptance as a measure for biomass Besides pH the most often applied measuring technique, should be calibrated in situ (installed) Mainly applied for physical characterization of culture conditions such as bubble size distribution analysis Sensitive to biomass concentration and morphology, tested only for resuspended yeast

Potentiometric measurement in comparison to a reference electrode: gas electrodes develop a gel layer with mobile hydrogen ions sensitive to pH changes in the medium

Different approaches possible: piezoresistive, capacitive, resistance strain gauges

Potentiometrical measurement by electrodes made of noble metals such as gold or platinum

Backward- and forward scattering and absorption of light, dependent on the size and number of particles

Resistance changes of several materials such a platinum, caused by temperature fluctuations

Frequency- and particle size-dependent ultrasound extinction

Rotational-, capillary flow- and impeller-type viscometers, oscillating crystals

pH

Pressure

Redox

Turbidity

Temperature

US

Viscosity

If not otherwise stated, all principles have been reported to be applied on line, but this is not necessarily standard.

Robust measuring technique, temperature compensation needed

Besides temperature the most often applied technique, main problems encountered with black clogging and spoiling of the reference electrode, calibration should be performed at appropriate temperature

sary

Standard exhaust gas analysis, gas cleaning and drying neces-

Paramagnetic properties of oxygen

Oxygen (gas phase)

Minor dependencies on membrane characteristics, aging and flow patterns, precipitations on anode and cathode, temperature dependency should be compensated, robust and valuable, established measuring technique

Amperometric reduction of oxygen behind a gaspermeable membrane at a polarizing potential of 600-750 mV

Oxygen (partial pressure)

44 Reports on investigated substances are widespread and cover applications such as the separation and characterization of proteins (Wahlund and Litzen, 1989), of viruses (Giddings et al., 1977), the separation of human and animal cells (Caldwell et al., 1984), the isolation of plasmid D N A (Schallinger et al., 1985), and the molecular weight distribution of polymers (Kirkland et al., 1988). The approach is relatively new in biotechnology, therefore, practical experiences are rare. High resolution, relatively gentle shear forces in comparison to other separation techniques, and a good theoretical tractability (e.g. calculate diffusion coefficient from elution time) are the advantages of FFF.

Conclusions A typical technical bioprocess is ill controlled due to the fact that on-line measurement of relevant biological variables is difficult (Table 1). Reliable quantification (and control) of physical and (a few) chemical variables is usually possible but the determination of biological conditions is very demanding. Most important drawbacks are caused by manifold interferences with irrelevant substances, falsifications due to the physical/chemical environment (e.g. pressure, gas bubbles, pH), non-sterilizability of the sensing materials (e.g. biosensors) and membrane selectivity and clogging due to complex and varying medium composition. This had and still has a significant influence on research in natural sciences and engineering. Development of technical processes as well as investigation of metabolism and physiology were hampered. Imprecise monitoring of bioprocesses promoted, unfortunately, a 'mystic' attitude towards life sciences because inappropriate measuring techniques and insufficiently controlled environmental conditions were in fact responsible for unexpected or badly reproducible experimental results. But, by this paper, we hope to have demonstrated that the era of mysticism is being overcome. The modern techniques of improved measurement and control of biological processes will provide efficient means to make biotechnology as objective as other disciplines.

References Ahlmann, N., Niehoff, A., Rinas, U., Scheper, T. and Schiigerl, K. (1986) Continuous on-line monitoring of intracellular enzyme activity. Anal. Chim. Acta 190, 221-226. Allen, D.G. and Robinson, C.W. (1990) Measurement of rheological properties of filamentous fermentation broths. Chem. Eng. Sci. 45, 37-48. Arnold, M.A. and Ostler, T.J. (1986) Fiber optic ammonia gas sensing probe. Anal. Chem. 58, 1137-1140. Arnold, M.A. and Meyerboff, M.E. (1988) Recent advances in the development and analytical applications of biosensing probes. CRC Crit. Rev. Anal. Chem. 20, 149-196. Attridge, J.W., Leaver, K.D. and Cozens, J.R. (1987) Design of a fibre-optic pH sensor with rapid response. J. Phys. E. Sci. Instr. 20, 548-553. Baker, M.R., Emery, A.N. and Nienow, A.W. (1988) Mass transfer and power characteristics of a simulated filamentous fermentation broth exhibiting biological activity. Bioreactor Fluid Dynamics, 79-93.

45 Bergveld, P. (1989) Exploiting the dynamic properties of FET-based chemical sensors. J. Phys. E. Sci. Instr. 22, 678-683. Berrada, R., Dauphin, G. and David, L. (1987) Epinigericin a new polyether carboxylic antibiotic, structural determination by 2D NMR methods. J. Org. Chem. 52, 2388-2391. Betz, J.W., Aretz, W. and H~irtel, W. (1984) Use of flow cytometry in industrial microbiology for strain improvement programs. Cytometry 5, 145-150. Beyeler, W., Einsele, A. and Fiechter, A. (1981) On-line measurements of culture fluorescence: method and application. Eur. J. Appl. Microbiol. Biotechnol. 13, 10-14. Birou, B. and von Stockar, U. (1989) Application of bench-scale calorimetry to chemostat cultures. Enzyme Microbiol. Technol. 11, 12-16. Blakebrough, N., McManamey, W.J. and Tart, K.R. (1978) Rheological measurements on Aspergillus niger fermentation systems. J. Appl. Chem. Biotechnol. 28, 453-461. Blake-Coleman, B.C., Clarke, D.J., Calder, M.R. and Moody, S.C. (1986) Determination of reactor biomass by acoustic resonance densitometry. Biotechnol. Bioeng. 28, 1241-1249. Boe, I. and Lovrien, R. (1990) Cell counting and carbon utilization velocities via microbial calorimetry. Biotechnol. Bioeng. 35, 1-7. Bongenaar, J.J.T.M., Kossen, N.W.F., Metz, B. and Meijboom, F.W. (1973) A method for characterizing the rheological properties of viscous fermentation broths. Biotechnol. Bioeng. 15, 201-206. Bormann, E.J. (1988) On the entropy balance of microbial product formation. J. Theor. Biol. 133, 215-221. Botr6, C. and Botr~, F. (1990) Determination of carbonic anhydrase activity by a pCO 2 sensor. Anal. Biochem. 185, 254-264. Briasco, C.A., Ross, D.A. and Robertson, C.R. (1990) Diffusional limitations of immobilized Escherichia coli in hollow fiber reactors: Influence on 31p-NMR spectroscopy. Biotechnol. Bioeng. 36, 887-901. Brookman, J.S.G. (1969) The design, construction, and characteristics of a new long-lived steam sterilizable oxygen electrode. Biotechnol. Bioeng. 6, 323-335. Bruinenberg, P.M. (1986) The NADP(H) redox couple in yeast metabolism. Antonie Leeuwenhoek J. Microbiol. 52, 411-429. Bugmann, G. and von Stockar, U. (1989) Characterizing bubbles in bioreactors by ultrasound. Tibtech. 7, 166-169. Caldwell, K.D., Cheng, Z.Q., Hradecky, P. and Giddings, J.C. (1984) Separation of human and animal cells by steric field flow fractionation. Cell Biophys. 6, 233-251. Camelbeeck, J.P., Comberbach, D.M., Goossens, J. and Roelants, P. (1988) On-line fermenter headspace gas analysis of methanol and ethanol by capillary inlet mass spectrometry. Biotechnol. Tech. 2, 183-188. Chisti, Y. and Moo-Young, M. (1989) On the calculation of shear rate and apparent viscosity in airlift and bubble column bioreactors. Biotechnol. Bioeng. 34, 1391-1392. Chowdhry, B.Z. and Cole, S.C. (1989) Differential scanning calorimetry: applications in biotechnology. Trends Biotechnol. 7, 11-18. Clarke, D.J., Calder, M.R., Carr, R.J.G., Blake-Coleman, B.C., Moody, S.C. and Collinge, T.A. (1985) The development and application of biosensing devices for bioreactor monitoring and control. Biosensors 1,213-320. Clarke, D.J., Blake-Coleman, B.C., Carr, R.J.G., Calder, M.R. and Atkinson, T. (1986) Monitoring reactor biomass. Trends Biotechnol. 4, 173-178. Cleland, N., Larsson, G. and Enfors, S.-O. (1990) Characterization of a biological test system for studies on insufficient mixing in bioreactors, H 2 evolution from E coli. Bioproc. Eng. 5, 79-84. Comberbach, D.M. and Bu'lock, J.D. (1983) Automatic on-line fermentation headspace gas analysis using a computer-controlled gas chromatograph. Biotechnol. Bioeng. 25, 2503-2518. Connolly, P., Lewis, S.J. and Corry, J.E.L. (1988) A medium for the detection of yeasts using a conductimetric method. Int. J. Food Microbiol. 7, 31-40. Copella, S.J. and Wang, D.I.C. (1990) Low cost and convenient method for off-gas flowrate determination in industrial fermenters. Biotechnol. Tech. 4, 161-164. Cordier, J.L., Butsch, B.M., Birou, B. and von Stockar, U. (1987) The relationship between elemental

46 composition and heat of combustion of microbial biomass. Appl. Microbiol. Biotechnol. 25, 305-312. Cox, R.P. (1987) Membrane inlets for on-line liquid-phase mass spectrometric measurements in bioreactors. Mass Spectrom. Biotechnol. Process Anal. Contr., 63-74. DaPra, E., Schneider, K. and Bachofen, R. (1989) Computer optimization of the performance of an anaerobic filter used for purification of highly polluted wastewater from a sugar refinery. Experientia 45, 1024-1029. Dean, S.W. and Rollings, J.E. (1989) Application of size-exclusion chromatography/low angle laser light scattering in fermentation processes. Biotechnol. Tech. 3, 161-166. Decristoforo, G. (1988) Flow-injection analysis for automated determination of /3-1actams using immobilized enzyme reactors with thermistor or ultraviolet spectrophotometric detection. Methods Enzymol. 137, 197-217. Dermoun, Z., Boussand, R., Cotten, D. and Belaich, J.P. (1985) A new batch calorimeter for aerobic growth studies. Biotechnol. Bioeng. 27, 996-1004. Dijkema, C., de Vries, S.C., Booij, H., Schaafsma, T.J. and von Kammen, A. (1988) Substrate utilization by suspension cultures and somatic embryos of Daucus carota L measured by 13C NMR. Plant Physiol. 88, 1332-1337. Ding, T. and Schmid, R.D. (1990) Rapid determination of microorganisms using a flow-injection system. Anal. Chim. Acta 234, 247-251. Donaldson, T.L. and Palmer, H.J. (1979) Dynamic response of the carbon dioxide electrode. AIChE 25, 143-151. Dullau, T., Reinhardt, B. and Schiigerl, K. (1989) High reliability and stability of enzyme cartridges in flow-injection analysis. Anal. Chim. Acta 225, 253-262. Ebina, Y., Ekida, M. and Hashimoto, H. (1989) Origin of changes in electrical impedance during the growth and fermentation process of yeast in batch culture. Biotechnol. Bioeng. 33, 1290-1295. Edwards, A.G. and Ho, C.S. (1988) Effects of carbon dioxide on Penicillium chrysogenum: an autoradiographic study. Biotechnol. Bioeng. 32, l-7. Endo, H., Sode, K. and Karube, I. (1990) On-line monitoring of the viscosity in dextran fermentation using piezoelectric quartz crystal. Biotechnol. Bioeng. 36, 636-641. Fardeau, M.-L., Plasse, F. and Belaich, J.-P. (1980) Microcalorimetry: a tool for the study of the biodegradability of straw by mixed bacterial cultures. Eur. J. Appl. Microbiol. Biotechnol. 10, 133-143. Farkas, V., Kerns, G., Liskova, M. and Bauer, S. (1986) ATP-levels and cellulase formation in batch and fed-batch cultures of Trichoderma L,iride grown on lactose. Folia Microbiol. 31, 277-281. Fatt, I. (1976) Polarographic oxygen sensors. CRC Press Inc., Cleveland, Ohio, U.S.A. Fernandez, E.J. and Clark, D.S. (1987) NMR spectroscopy: a non-invasive tool for studying intracellular processes. Enzyme Microbiol. Technol. 9, 259-271. Fernandez, E.J., Mancuso, A. and Clark, D.S. (1988) NMR spectroscopy studies of hybridoma metabolism in a simple membrane reactor. Biotechnol. Prog. 4, 173-183. Filippini, C., Sonnleitner, B., Fiechter, A., Bradley, J. and Schmid, R.D. (1991) On-line determination of glucose in biotechnological processes: comparison between FIA and an in situ enzyme electrode. J. Biotechnol. 18, 153-160. Fleischaker, R.J., Weaver, J.C. and Sinskey, A.J. (1981) Instrumentation for process control in cell culture. Adv. Appl. Microbiol. 27, 137-167. Fujita, T. and Nunomura, K. (1968) New turbidimetric device for measuring cell concentrations in thick microbial suspensions. Appl. Microbiol. 16, 212-215. Furukawa, K., Heinzle, E. and Dunn, IJ. (1983) Influence of oxygen on the growth of Saccharomyces cerevisiae in continuous culture. Biotechnol. Bioeng. 25, 2293-2317. Galazzo, J.L., Shanks, J.V. and Bailey, J.E. (1990) Comparison of intracellular sugar-phosphate levels from 31p NMR spectroscopy of intact cells and cell-free extracts. Biotechnol. Bioeng. 35, 1164-1168. Garn, M., Cevey, P., Gisin, M. and Thommen, C. (1989) A flow injection analysis system for fermentation monitoring and control. Biotechnol. Bioeng. 34, 423-428. Gary, K., Meier, P. and Ludwig, K. (1988) General aspects of the use of sensors in biotechnology with special emphasis on cell cultivation. Canbiocon 1988 Biotechnol. Res. Appl., 155-164.

47 Gencer, M.A. and Mutharasan, R. (1979) Determination of biomass concentration by capacitance measurement. Biotechnol. Bioeng. 21, 1097-1103. Geppert, G. and Thielemann, H. (1984) Streulichtphotometer zur kontinuierlichen Bestimmung der Biomassekonzentration in Fermentationsmedien. Acta Biotechnol. 4, 361-367. Geppert, G., Thielemann, H. and Langkopf, G. (1989) Industrielle Mel3ger~ite zur Bestimmung der Triibung yon Fliissigkeiten. Acta Biotechnol. 9, 541-545. Giddings, J.C., Yang, F.J. and Myers, M.N. (1977) Flow field-flow fractionation: new method for separating, purifying and characterizing the diffusivity of viruses. J. Virol. 21, 131-138. Giddings, J.C. (1989) Field-flow fractionation of macromolecules. J. Chromatogr. 470, 327-335. Gillies, R.J., MacKenzie, N.E. and Dale, B.E. (1989) Analyses of bioreactor performance by nuclear magnetic resonance spectroscopy. Bio/Technology 7, 50-54. Griot, M., Heinzle, E., Dunn, l.J. and Bourne, J.R. (1987) Optimisation of a MS-membrane probe for the measurement of acetoin and butanediol. Mass Spectrom. Biotechnol. Process Anal. Contr., 75-90. Groboillot, A., Pons, M.-N. and Engasser, J.-M. (1989) Monitoring of volatiles in alcoholic fermentations on molasses via a gas membrane sensor. Appl. Microbiol. Biotechnol. 32, 37-44. Gschwend, K., Beyeler, W. and Fiechter, A. (1983) Detection of reactor nonhomogenities by measuring culture fluorescence. Biotechnol. Bioeng. 25, 2789-2793. Guilbault, G.G. and Luong, J.H.T. (1989) Biosensors: current status and future possibilities. Selective Electrode Rev. 11, 3-16. Hall, E.A.H. (1986) The developing biosensor arena. Enzyme Microbiol. Technol. 8, 651-658. Hancher, C.W., Thacker, L.H. and Phares E.F. (1974) A fiber-optic retroreflective turbidimeter for continuously monitoring cell concentration during fermentation. Biotechnol. Bioeng. 16, 475-484. Harper, S.R. and Pohland, F.G. (1986) Recent developments in hydrogen management during anaerobic biological wastewater treatment. Biotechnol. Bioeng. 28, 585-602. Harris, C.M. and Kell, D.B. (1985) The estimation of microbial biomass. Biosensors 1, 17-84. Harris, C.M., Todd, R.W., Bungard, S.J., Lovitt, R.W., Morris, J.G. and Kell, D.B. (1987) Dielectric permittivity of microbial suspensions at radio frequencies: a novel method for the real-time estimation of microbial biomass. Enzyme Microbiol. Technol. 9, 181-186. Hayward, M.J., Kotiaho, T., Lister, A.K., Cooks, R.G., Austin, G.D. and Narayan, R. (1990) On-line monitoring of bioreactions of Bacillus polymyxa and Klebsiella oxytoca by membrane introduction tandem mass spectrometry with flow injection analysis sampling. Anal. Chem. 62, 1798-1804. Heinzle, E., Kramer, H. and Dunn, l.J. (1985) State analysis of fermentation using a mass spectrometer with membrane probe. Biotechnol. Bioeng. 27, 238-246. Heinzle, E., Moes, J., Griot, M., Sandmeier, E., Dunn, I.J. and Bucher, R. (1986) Measurement and control of dissolved oxygen below 100 ppb. Ann. N.Y. Acad. Sci. 469, 178-189. Heinzle, E. (1987) Mass spectrometry for on-line monitoring of biotechnological processes. Adv. Biochem. Eng./Biotechnol. 35, 1-45. Heinzle, E. (1992) Present and potential applications of mass spectrometry for bioprocess research and control. J. Biotechnol. 25, 81-114. Henschke, P.A. and Thomas, D.S. (1988) Detection of wine-spoiling yeasts by electronic methods. J. Appl. Bacteriol. 64, 123-133. Higgins, I.J., Cardosi, M.F. and Turner, A.P.F. (1988) Harnessing biology-catalyzed electron transfer reactions for biosensors. NATO ASI Ser. A, 128 Perspectives Biotechnol. 55-66. Hirose, Y. (1986) Biochemical effects of oxygen supply and carbon dioxide removal. Prog. Ind. Microbiol. 24, 67-80. Holzhauer-Rieger, K., Zhou, W. and SchiJgerl, K. (1990) On-line high performance liquid chromatography for the determination of cephalosporin-C and by-products in complex fermentation broths. J. Chromatogr. 499, 609-615. Hong, K., Tanner, R.D., Malaney, G.W. and Wilson, D.J. (1987) A spectrophotometric method for estimating the yeast cell concentration in a semi-solid state fermentation. Process Biochem. 22, 149-153. Horvath, J.J., Enriquez-Ortiz, A.B. and Semerjian, H.G. (1989) On-line monitoring of S. cerevisiae fermentations using multiple excitation wavelength fluorescence measurements. Abstr. Pap. Am. Chem. Soc. 198 Meet., MBTD65.

48 Huang, Z., Poulter, C.D., Wolf, F.R., Somers, T.C. and White, J.D. (1988) Braunicene a novel cyclic C32 isoprenoid from Botryococcus braunii. J. Am. Chem. Soc. 110, 3959-3964. Humphrey, A.E. (1988) The potential of on-line fluorometric measurements for the monitoring and control of fermentation systems. Aust. J. Biotechnol. 141-147. Iijima, S., Yamashita, S., Matsunaga, K., Miura, H. and Morikawa, M. (1987) Use of a novel turbidimeter to monitor microbial growth and control glucose concentration. J. Chem. Technol. Biotechnol. 40, 203-213. Ishihara, K., Suzuki, T., Yamane, T. and Shimizu, S. (1989) Effective production of Pseudomonas fluorescens lipase by semi-batch culture with turbidity-dependent automatic feeding of both olive oil and iron ion. Appl. Microbiol. Biotechnol. 31, 45-48. Ishikawa, Y. and Shoda, M. (1981) Design and performance of a new microcalorimetric system for aerobic cultivation of microorganisms. Biotechnol. Bioeng. 23, 2629-2640. Jee, H.S., Nishio, N. and Nagai, S. (1987) Influence of redox potential on biomethanation of H 2 and CO 2 by Methanobacterium thermoautotrophicum in Eh-stat batch cultures. J. Gen. Appl. Microbiol. 33, 401-408. Jolicoeur, C., To T.C., Beaubien, A. and Samson, R. (1988) Flow microcalorimetry in monitoring biological activity of aerobic and anaerobic waste-water treatment processes. Anal. Chim. Acta 213, 165-176. Jones, R.P. and Greenfield, P.F. (1982) Effect of carbon dioxide on yeast growth and fermentation. Enzyme Microbiol. Technol. 4, 210-223. Ju, L.-K and Ho, C.S. (1988) Simultaneous measurements of oxygen diffusion coefficients and solubilities in fermentation media with polarographic oxygen electrodes. Biotechnol. Bioeng. 31, 995-1005. Junker, B.H., Wang, D.I.C. and Hatton, T.A. (1988) Fluorescence sensing of fermentation parameters using fiber optics. Biotechnol. Bioeng. 32, 55-63. Kachel, V., Schneider, H., Bauer, J. and Malin-Berdel J. (1983) Application of the CYTOMIC 12 flow cytometric compact analyzer for automatic kinetic measurements. Cytometry 3, 244-250. Kanazawa, K.K. and Gordon, J.G., II (1985) The oscillation frequency of a quartz resonator in contact with a liquid. Anal. Chim. Acta 175, 99-105. Kang, S.J., Pugh, L.B. and Borchardt, J.A. (1983) ATP as a measure of active biomass concentration and inhibition in biological wastewater treatment processes. Proc. Ind. Waste Conf., 751-759. Karube, I., Tamiya, E., Sode, K., Yokoyama, K., Kitagawa, Y. and Suzuki, H. (1988) Application of microbiological sensors in fermentation processes. Anal. Chim. Acta 213, 69-77. Karube, I., Sode, K. and Tamiya, E. (1989) Current trends in microbiosensor development. Swiss Biotech 7, 25-32. Kell, D.B., Markx, G.H., Davey, C.L. and Todd, R.W. (1990) Real-time monitoring of cellular biomass: methods and applications. Trends Anal. Chem. 9, 190-194. Kilburn, D.G., Fitzpatrick, P., Blake-Coleman, B.C., Clarke, D.J. and Griffiths, J.B. (1989) On-line monitoring in mammalian cell cultures by acoustic densitometry. Biotechnol. Bioeng. 33, 1379-1384. Kirkland, J.J., Rementer, S.W. and Yau, W.W. (1988) Molecular-weight distributions of polymers by thermal field flow fractionation with exponential temperature programming. Anal. Chem. 60, 610-616. Kjaergaard, L. (1977) The redox potential: its use and control in biotechnology. Adv. Biochem. Eng. 7, 131-150. Kroner, K.H. (1988) On-line-Bestimmung yon Enzymen in der BioprozeBanalytik unter Beriicksichtigung yon Probenahme und FlieBinjektionstechnik. Fresenius' Z. Anal. Chem. 329, 718-725. Kruth, H.S. (1982) Flow cytometry: rapid biochemical analysis of single cells. Anal. Biochem. 125, 225-242. Lacoursiere, A., Thompson, B.G., Kole, M.M., Ward, D. and Gerson, D.F. (1986) Effects of carbon dioxide concentration on anaerobic fermentations of Escherichia coli. Appl. Microbiol. Biotechnol. 23, 404-406. Lee, C. and Lira, H. (1980) New device for continuously monitoring the optical density of concentrated microbial cultures. Biotechnol. Bioeng. 22, 639-642. Lee, C. (1981) Pulsed light probe for cell density measurement. Biotechnol. Bioeng. 23, 1903-1906.

49 Li, J., Gomez, P. and Humphrey, A. (1990) The use of fluorimetry for on-line measurement of mixing time and hold-up in fermentations. Biotechnol. Tech. 4, 293-298. Lid~n, G., Larsson, C., Gustafsson, L. and Niklasson, C. (1989) A calorimetric and fluorescence study of batch cultures of Saccharomyces cereuisiae. Appl. Microbiol. Biotechnol. 31,355-357. L'Italien, Y., Thibault, J. and LeDuy, A. (1989) Improvement of ethanol fermentation under hyperbaric conditions. Biotechnol. Bioeng. 33, 471-476. Lloyd, D. and Whitmore, T.N. (1988) Hydrogen-dependent control of the continuous thermophilic anaerobic digestion process using membrane inlet mass spectrometry. Lett. Appl. Microbiol. 6, 5-10. Locher, G., Sonnleitner, B. and Fiechter A. (1992) On-line measurement in biotechnology: Exploitation, objectives and benefits. J. Biotechnol. 25, 55-73. Lohmeier-Vogel, E., Skoog, K., Vogel, H. and Hahn-H~igerdal, B. (1989) 31p-nuclear magnetic resonance study of the effect of azide on xylose fermentation by Candida tropicalis. Appl. Env. Microbiol. 55, 1974-1980. Lorenz, T., Diekmann, J., Friih, K., Hiddessen, R., M611er, J. and Schiigerl, K. (1987) On-line measurement and control of penicillin V production in a tower loop reactor. J. Chem. Technol. Biotechnol. 38, 41-53. Lovrien, R.E., Williams, K.K., Ferrey, M.L. and Ammend, D.A. (1987) Calorimetric versus growth microbial analysis of cellulase enzymes acting on cellulose. Appl. Env. Microbiol., 53, 2935-2941. Liidi, H., Garn, M.B., Bataillard, P. and Widmer, H.M. (1990) Flow injection analysis and biosensors: applications for biotechnology and environmental control. J. Biotechnol. 14, 71-79. Luong, J.H.T. and Volesky, B. (1982) A new technique for continuous measurement of the heat of fermentation. Eur. J. Appl. Microbiol. Biotechnol. 16, 28-34. Luong, J.H.T. and Carrier, D.J. (1986) On-line measurement of culture fluorescence during cultivation of Methylomonas mucosa. Appl. Microbiol. Biotechnol. 24, 65-70. McLaughlin, J.K., Meyer, C.L. and Papoutsakis, E.T. (1985) Gas chromatography and gateway sensors for on-line state estimation of complex fermentations (butanol-acetone fermentation). Biotechnol. Bioeng. 27, 1246-1257. Meiners, M. and Schaller, K. (1986) Einsatz von Auto-Analysatoren zur Prozef3analytik in der Biotechnologie. BTF-Biotech.-Forum 3, 193-202. Memmert, K. and Wandrey, C. (1987) Continuous production of Bacillus exoenzymes through redoxregulation. Ann. N.Y. Acad. Sci. 506, 631-636. Merchuk, J.C., Yona, S., Siegel, M.H. and Zvi, A.B. (1990) On the first-order approximation to the response of dissolved oxygen electrodes for dynamic KLa estimation. Biotechnol. Bioeng. 35, 1161-1163. Merten, O.W., Palfi, G.E. and Steiner, J. (1986) On-line determination of biochemical/physiological parameters in the fermentation of animal cells in a continuous or discontinuous mode. Adv. Biotechnol. Processes 6, 111-178. Meschke, J., Bennemann, H., Herbst, H., Dormeier, S. and Hempel, D.C. (1988) On-line HPLC-measurement and control of substrate in a continuously operated biological tank reactor. Bioproc. Eng. 3, 151-157. Meyer, C. and Beyeler, W. (1984) Control strategies for continuous bioprocesses based on biological activities. Biotechnol. Bioeng. 26, 916-925. Meyer, H.P., Beyeler, W. and Fiechter, A. (1984) Experiences with the on-line measurement of culture fluorescence during cultivation of Bacillus subtilis, Escherichia coli and Sporotrichum thermophile. J. Biotechnol. 1,341-349. Moes, J., Griot, M., Keller, J., Heinzle, E., Dunn, I.J. and Bourne, J.R. (1985) A microbial culture with oxygen-sensitive product distribution as a potential tool for characterizing bioreactor oxygen transport. Biotechnol. Bioeng. 27, 482-489. Molin, G. (1983) The resistance to carbon dioxide of some food related bacteria. Eur. J. Appl. Microbiol. Biotechnol. 18, 214-217. MSller, J., Hiddessen, R., Niehoff, J. and SchiJgerl, K. (1986) On-line high-performance liquid chromatography for monitoring fermentation processes for penicillin production. Anal. Chim. Acta 190, 195-203.

50 Monzambe, K.M., Naveau, H.P., Nyns, E.J., Bogaert, N. and Buehler, H. (1988) Problematics and stability of on-line pH measurements in anaerobic environments: the jellied combined electrode. Biotechnol. Bioeng. 31, 659-665. Munkholm, C., Walt, D.R. and Milanovich, F.P. (1988) A fiber optic sensor for CO 2 measurement. Talanta 35, 109-112. Nestaas, E. and Wang, D.I.C. (1983) A new sensor - the 'filtration probe' - for quantitative characterization of penicillin fermentation. Ill. An automatically operating probe. Biotechnol. Bioeng. 25, 1981-1987. Nielsen, J., Nikolajsen, K., Benthin, S. and Villadsen, J. (1990) Application of flow-injection in the on-line monitoring of sugars, lactic acid, protein and biomass during lactic acid fermentations. Anal. Chim. Acta 237, 165-175. Nipkow, A., Sonnleitner, B. and Fiechter, A. (1985) Effect of carbon dioxide on growth of Zymomonas mobilis in continuous culture. Appl. Microbiol. Biotechnol. 21,287-291. Ogbomo, I., Prinzing, U. and Schmidt, H.L. (1990) Prerequisites for the on-line control of microbial processes by flow injection analysis. J. Biotechnol. 14, 63-70. Ohashi, M., Watabe, T., Ishikawa, T., Watanabe, Y., Miwa, K., Shode, M., Ishikawa, Y., Ando, T., Shibata, T., Kitsunai, T., Kamiyama, N. and Oikawa, Y. (1979) Sensors and instrumentation: steam-sterilizable dissolved oxygen sensor and cell mass sensor for on-line fermentation system control. Biotechnol. Bioeng. Symp. 9, 103-116. Olsson, B., Orgren, L. and Johanson, G. (1983) An enzymatic flow injection method for the determination of oxygen. Anal. Chim. Acta 145, 101-108. Owen, V.M. and Turner, A.P.F. (1987) Biosensors, a revolution in clinical analysis? Endeavour 11, 100-104. Owens, J.D. and Wacher-Viveros, M.C. (1986) Selection of pH buffers for use in conductimetric microbial assays. J. Appl. Bacteriol. 60, 395-400. Owens, J.D., Thomas, D.S., Thompson, P.S. and Timmermann, J.W. (1989) Indirect conductimetric enumeration of microbial populations. Lett. Appl. Microbiol. 9, 245-249. Owusu, R.K. and Finch, A. (1986) Flow microcalorimetric study of immobilized enzyme kinetics using the co-immobilized glucose-oxidase-catalase system. Biochim. Biophys. Acta 872, 83-91. Park, S.H., Hong, K.T., Lee, J.H. and Bae, J.C. (1983) Onqine estimation of cell growth for glutamic acid fermentation system. Eur. J. Appl. Microbiol. Biotechnol. 17, 168-172. Pauss, A., Samson, R., Guiot, S. and Beauchemin, C. (1990) Continuous measurement of dissolved H 2 in an anaerobic reactor using a new hydrogen/air fuel-cell detector. Biotechnol. Bioeng. 35, 492-501. Perley, C.R., Swartz, J.R. and Cooney, C.L. (1979) Measurement of cell mass concentration with a continuous-flow viscometer. Biotechnol. Bioeng. 21,519-523. Peterson, J.I., Fitzgerald, R.V. and Buckhold, D.K. (1984) Fiber-optic probe for in vivo measurement of oxygen partial pressure. Anal. Chem. 56, 62-67. Picque, D. and Corrieu, G. (1988) New instrument for on-line viscosity measurement of fermentation media. Biotechnol. Bioeng. 31, 19-23. Pih, N., De Bernardez, E. and Dhurjati, P. (1988) Elucidation of enzyme control mechanisms using macroscopic measurements in a mixed substrate fermentation system. Biotechnol. Bioeng. 31, 311-320. Pohlmann, A., Stamm, W.W., Kusakabe, H. and Kula, M.-R. (1990) Enzymatic determination of l-lysine by flow-injection techniques. Anal. Chim. Acta 235, 329-335. Prior, B.A., Holder, N.H.M., Kilian, S.G. and Du Preez, J.C. (1988) Measurement of Candida utilis growth using the adenosine triphosphate bioluminescent assay. Systematic Appl. Microbiol. 10, 191-194. Puhar, E., Einsele, A., Biihler, H. and Ingold, W. (1980) Steam-sterilizable pCO 2 electrode. Biotechnol. Bioeng. 22, 2411-2416. Pungor E., Jr., Schaefer, E.J., Cooney, C.L. and Weaver, J.C. (1983) Direct monitoring of the liquid and gas phases during fermentation in a computer-mass-spectrometer-fermentor system. Eur. J. Appl. Microbiol. Biotechnol. 18, 135-140. Ranzi, B.M., Compagno, C. and Martegani, E. (1986) Analysis of protein and cell volume distribution in glucose-limited continuous cultures of budding yeast. Biotechnol. Bioeng. 28, 185-190.

51 Rao, G. and Mutharasan, R. (1989) NADH levels and solventogenesis in Clostridium acetobutylicum: new insights through culture fluorescence. Appl. Microbiol. Biotechnol. 30, 59-66. Reardon, K.F., Scheper, T.H. and Bailey, J.E. (1987) Metabolic pathway rates and culture fluorescence in batch fermentations of Clostridium acetobutylicurn. Biotechnol. Prog. 3, 153-167. Reuss, M., Boelcke, C., Lenz, R. and Peckmann, U. (1987) Entwicklung und Einsatz einer Apparatur zur automatischen Erfassung der Filtrationseigenschaften von Biosuspensionen und zur Ankoppelung von Analysenautomaten bei industriellen Fermentationsprozessen. BTF - Biotech. Forum 4, 3-12. Rohner, M., Locher, G., Sonnleitner, B. and Fiechter, A. (1988) Kinetics and modeling of the stereoselective reduction of acetoacetic acid esters by continuously growing cultures of Saccharomyces cerevisiae. J. Biotechnol. 9, 11-28. Roy, D. and Samson, R. (1988) Investigation of growth and metabolism of Saccharomyces cerevisiae (baker's yeast) using microcalorimetry and bioluminometry. J. Biotechnol. 8, 193-206, Ruzicka, J. and Hansen, E.H. (1981) Flow Injection Analysis. Wiley, New York. Salmon, J.M. (1987) Determination of malic enzyme activity on permeabilized cells of Saccharomyces cerevisiae using a dissolved CO 2 probe. Biotechnol. Tech. 1, 7-10. Samson, R., Beaumier, D. and Beaulieu, C. (1987) Simultaneous evaluation of on-line microcalorimetry and fluorometry during batch culture of Pseudomonas putida ATCC 11172 and Saecharomyces cerevisiae ATCC 18824. J. Biotechnol. 6, 175-190. Sandmeier, E.P., Keller, J., Heinzle, E., Dunn, I.J. and Bourne J.R. (1987) Development of an on-line pyrolysis mass spectrometry system for the on-line analysis of fermentations. Mass Spectrom. Biotechnol. Process Anal. Contr., 209-215. Sattur, A.P., Karanth, N.G. and Divakar, S. (1988) A rapid method for monitoring intracellular lipid production using 1H NMR spectroscopy. Biotechnol. Tech. 2, 73-76. Schallinger, L.E., Gray, J.E., Wagner, L.W., Knowlton, S. and Kirkland, J.J. (1985) Preparative isolation of plasmid DNA with sedimentation field flow fractionation. J. Chromatogr. 342, 67-77. Scheper, T. and Schiigerl, K. (1986) Characterization of bioreactors by in-situ fluorometry. J. Biotechnol. 3, 221-229. Scheper, T., Hoffmann, H. and Schiigerl, K. (1987a) Flow cytometric studies during culture of Saccharomyces cerevisiae. Enzyme Microbiol. Technol. 9, 399-405. Scheper, T., Lorenz, T., Schmidt, W. and Schiigerl, K. (1987b) On-line measurement of culture fluorescence for process monitoring and control of biotechnological processes. Ann. N.Y. Acad. Sci. 506, 431-445. Schmid, R.D. and Kiinneke, W. (1990) Flow injection analysis (FIA) based on enzymes or antibodies applications in the life sciences. J. Biotechnol. 14, 3-31. Schmidt, E. (1988) Bioconversion of 3-chlorobenzoate to 2-chloromuconate controlled by on line HPLC. Appl. Microbiol. Biotechnol. 27, 347-350. Schiigerl, K., Liibbert, A. and Scheper, T. (1987) Online-ProzeBanalyse in Bioreaktoren. Chem.-Ing.Tech. 59, 701-714. Schiigerl, K. (1990a) On-line measurements in biotechnology. Proc. 5th Eur. Congr. Biotechnol., 1010-1016. Schiigerl, K. (1990b) Aseptic sampling. Proc. 5th Eur. Congr. Biotechnol., 1188-1193. Schiigerl, K. (Ed.) (1991) Analytische Methoden in der Biotechnologie. Vieweg, Braunschweig, 1991. Severin, E., Wiezorek, C. and Stellmach, J. (1987) Die halbautomatische Messung der RedoxaktivitS.t in Einzelzellen mit einem flnoreszierenden Formazan. Acta Histochem. Suppl. 34, 169-176. Shimmons, B.W., Svrcek, W.Y. and Zajic, J.E. (1976) Cell concentration control by viscosity. Biotechnol. Bioeng. 18, 1793-1805. Silman, R.W. (1988) Control of feed rate to a fed-batch culture using a heat flux sensor. Abstr. Pap. Am. Chem. Soc. 196 Meet., MBTD 81. Siro, M.-R., Romar, H. and L6vgren, T. (1982) Continuous flow method for extraction and bioluminescence assay of ATP in baker's yeast. Eur. J. Appl. Microbiol. Biotechnol. 15, 258-264. Smolenski, W. and Suflita, J.M. (1987) An HPLC system for the automatic sampling and analysis of time-series kinetic studies. J. Microbiol. Methods 6, 71-79. Sonnleitner, B., Fiechter, A. and Giovannini, F. (1984) Growth of Thermoanaerobium brockii in batch and continuous culture at supraoptimal temperatures. Appl. Microbiol. Bioteehnol. 19, 326-334.

52 Sonnleitner, B., Locher, G. and Fiechter, A. (1992) Biomass determination. J. Biotechnol. 25, 5-22. Spinnler, H.E., Bouillanne, C., Desmazeaud, M.J. and Corrieu, G. (1987) Measurement of the partial pressure of dissolved CO 2 for estimating the concentration of Streptococcus thermophilus in coculture with Lactobacillus bulgaricus. Appl. Microbiol. Biotechnol. 25, 464-470. Spohn, U. and VoB, H. (1989) Probenahmesysteme in der Online-BioprozeBanalytik. BTF Biotech. Forum 6, 274-288. Srienc, F., Campbell, J.L. and Bailey, J.E. (1986) Flow cytometry analysis of recombinant Saccharomyces cerevisiae populations. Cytometry 7, 132-141. Srinivas, S.P. and Mutharasan, R. (1987a) Culture fluorescence characteristics and its metabolic significance in batch cultures of Clostridium acetobutylicum. Biotechnol. Lett. 9, 139-142. Srinivas, S.P. and Mutharasan, R. (1987b) Inner filter effects and their interferences in the interpretation of culture fluorescence. BiotechnoL Bioeng. 30, 769-774. Stamm, W.W., Pommerening, G., Wandrey, C. and Kula, M.-R. (1989) On-line measurement of extracellular proteins in the continuous cellulase production by flow injection analysis (FIA). Enzyme Microbiol. Technol. 11, 96-105. St6cklein, W. and Schmid, R.D. (1990) Flow-injection immunoanalysis for the on-line monitoring of monoclonal antibodies. Anal. Chim. Acta 234, 83-88. Strays, A.A., Pittet, A., von Stockar, U. and Reilly, P.J. (1986) Measurement of interracial areas in aerobic fermentations by ultrasonic pulse transmission. Biotechnol. Bioeng. 28, 1302-1309. Suzuki, T., Yamane, T. and Shimizu, S. (1986) Control of carbon-source supply and dissolved oxygen by use of carbon dioxide concentration of exhaust gas in fed-batch culture. J. Ferment. Technol. 64, 317-326. Taya, M., Hegglin, M., Prenosil, J.E. and Bourne, J.R. (1989) On-line monitoring of cell growth in plant tissue cultures by conductometry. Enzyme Microbiol. Technol. 11, 170-176. Tellier, C., Guillou-Charpin, M., Grenier, P. and Le Botlan, D. (1989) Monitoring alcoholic fermentation by low-resolution pulsed nuclear magnetic resonance. J. Agric. Food Chem. 37, 988-991. Thomas, D.C., Chittur, V.K., Cagney, J.W. and Lim, H.C. (1985) On-line estimation of mycelial cell mass concentrations with a computer-interfaced filtration probe. Biotechnol. Bioeng. 27, 729-742. Ur, A. and Brown, D.F.J. (1974) Rapid detection of bacterial activity using impedance measurements. Biomed. Eng. 9, 18-20. Valero, F., Lafuente J., Poch, M. and Sola, C. (1990) Biomass estimation using on-line glucose monitoring by flow injection analysis application to Candida rugosa batch growth. Appl. Biochem. Biotechnol. 24-25, 591-602. Van Hemert, P., Kilburn, D.G., Righelato, R.C. and Van Wezel, A.L. (1969) A steam sterilizable electrode of the galvanic type for the measurement of dissolved oxygen. Biotechnol. Bioeng. 6, 549-560. Virolle, J.-J., Morris, V.J. and Bibb, M.J. (1990) A simple and reliable turbidimetric and kinetic assay for alpha-amylase that is readily applied to culture supernatants and cell extracts. J. Ind. Microbiol. 5, 295-302. Volk, K.J., Yost, R.A. and Brajter-Toth, A. (1990) On-line mass spectrometric investigation of the peroxidase-catalysed oxidation of uric acid. J. Pharm. Biomed. Anal. 8, 205-215. von Stockar, U. and Birou, B. (1989) The heat generated by yeast cultures with a mixed metabolism in the transition between respiration and fermentation. Biotechnol. Bioeng. 34, 86-101. von Stockar, U. and Marison, I.W. (1989) The use of calorimetry in bioteehnology. Adv. Bioehem. Eng. Biotechnol. 40, 93-136. Wahlund, K.G. and Litzen, A. (1989) Application of an asymmetrical flow field-flow fractionation channel to the separation and characterization of proteins plasmid fragments polysaccbarides and unicellular algae. J. Chromatogr. 461, 73-87. Wang, N.S. and Simmons, M.B. (1987) Dependence of the measured fluorescence signal on the fluorophore concentration. Abstr. Pap. Am. Chem. Soc. 194 Meet, MBTD 84. Wang, H.Y. and Li, X.M. (1989) Transient measurement of dissolved oxygen using membrane electrodes. Biosensors 4, 273-285. Whitmore, T.N., Jones, G., Lazzari, M. and Lloyd, D. (1987) Methanogenesis in mesophilic and

53 thermophilic anaerobic digestors: monitoring and control based on dissolved hydrogen. Mass Spectrom. Biotechnol. Process Anal. Contr., 143-162. Wilkins, J.R. (1978) Use of platinum electrodes for the electrochemical detection of bacteria. Appl. Env. Microbiol. 36, 683-687. Wilkins, J.R., Young, R.N. and Boykin, E.H. (1978) Multichannel electrochemical microbial detection unit. Appl. Env. Microbiol. 35, 214-215. Wilson, D.F. and yon Westerhoff, H.V. (1982) Should irreversible thermodynamics be applied to metabolic systems? TIBS, 275-279. Wilson, P.D.G. (1987) On-line estimation of biomass using dynamic oxygen balancing. Biotechnol. Tech. 1, 151-156. Winter, E.L., Rao, G. and Cadman, T.W. (1988) Relationship between culture redox potential and culture fluorescence in Corynebacteriumglutamicum. Biotechnol. Tech. 2, 233-236. Wolfbeis, O.S. (1987) Fibre-optics sensors for chemical parameters of interest in biotechnology. GBF Monograph. 10, Biosensors Int. Workshop 197-205. Yao, T., Matsumoto, Y. and Wasa, T. (1990) Development of a FIA system with immobilized enzymes for specific post-column detection of purine bases and their nucleosides separated by HPLC column. J. Biotechnol. 14, 89-97. Yegneswaran, P.K., Gray, M.R. and Thompson, B.G. (1990) Kinetics of CO 2 hydration in fermentors: pH and pressure effects. Biotechnol. Bioeng. 36, 92-96. Zabriskie, D.W. and Humphrey, A.E. (1978) Estimation of fermentation biomass concentration by measuring culture fluorescence. Enzyme Microbiol. Technol. 35, 337-343.