University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Journal of Biotechnology

  • Publisher: Elsevier
  • FAD Code: JBTC
  • CODEN: JBITD4
  • ISSN: 0168-1656
  • Abbreviation: J. Biotechnol.
  • DOI Prefix: 10.1016/j.jbiotec,10.1016/S0168-1656
  • Language: English
  • Comments: Fulltext from 1984 V1

Citations 70

"Polyketone Polymer: A New Support For Direct Enzyme Immobilization"
J. Biotechnol. 2007 Volume 127, Issue 4 Pages 670-678
E. Agostinelli, F. Belli, G. Tempera, A. Mura, G. Floris, L. Toniolo, A. Vavasori, S. Fabris, F. Momo and R. Stevanato

Abstract: Polyketone polymer {single bond}[{single bond}CO{single bond}CH2CH2{single bond}]n{single bond}, obtained by copolymerization of ethene and carbon monoxide, is utilized for immobilization of three different enzymes, one peroxidase from horseradish (HRP) and two amine oxidases, from bovine serum (BSAO) and lentil seedlings (LSAO). The easy immobilization procedure is carried out in diluted buffer, at pH 7.0 and 3°C, gently mixing the proteins with the polymer. No bifunctional reagents and spacer arms are required for the immobilization, which occurs exclusively via a large number of hydrogen bonds between the carbonyl groups of the polymer and the {single bond}NH groups of the polypeptidic chain. Experiments demonstrate a high linking capacity of polymer for BSAO and an extraordinary strong linkage for LSAO. Moreover, activity measurements demonstrate that immobilized LSAO totally retains the catalytic characteristics of the free enzyme, where only a limited increase of KM value is observed. Finally, the HRP-activated polymer is successfully used as active packed bed of an enzymatic reactor for continuous flow conversion and flow injection analysis of hydrogen peroxide containing solutions. © 2006 Elsevier B.V. All rights reserved.

"Two-dimensional Fluorescence Spectroscopy: A Novel Approach For Controlling Fed-batch Cultivations"
J. Biotechnol. 2006 Volume 121, Issue 3 Pages 410-417
K. Hantelmann, M. Kollecker, D. Hüll, B. Hitzmann and T. Scheper

Abstract: In industrial fed-batch cultivations it is often necessary to control substrate concentrations at a low level to prevent the production of overflow metabolites and thus optimize the biomass yield. A new method for on-line monitoring and fed-batch control based on fluorescence measurements has been developed. Via instantaneous in situ measurements and multivariate data analysis a chemometric model has been established, which enables the rapid detection of ethanol production at aerobic Saccharomyces cerevisiae fed-batch cultivations. The glucose feed rate is controlled by predicting the metabolic state directly from the fluorescence intensities. Thus, ethanol production could be avoided completely while increasing the biomass yield accordingly. The robust instrumentation is suitable for industrial applications.

"Amperometric Biosensor For The Detection Of Hydrogen Peroxide Using Catalase Modified Electrodes In Polyacrylamide"
J. Biotechnol. 2005 Volume 119, Issue 2 Pages 172-180
Shailly Varma and Bo Mattiasson

Abstract: A simple biosensor for the detection of hydrogen peroxide in organic solvents has been developed and coupled to a flow injection analysis (FIA) system. Catalase was entrapped in polyacrylamide gel and placed on the surface of platinum (working electrode) fixed in a Teflon holder with Ag-wire (auxiliary electrode), followed by addition of filter paper soaked in KCl. The entrapped catalase gel was held on the electrode using membranes. The effects of cellulose and polytetrafluoroethylene (PTFE) membranes on the electrode response towards hydrogen peroxide have been studied. The modified electrode has been used to study the detection of hydrogen peroxide in solvents like water, dimethyl sulfoxide (DMSO), and 1,4-dioxane using amperometric techniques like cyclic voltammetry (CV) and FIA. The CV of modified catalase electrode showed a broad oxidation peak at -150 mV and a clear reduction peak at -212 mV in the presence of hydrogen peroxide. Comparison of CV with hydrogen peroxide in various solvents has been carried out. The electrode showed an irreversible kinetics with DMSO as the solvent. A flow cell has been designed in order to carry on FIA studies to obtain calibration plots for hydrogen peroxide with the modified electrode. The calibration plots in several solvents such as water, dimethyl sulfoxide, 1,4-dioxane have been obtained. The throughput of the enzyme electrode was 10 injections per hour. Due to the presence of membrane the response time of the electrode is concentration dependent.

"Monitoring And Control Of Gluconacetobacter Xylinus Fed-batch Cultures Using In Situ Mid-IR Spectroscopy"
J. Biotechnol. 2004 Volume 113, Issue 1-3 Pages 231-245
Henri Kornmann, Sergio Valentinotti, Philippe Duboc, Ian Marison and Urs von Stockar

Abstract: A partial least-squares calibration model, relating mid-infrared spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concentrations has been designed to monitor and control cultivations of Gluconacetobacter xylinus and production of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solutions containing a mixture of the major components of culture media have been used to calibrate the spectrometer. A factorial design has been applied to determine the composition and concentration in the calibration matrix. This approach guarantees a complete and intelligent scan of the calibration space using only 55 standards. This calibration model allowed standard errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concentrations of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, respectively.With G. xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass. However, residual ethanol in the culture medium prevents bacterial growth. On-line spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermentation. Acetate concentration was controlled at a constant value by feeding ethanol into the bioreactor. The designed fed-batch process allowed biomass production on ethanol. This was not possible in a batch process due to ethanol inhibition of bacterial growth. In this way, the productivity of gluconacetan was increased from 1.8 X 10^-3 [C-mol/C-mol substrate/h] in the batch process to 2.9 X 10^-3 [C-mol/C-mol substrate/h] in the fed-batch process described in this study.

"On-line Monitoring Of A Two-stage Anaerobic Digestion Process Using A BOD Analyzer"
J. Biotechnol. 2004 Volume 109, Issue 3 Pages 263-275
Jing Liu, Gustaf Olsson and Bo Mattiasson

Abstract: A computer-controlled biochemical oxygen demand (BOD) analyzer has been developed for fast estimation of biochemical oxygen demand (BODst) automatically with the purpose of on-line monitoring of a process for conversion of biomass under field conditions. The instrument was tested by on-line monitoring of the connecting stream between two stages of a two-stage anaerobic process in laboratory scale. In the first stage, hydrolysis of sugar beet leaves and its conversion into volatile fatty acids and other low molecular weight substrates took place. The effluent from the first reactor was used as a feed stream to the second stage, i.e. an anaerobic contact reactor. The feed stream was sampled intermittently, diluted and analyzed by the BOD analyzer automatically in order to estimate the organic loading rate to the reactor. The results from this study demonstrated that the BOD analyzer could be a stand-alone and promising sensor device for rapid on-line monitoring of easily biodegradable organic substances in biological treatment processes.

"Single-cell Variability In Growing Saccharomyces Cerevisiae Cell Populations Measured With Automated Flow Cytometry"
J. Biotechnol. 2004 Volume 109, Issue 3 Pages 239-254
James Kacmar, Abdelqader Zamamiri, Ross Carlson, Nicholas R. Abu-Absi and Friedrich Srienc

Abstract: Cell cultures normally are heterogeneous due to factors such as the cell cycle, inhomogeneous cell microenvironments, and genetic differences. However, distributions of cell properties usually are not taken into account in the characterization of a culture when only population averaged values are measured. In this study, the cell size, green fluorescence protein (Gfp) content, and viability after automated staining with propidium iodide (PI) are monitored at the single-cell level in Saccharomyces cerevisiae cultures growing in a batch bioreactor using an automated flow injection flow cytometer system. To demonstrate the wealth of information that can be obtained with this system, three cultures containing three different plasmids are compared. The first plasmid is a centromeric plasmid expressing under the control of a TEF2 promoter the S65T mutant form of Gfp. The other two plasmids are 2 µm plasmids and express the FM2 mutant of Gfp under the control of either the TEF1 or the TEF2 promoter. The automated sampling, cell preparation, and analysis permitted frequent quantification of the culture characteristics. The time course of the data representing not only population average values but also their variability, provides a detailed and reproducible 'fingerprint' of the culture dynamics. The data demonstrate that small changes in the genetic make up of the recombinant system can result in large changes in the culture Gfp production and viability. Thus, the developed instrumentation is valuable for rapidly testing promoter strength, plasmid stability, cell viability, and culture variability.

"Evaluation Of The GFP Signal And Its Aptitude For Novel On-line Monitoring Strategies Of Recombinant Fermentation Processes"
J. Biotechnol. 2004 Volume 108, Issue 2 Pages 115-125
Helga Reischer, Irene Schotola, Gerald Striedner, Florentina Pötschacher and Karl Bayer

Abstract: A high number of economically important recombinant proteins are produced in Escherichia coli based host/vector systems. The major obstacle for improving current processes is a lack of appropriate on-line in situ methods for the monitoring of metabolic burden and critical state variables. Here, a pre-evaluation of the reporter green fluorescent protein (GFP) was undertaken to assess its use as a reporter of stress associated promoter regulation. The investigation of GFP and its blue fluorescent variant BFP was done in model fermentations using E. coli HMS174(DE3)/pET11aGFPmut3.1and E. coli HMS174(DE3)/pET11aBFP host/vector systems cultured in fed-batch and chemostat regime. Our results prove the suitability of the fluorescent reporter proteins for the design of new strategies of on-line bioprocess monitoring. GFPmut3.1 variant can be detected after a short lag-phase of only 10 min, it shows a high fluorescence yield in relation to the amount of reporter protein, a good signal to noise ratio and a low detection limit. The fluorescence-signal and the amount of fluorescent protein, determined by ELISA, showed a close correlation in all fermentations performed. A combination of reporter technology with state of the art sensors helps to develop new strategies for efficient on-line monitoring needed for industrial process optimization. The development of efficient monitoring will contribute to advanced control of recombinant protein production and accelerate the development of optimized production processes.

"Experimental Modelling Of Thermal Inactivation Of Urease"
J. Biotechnol. 2003 Volume 105, Issue 3 Pages 235-243
Viera Illeová, Milan Polakovic, Vladimír tefuca, Pavel Aai and Mohammad Juma

Abstract: Thermal inactivation of jack bean urease (EC 3.5.1.5) was investigated in a 0.1 M phosphate buffer with pH 7. An injection flow calorimetry method was adapted for the measurement of the enzyme activity. The inactivation curves were measured in the temperature range of 55 to 87.5°C. The curves exhibited a biphasic pattern in the whole temperature range and they were well fitted with a biexponential model. A simultaneous fit of all inactivation data was based on kinetic models that were derived from different inactivation mechanisms and comprised the material balances of several enzyme forms and the enthalpy balance characterizing the initial heating period of enzyme solution. The multitemperature evaluation revealed that an adequate model had to incorporate at least three reaction steps. It was concluded that the key reaction steps at urease thermal inactivation were the reversible dissociation/denaturation of native form into an inactive denatured form, and irreversible association reactions of both the denatured and native forms.

"State And Parameter Estimation Of Microalgal Photobioreactor Cultures Based On Local Irradiance Measurement"
J. Biotechnol. 2003 Volume 105, Issue 1-2 Pages 165-178
Wei Wen Su, Jian Li and Ning-Shou Xu

Abstract: Local photosynthetic photon flux fluence rate (PPFFR) determined by a submersible 4[pi] quantum micro-sensor was used in developing a versatile on-line state estimator for stirred-tank microalgal photobioreactor cultures. A marine micro-alga Dunaliella salina was used as a model organism in this study. On-line state estimation was realized using the extended Kalman filter (EKF), based on a state model of the photobioreactor and on-line local PPFFR measurement. The dynamic state model for the photobioreactor was derived based on mass-balance equations of the relevant states. The measurement equation was established based on an empirical correlation between the microalgal biomass concentration and the local PPFFR measured at a fixed point inside the photobioreactor. An internal model approach was used to estimate the specific growth rate without the need of state-based kinetic expression. The estimator was proven to be capable of estimating biomass concentration and specific growth rate, as well as phosphate and dissolved oxygen concentrations in a photobioreactor illuminated with either fixed or time-varying incident radiation. The quantum sensor was shown to be robust and able to quickly respond to dynamic changes in local PPFFR. In addition, the quantum sensor outputs were not affected by bubble aeration or agitation within the typical operating range. The strong filtering capacity of EKF gives the state estimator superior performance compared to direct calculation from the empirical biomass/local PPFFR correlation. This state estimation system makes use of inexpensive and reliable sensor hardware to report key process dynamics of microalgal photobioreactor cultures on-line, enabling improved operation of such a process.

"On-line Bioprocess Monitoring With A Multi-wavelength Fluorescence Sensor Using Multivariate Calibration"
J. Biotechnol. 2001 Volume 88, Issue 1 Pages 47-57
Erik Skibsted, Carsten Lindemann, Christophe Roca and Lisbeth Olsson

Abstract: Cultivations of Pseudomonas fluorescens were monitored with a multi-wavelength on-line fluorescence sensor. The multi-wavelength fluorometer used excitation light from 270 to 550 nm with 20 nm steps and measured fluorescence emission from 310 to 590 nm. The fluorescence, on-line exhaust gas measurements and off-line analysis of nitrate, succinate, optical density and protein were compared chemometrically by multivariate calibration, i.e. computing partial least square (PLS) regression models. Based on the multivariate regression models, it was possible to determine CO2 and O2 composition in the exhaust gas (the correlation coefficients, R2 between the predicted values by the PLS model and the measured values was 0.97 for CO2 and 0.97 for O2, respectively). Also to make quantitative determinations of succinate (R2=0.97), protein (R2=0.94), optical density (R2=1.0) and nitrate (R2=0.98) in the medium based on the fluorescence spectra. Only a limited data set was available but the results indicated that the sensor could indirectly determine non-fluorescent compounds, i.e. nitrate and succinate, which probably is due to the stoichiometric relationship between fluorescent cellular components and non-fluorescent compounds. Consequently multi-wavelength fluorescence is an interesting technique for a wide range of applications.

"Progress In Monitoring, Modeling And Control Of Bioprocesses During The Last 20 Years"
J. Biotechnol. 2001 Volume 85, Issue 2 Pages 149-173
Karl Schügerl

Abstract: The paper gives a review on the recent development of bioprocess engineering. It includes monitoring of product formation processes by flow injection analysis, Various types of chromatographic and spectroscopic methods as well as by biosensors. The evaluation of mycelial morphology and physiology by digital image analysis is discussed also. It deals with advanced control of indirectly evaluated process variables by means of state estimation/observer, with the use of structured and hybrid models, expert systems and pattern recognition for process optimization and gives a short report on the state of the art of metabolic flux analysis and metabolic engineering. (C) 2001 Elsevier Science B.V. All rights reserved.
Process monitoring

"The Use Of Elements Of The E-coli Ntr-system For The Design Of An Optimized Recombinant Expression System For High Cell Density Cultivations"
J. Biotechnol. 1999 Volume 75, Issue 2-3 Pages 241-250
Volker Schroeckh, Rolf Wenderoth, Marian Kujau, Uwe Knüpfer and Dieter Riesenberg

Abstract: The inducible glnA promoter 2 of the E. coli glutamine synthetase gene is suitable as an expression unit for the production of recombinant proteins at low and high cell densities. It is active when the concentration of ammonium as the sole nitrogen source in the culture medium is below 1 mM. This nitrogen regulatory system was optimized by introduction of expression cassettes consisting of additional elements of the ntr-system These artificial constructions result in enhanced recombinant gene expression in the production phase. Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control. A three-to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter protein) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC similar to P via plasmidal co-expression of the ntrC gene. The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half. It is assumed that under nitrogen excess conditions sigma(70)-RNA polymerase binds at glnA pi and thereby prevents most of the binding of sigma(54)-RNA polymerase at glnAp2. The optimized expression systems were successfully applied in low and high cell density cultivations. In the fed-batch phase of high cell density cultivations recombinant protein formation was induced through external nitrogen limitation under FIA-controlled concentration of glucose as carbon source.

"Development Of A Highly Sensitive Chemiluminescence Flow Injection Analysis Sensor For Phosphate-ion Detection Using Maltose Phosphorylase"
J. Biotechnol. 1999 Volume 75, Issue 2-3 Pages 127-133
Hideaki Nakamura, Mami Hasegawa, Yoko Nomura, Yoshiko Arikawa, Ritsuko Matsukawa, Kazunori Ikebukuro and Isao Karube

Abstract: A chemiluminescence flow injection analysis biosensor has been constructed for phosphate-ion detection. This system is coenzymeless and employs a maltose phosphorylase, mutarotase, and glucose-oxidase (MP-MUT-GOD) reaction system combined with an Arthromyces ramosus peroxidase-luminol reaction system. The system consists of a column packed with MP-MUT-GOD immobilized on N-hydroxysuccinimide beads, a mixing joint for the chemiluminescence reaction, and a photomultiplier. The response provided by this system was linear, with a wide range between 10 nM and 30 µM phosphate ion, and a measuring time of 3 min per sample. Under the optimal condition, the sensor was able to detect 1.0 µM phosphate ion for at least 2 weeks. For a practical application, the determination of phosphate ion in river water was examined using ethylenediaminetetraacetic acid, and the results were estimated by comparing with the molybdenum-blue method.
Apparatus

"High Performance Flow Injection Analysis Of Recombinant Protein G"
J. Biotechnol. 1999 Volume 69, Issue 1 Pages 1-7
Jörg Hagedorn, Cornelia Kasper, Ruth Freitag and Tatiana Tennikova

Abstract: Chromatographic discs were investigated for their potential to substitute for the hitherto used cartridges in heterogeneous Bow injection analysis. Originally designed for fast high performance liquid chromatography (HPLC) of biopolymers, the discs combine reliability with speed and resolution. This together with their price and their long-standing time made them attractive for use in flow injection analysis, The base material of the discs is a glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) co-polymer. The epoxy groups inherent to this base structure can be used for immobilization purposes. In this first demonstration, antibodies were immobilized and the resulting affinity discs used for the fast analysis (< 5 min) of protein G from cell lysate of recombinant Escherichia coli. A linear calibration curve over several orders of magnitude as well as excellent reproducibility and correlation with data produced by conventional protein assay were obtained.

"Neural Networks As A Modeling Tool For The Evaluation And Analysis Of FIA Signals"
J. Biotechnol. 1998 Volume 65, Issue 1 Pages 15-22
Bernd Hitzmann*, Adnan Ritzka, Roland Ulber, Karsten Sch&ouml;ngarth and Olaf Broxtermann

Abstract: Three different application examples are discussed, where neural networks are used as a modeling tool for the evaluation of FIA measurements. In the first example, the ability of a neural network is presented to relate the characteristic shape of a FIA measurement with the corresponding analyte, i.e. penicillin, concentration. Using classical evaluation techniques, based on peak height, area or width, these signals cannot be evaluated reliably. The second application demonstrates a multiple injection FIA system for glucose detection, whose measurement signals are a superposition caused by a fast triplicate injection. The neural network is used for the deconvolution process, which makes possible a simultaneous measurement and calibration. In the last application, neural networks are presented as a tool to detect distorted FIA measurement signals.
Penicillin Glucose Theory Neural network Deconvolution Detector

"Fast Online Data Evaluation Of Flow Injection Analysis Signals Based On Parameter Estimation By An Extended Kalman Filter"
J. Biotechnol. 1998 Volume 62, Issue 1 Pages 11-28
X. Wu and K. -H. Bellgardt*

Abstract: The present paper is concerned with the fast evaluation of the flow injection analysis (FIA) signals and the automatic correction of the analytical values interfered by systematic and stochastic disturbances. With the application of the extended Kalman filter, the highest amount of information for the data evaluation of analytical signals can be estimated from FIA peaks. The concentration of the analyte and the offset of the baseline are estimated as time-variable parameters by filtering. The results of the application to simulated and real FIA data show that the parameters corresponded to the evaluation of the analytical data, but is already available before the maximum of the FIA peak, i.e. the evaluation of the data with the Kalman filter can be done during the running peak. Good estimates of the measured values are already obtained short after start of the peak. For this reason, the measuring dead-time of the FIA system can be reduced by the use of this method. The evaluation of FIA signals disturbed often by fluctuation of the baseline and other noise can be corrected by estimation of the offset of FIA peaks and smoothing of real FIA signals. Therefore, the application of the extended Kalman filter can also improve conventional evaluation methods.
Fermentation broth Kalman filter Signal processing Quality

"A Bienzyme Electrode For L-malate Based On A Novel And General Design"
J. Biotechnol. 1998 Volume 61, Issue 2 Pages 129-133
Nenad Gajovic*, Axel Warsinke and Frieder W. Scheller

Abstract: The coimmobilization of a NAD(P)(+)-dependent dehydrogenase with salicylate hydroxylase (SHL, EC 1.14.13.1) in front of a Clark-electrode yields a flexible new design for dehydrogenase based biosensors. The feasibility of the approach has been tested with malic enzyme (MDH; EC 1.1.1.40) as the dehydrogenase, resulting in a novel L-malate sensor. It had substantial advantages over the biosensor approaches reported earlier: effective re-oxidation of NADPH by SHL yielded an extended linear range from 0.01 to 1.2 mmol L-1 L-malate and strongly reduced NADP(+)-requirement (< 0.025 mmol l-1), while the working stability was increased to more than 30 days. The results obtained from six real samples showed a close correlation with the standard enzymatic method. The presented scheme with SHL and the Clark-electrode can be employed together with any NAD(P)(+)-dependent dehydrogenase.
l-Malate Electrode Electrode Apparatus Linear dynamic range

"Single Turnover Mechanism Of A Trypsin-reactor With High Enzyme Concentration"
J. Biotechnol. 1998 Volume 60, Issue 1-2 Pages 81-95
Paola Fermi, Riccardo Biffi, Virna Conti, Roberto Ramoni, Stefano Grolli, Paolo Accornero and Enrico Bignetti*

Abstract: A small column containing 2 mM CH-Sepharose 4B-immobilized trypsin was connected to a flow injection device equipped for potentiometric measurements (0.01-2 mM protons) and for post-column analysis by spectrophotometry and capillary electrophoresis (CE). The device was engaged with N-α-benzoyl-l-arginine pNO2-anilide (BAPNA), β-lactoglobulin (β-Lac) and peptides of V8-protease predigested β-Lac. At a given flow rate, the reaction with BAPNA or β-Lac (below 2 mM) produced about 1 proton per substrate mol. in each sample (linear relation to substrate amount) with peptides (below 22 mM), the reaction did not exceed 0.17 acid equivalent per substrate molecule. (hyperbolic dependence). Final experiments demonstrated that the reactor gave a correct estimate of available lysine in peptides of β-Lac modified with 5-nitrosalicylaldehyde. The data could be predicted by a kinetic model describing the reactor performance in 'single turnover' conditions. The interplay between resident time and the non-catalytic amount of trypsin prevented each enzyme molecule from recycling as well as each substrate molecule (containing one or more cleavage sites) from encountering the enzyme more than once. In conclusion, both from the experimental and the theoretical point of view, this work permitted the analysis of trypsin behavior in some extreme working conditions and indicates how to modulate the performance of an endoprotease-based reactor. A brief discussion on potential applications in protein mapping and tagging and in the quanitative analysis of protein bioavailability by means of a biosensorial strategy is also described.
Enzyme, trypsin Lysine Mucus Potentiometry Electrode Spectrophotometry Electrophoresis Reaction order Kinetic Modeling

"Fast Online Flow Injection Analysis System For IgG Monitoring In Bioprocesses"
J. Biotechnol. 1997 Volume 59, Issue 1-2 Pages 145-153
Martin Reinecke and Thomas Scheper*

Abstract: An automated immunoassay, with one affinity component immobilized on a solid surface, has been developed to monitor the production of different immunoglobulins during mammalian cell cultivation processes. The whole analysis device is based on the principle of flow injection analysis (FIA) and a cartridge with the immobilized affinity component is implemented into the FIA system. This cartridge is filled with a carrier material to which protein G is covalently bound. After sample injection, binding of the IgG on the protein G within the cartridge takes place while after a washing step, the IgGs are eluted by a pH shift, and the IgG concentration is monitored via fluorescence. In the automated immunoassay, undiluted cell free samples from the reactor or from down-stream processing can be analyzed directly. Due to the separation the IgG can be detected without interference from other sample components by protein fluorescence. The results are obtained with analysis times below 6 min. Sample volumes of less than 100 µL may be used. The assay is sensitive to concentrations from 5 up to 500 µg mL-1. Using this FIA-System, immunoglobulins G, produced in different media, were successfully monitored. The results of the assay were validated by ELISA.
Immunoglobulin G Cell Fluorescence Immunoassay Method comparison Immobilized protein Interferences Process monitoring

"Bioaffinity Layering: A Novel Strategy For The Immobilization Of Large Quantities Of Glycoenzymes"
J. Biotechnol. 1997 Volume 55, Issue 3 Pages 171-179
Mariya Farooqi, Mohammed Saleemuddin, Roland Ulber, Peter Sosnitza and Thomas Scheper*

Abstract: A simple strategy for increasing considerably the quantities of glycoenzymes immobilized on insoluble supports is described. The strategy that we call bioaffinity layering makes use of the multivalent nature of concanavalin A (Con A) and the multiple oligosaccharide chains of most glycoenzymes to build alternating lectin and glycoenzyme layers on a Sepharose matrix with precoupled Con A. Using this procedure, it was possible to increase the amounts of several glycoenzymes immobilized on Sepharose and 19.0 mg glucose oxidase could be associated with one mL Sepharose matrix after seven Con A/glucose oxidase incubation cycles. Bioaffinity layered preparations of glycoenzymes exhibited high activities as indicated by very high effectiveness factor (eta) values and those of glucose oxidase and invertase exhibited a layer-by-layer increase in thermostability. The sensitivity of a flow-through glucose monitoring cartridge integrated into a flow injection analysis (FIA) system was enhanced significantly by increasing the amount of immobilized glucose oxidase via bioaffinity layering. A cartridge bearing six layers of glucose oxidase on Sepharose support was used effectively and repeatedly for analysis of medium glucose concentration during a fed-batch cultivation of the yeast Saccharomyces cerevisiae.
Glucose Fermentation broth Sensor Immobilized enzyme Kinetic Reactor

"Bioprocess Automation And Bioprocess Design"
J. Biotechnol. 1997 Volume 52, Issue 3 Pages 175-179
Bernhard Sonnleitner*

Abstract: The importance of automation in bioprocessing is highlighted from several points of view, namely under scientific, practical and routine aspects. Valuable opportunities opened by automation are shown and some requirements for realization of a robust automated system are discussed; some potential pit-falls are addressed. A standard operating procedure useful for bioprocess development and routine operation is proposed. (C) 1997 Elsevier Science B.V. 11 References
Automation Process control Process monitoring Review

"Development Of An Online Control System For The Cultivation Of Animal Cells In A Hollow-fiber Reactor Using Flowing Injection Analysis And A Visual Programming Language"
J. Biotechnol. 1996 Volume 51, Issue 1 Pages 37-48
T. S. Stoll, P. -A. Ruffieux, U. Von Stockar and I. W. Marison*

Abstract: A system for the independent control of ammonia and glutamine in a hollow-fiber reactor has been developed. Online analysis of the two chemical species was performed using FIA. A custom-made program has been written to fully automate the FIA, to perform measurements, calibrations and data acquisition, and to activate two peristaltic feed pumps. Diagnostic tests were also automatically performed in order to identify, and in some cases eliminate, erroneous measurements and calibrations due to instrument failures. For this purpose, a visual programming language (LabVIEW) has been chosen. Ammonia concentration was controlled by varying the medium supply rate to the hollow-fiber reactor which was shown to behave like an ideal-stirred tank reactor. The glutamine concentration was controlled through the intermittent activation of a pump delivering a concentrated solution of this species to the reactor. This control system was tested with a culture of hybridoma cells at three different ammonia and one glutamine set- points. The hollow-fiber reactor was successfully operated automatically for more than 1200 h with ammonia and glutamine concentrations accurately controlled at non-limiting levels. The visual programming environment was found to be reliable and highly suitable for the development of custom programs for bioprocess control.
Ammonia Glutamine Process control Computer

"Online Simultaneous Monitoring Of Ammonia And Glutamine In A Hollow-fiber Reactor Using Flow Injection Analysis"
J. Biotechnol. 1996 Volume 51, Issue 1 Pages 27-35
T. S. Stoll, P. -A. Ruffieux, M. Schneider, U. Von Stockar and I. W. Marison*

Abstract: A FIA method has been developed and fully characterized for the simultaneous detection of ammonia and glutamine in culture media. Ammonia detection is based upon a chemical method and does not require an electrode. This species diffuses across a hydrophobic porous membrane into an indicator solution, the absorbance of which is continuously monitored using a LED and a phototransistor. Glutamine was determined by a difference method in which ammonia was measured before and after passage through a module containing immobilized glutaminase. Very good linearity was observed in the range 0-4 mM for both species. Total analysis time was 22 min. This FIA method was used to accurately monitor ammonia and glutamine on line for over 600 h for a hybridoma culture performed in a hollow-fiber reactor. FIA measurements were in good agreement with off-line measurements. No instrument failures occurred, thanks to systematic cleaning and maintenance procedures. This FIA method is a very attractive tool for the monitoring, and possibly control, of continuous cultures over extended periods.
Ammonia Glutamine Fermentation broth Spectrophotometry Method comparison Photodiode Light emitting diode

"Analysis Of Various Sugars By Means Of Immobilized Enzyme Coupled Flow Injection Analysis"
J. Biotechnol. 1996 Volume 50, Issue 2-3 Pages 93-106
B. Weigela, B Hitzmanna, G. Kretzmera, K. Sch&uuml;gerla,*, A. Huwigb and F. Giffhornb

Abstract: Glucose, maltose, sucrose, lactose, xylose, sorbose, galactose, fructose and gluconolactone were analyzed by means of immobilized pyranose oxidase as well as by the combination of immobilized glucose oxidase with immobilized glycoamylase, invertase, mutarotase, maltase (α-glucosidase) and glucose isomerase by flow injection analysis (FIA). For the simultaneous analysis of glucose and other sugars three different flow injection configurations were applied and compared. The average error of prediction of the analyzes were better than 3% in model media and better than 6% in yeast extract containing media.
Sugars Glucose Sucrose Lactose Xylose Sorbose Galactose Fructose Gluconolactone Chemiluminescence Immobilized enzyme Simultaneous analysis Manifold comparison

"Glucose Uptake Kinetics Of Saccharomyces Cerevisiae Monitored With A Newly Developed FIA"
J. Biotechnol. 1996 Volume 50, Issue 1 Pages 1-12
S. A. Rothen, M. Saner, S. Meenakshisundaram, B. Sonnleitner and A. Fiechter*

Abstract: The glucose content of the culture liquid during shift experiments and synchronized cultures of Saccharomyces cerevisiae H1022 (ATCC 32167) was monitored using a greatly improved and highly precise FIA. During shift-up experiments on the dilution rate, an overshoot of the glucose- concentration was observed. The amplitude of the overshoot showed a dependency on the duration of undisturbed cultivation before application of the shift. Mutarotational non-equilibrium was excluded as the cause of the observed overshoot. For the first time glucose measurements of oscillating cultures of Saccharomyces cerevisiae are demonstrated with high accuracy and reproducibility. The data strongly support the proposals by Munch et al. (1992a, b) that faint oscillations in glucose concentration are responsible for the persistence of the synchronization. Analytical subsystems prove to be a powerful tool for investigation of the dynamics of metabolic pathways of microbial organisms. Accurate glucose measurements at low concentrations point out the limits and allow refinements of commonly used models.
Glucose Fermentation broth Biotechnology Process monitoring Kinetic

"High Speed Centrifugal Separator For Rapid Online Sample Clarification In Biotechnology"
J. Biotechnol. 1996 Volume 49, Issue 1-3 Pages 111-118
P. Richardson, J. Molloy, R. Ravenhall, I. Holwill*, M. Hoare and P. Dunnill

Abstract: Sample clarification is a common operation in biochemical analytical methods for removing interfering or unwanted particulates from an analyte sample. Filtration provides one option for removal of particulates. However, in many cases the loss of soluble protein due to filter adsorption is unacceptable and an alternative must be sought. In this paper a microcentrifuge designed to automatically sample, spin, deliver supernatant to an analyzer. and wash out solids from the bowl is described. The performance of the system is assessed in terms of its clarification efficiency and the time required to achieve satisfactory clarification. Additionally, the effects of different protein precipitating agents on yeast homogenate samples separated using the microcentrifuge are studied where the system is used to deliver supernatant to a flow injection analyzer.. The paper demonstrates that the microcentrifuge may be used to separate rapidly such samples on a time scale between 10^-60 s depending upon the type and size of sample and be successfully used as a component of an at-line monitoring system.
Biotechnology Interferences

"Improvement Of The Production Of Subtilisin Carlsberg Alkaline Protease By Bacillus Licheniformis By Online Process Monitoring And Control In A Stirred Tank Reactor"
J. Biotechnol. 1996 Volume 49, Issue 1-3 Pages 83-93
A. B. Van Puttena, F. Spitzenbergerb, G. Kretzmerb, B. Hitzmannb, M. Dorsb, R. Simutisb and K. Sch&uuml;gerlb,*

Abstract: The cultivation of Bacillus licheniformis and the production of subtilisin Carlsberg serine protease were investigated in complex medium with starch and glucose, respectively, and Na-caseinate as substrates to maximize the protease concentration. The turbidity and culture fluorescence were monitored in situ, the optical density online and the (dry) sediment and (dry) cell mass concentrations as well as the cell count and the DNA content were monitored off-line. These values are closely interrelated and were quantified by particular relationships. By means of the six-channel flow injection analyzer. (FIA) system, the following medium components were monitored online: glucose, maltose, starch, ammonium, urea, phosphate and protease activity. The same components as well as protein, intracellular phosphate and α-amylase activity were evaluated off-line. The off-gas composition was analyzed online as well. Various control strategies were tested in order to maximize the protease concentration: On one hand, starch in various concentrations was used as substrate. These runs were performed at non-controlled starch decomposition, at controlled and non-controlled pH-values, respectively, and non-controlled Po-2-values. On the other hand, glucose was used as substrate in fed-batch mode. These runs were performed with closed loop controlled pH- and Po-2-values and open-loop and closed-loop controlled glucose concentrations, respectively. The latter strategy yielded a higher protease concentration than the former. With complex medium and closed-loop controlled process, extremely high protease activities (24000 EPE mL-1) were obtained.
Glucose Maltose Starch Ammonium Urea Phosphate Enzyme, protease Fermentation broth Turbidimetry Fluorescence Well stirred mixing chamber Multichannel Process monitoring Closed loop

"A Novel Biosensor System For The Determination Of Phosphate"
J. Biotechnol. 1996 Volume 48, Issue 1-2 Pages 67-72
Kazunori Ikebukuro, Ryoko Nishida, Hiroyuki Yamamoto, Yoshiko Arikawa, Hideaki Nakamura, Masayasu Suzuki, Izumi Kubo, Toshifumi Takeuchi and Isao Karube*

Abstract: A highly sensitive biosensor system has been developed for phosphate detection. This biosensor system is based on the pyruvate oxidase reaction and a subsequent luminol chemiluminescence reaction. Hydrogen peroxide generated by pyruvate oxidase reacts with luminol catalyzed by an immobilized peroxidase. The resulting chemiluminescence is detected by a photomultiplier. This system uses a flow injection analysis (FIA) system resulting in the rapid determination of phosphate, taking approximately 3 min for one measurement. A linear response was observed from 0.37 µM to 7.4 µM phosphate while the detection limit was 74 nM. This sensitivity is sufficient to determine the maximum permissible phosphate concentration of the natural waters of Japan (0.32 µM). The pyruvate oxidase immobilized column gave a stable response over a period of 2 weeks when stock solutions were analyzed.
Phosphate Environmental Sensor Chemiluminescence Immobilized enzyme

"Online Control Of An Immobilized Hybridoma Culture With Multi-channel Flow Injection Analysis"
J. Biotechnol. 1995 Volume 43, Issue 3 Pages 229-242
Jens J. van der Pol*, Burkhard Joksch, Jochem Galgens, Manfred Biselli, Cornells D. de Gooijer, Johannes Tramper and Christian Wandrey

Abstract: An immobilized hybridoma cell line was cultivated at controlled glucose and glutamine concentrations. Online analysis of the substrates was carried out with a multi-channel flow injection analysis system. The analysis system also determined online the lactate and ammonium concentration. The substrate concentrations were controlled using an adaptive-control strategy. This strategy consisted of the estimation of the real-time concentrations and volumetric substrate consumption rates by an Extended Kalman Filter, and a minimum variance controller, which used the estimated parameters to set the feed rates of the substrates. The closed-loop control was used to start-up two cultures with either glucose or glutamine as control-substrate for the medium feed rate. The controller kept the concentration of the control-substrate constant by enhancing the medium feed rate simultaneously to the increasing volumetric consumption rate of the substrate. When glutamine was used as control-substrate, the glucose concentration remained relatively constant, whereas the glutamine concentration decreased during the start-up at a constant glucose concentration. This indicates that glutamine is consumed faster than glucose and will be a better control- substrate to avoid limitation during the start-up of a culture with the applied hybridoma cell line. During the colonization of the microcarriers, the yield of ammonium on glutamine decreased from 0.80 to 0.55 (mol mol-1), indicating a change in the glutamine metabolism. The yield of lactate on glucose stayed constant for both experiments. During long-term culture of more than 800 h, the controller kept both the glucose and glutamine concentrations constant at perfusion rates between 0.50 h-1 and 0.15 h-1. The medium, glucose and glutamine feed rate were independently controlled. Both the specific glutamine and glucose consumption rates remained constant for all perfusion rates, which was probably as a result of the constant concentrations. The specific monoclonal antibody production rate decreased with the perfusion rate decreasing from 0.40 h-1 to 0.20 h-1. The immobilized- cell concentration decreased only at the lowest perfusion rate. Both effects could not be explained directly by the increasing ammonium and lactate concentrations nor by the decreasing amino-acid concentrations. (37 references)
Glucose Glutamine Lactate Ammonium Fermentation broth Biotechnology Immobilized cell Kalman filter Perfusion Interferences Reactor Multichannel

"Silica Immobilized Enzyme-catalyzed Removal Of Chlorolignins From Eucalyptus Kraft Effluent"
J. Biotechnol. 1995 Volume 43, Issue 3 Pages 161-167
Marcia Dezott, Lucia H. Innocentini-Mei and Nelson Dur&aacute;n*

Abstract: Immobilization of lignin peroxidases type I, II, III, lyophilized fungal culture from Chrysonilia sitophila (TFB-27441) and horseradish peroxidase (HRP) on activated silica were carried out. Immobilized HRP gave 37% decolorization, with 60% mineralization. The most efficient lignin peroxidase was Lip type III with 20% mineralization, 65% COD reduction and 12% decolorization. None of the enzymes which were immobilized exhibited loss of activities after being frozen for 2 months or after 5 d contact with a kraft effluent. Silica appeared as an important immobilizing support in the industrial use of oxidoreductases in environmental studies. (27 References)
Chlorolignins Plant Immobilized enzyme Silica

"L-glutamate Biosensor Using A Novel L-glutamate Oxidase And Its Application To Flow Injection Analysis System"
J. Biotechnol. 1995 Volume 42, Issue 1 Pages 45-52
Bang-Ce Ye, Qing-Shan Li*, You-Rong Li, Xiao-Bo Li and Jun-Tang Yu

Abstract: Streptomyces P-106, isolated from soil samples, produced extracellular -glutamate oxidase in liquid culture. About 10 units enzyme activity per mL medium could be reached after 48-60 h incubation at 28°C. A micro-enzyme electrode was prepared by cross linking the purified novel -glutamate oxidase, produced in our lab, with glutaraldehyde on an aminopropyl-platinized-platinum wire (0.5 mm). It was used for the determination of L-glutamate in a flow injection analysis system with good performance: accuracy (CV = 0.4%), fast response (<40 s) and stability (>30 d). The system showed linear response to the L-glutamate concentration, ranging from 3.0 mg L-1 to 300 mg L-1 (~0.02 mol L-1 to 2.0 mmol L-1). The system was applied to determine the concentration of L-glutamic acid during synthesis. Good correlations were achieved between results obtained with the system and with Warburg method.
l-Glutamate Sensor Electrode

"Online Monitoring Of An Animal Cell Culture With Multi-channel Flow Injection Analysis"
J. Biotechnol. 1994 Volume 37, Issue 3 Pages 253-264
Jens J. van der Pola,*, Uwe Spohnb, Rolf Eberhardta, Jochem Gaetgensa, Manfred Bisellia, Christian Wandreya and Johannes Tramperc

Abstract: A multi-channel flow injection analysis system was used for online monitoring of a continuous animal cell culture with high cell density. With this system, the glucose, lactate and glutamine concentration were determined using immobilized dehydrogenases, ammonium using an aqueous o-phthaldialdehyde solution. Glutamine concentration was determined on the basis of the difference between a glutamine and a glutamate measurement. To prevent disturbance of the measurement and pollution of the system, the analytes in the sample were separated from high molecular compounds by online dialysis. Online gas dialysis was used to avoid interference of other amino groups with the ammonium determination. In addition, dialysis was used as a dilution step. The measurement time for all four components was 42 min. This time included a final washing period after the analysis cycle. The system was calibrated once a day. Two continuous cultivations of a hybridoma cell line immobilized in open-porous glass carriers were monitored, using a fluidized bed reactor as cultivation system. The concentration of glutamine, glucose and ammonium determined with the online FIA system were in good agreement with the off-line data determined once a day. Only the lactate data showed some deviation. The immobilized enzyme reactors could be used for up to 3000-5000 injections. During the first cultivation, lasting 200 h, the start up period of the reactor was monitored. The online measurements described much better the time- course of the concentrations than the off-line data. It was possible to estimate the growth rate of the cells in the micro-carriers by the on- line data. In the course of the second cultivation, which lasted almost 1000 h, the influence of the dissolved oxygen concentration on the cell metabolism was monitored. It was noted that a sudden change of the glutamine concentration in the feed caused a fast change of the consumption and production rate of the measured metabolites.
Glucose Lactate Glutamine Feed Fermentation broth Biotechnology Immobilized enzyme Method comparison Process monitoring Interferences Dialysis Multichannel

"A Fluorometric Fiber-optic Biosensor For Dual Analysis Of Glucose And Fructose Using Glucose-fructose-oxidoreductase Isolated From Zymomonas Mobilis"
J. Biotechnol. 1994 Volume 36, Issue 1 Pages 39-44
Sei-Jin Leea, Mohammed Saleemuddinb, Thomas Scheperb,*, Heidi Loosc and Hermann Sahmc

Abstract: A fluorometric fiber-optic biosensor was used to determine the content of glucose and fructose according to the measurement principle of flow injection analysis (FIA) The enzyme glucose-fructose-oxidoreductase (GFOR) isolated from Zymomonas mobilis was confined in a measurement cell behind an ultrafiltration membrane. The GFOR catalyzes the oxidation of glucose to gluconolactone and the reduction of fructose to sorbitol according to the ping-pong mechanism. This unique enzyme contains NADPH tightly confined in the enzyme complex. The change of NADPH fluorescence was detected by a fluorosensor. The stability could be increased remarkably by crosslinking with glutaraldehyde. The detection range for glucose was within a concentration range of 0.055-55.5 mM and that for fructose within 0.278-331 mM. The response time for glucose and fructose was in the range of 40 s and 1 min, respectively. Steady-state was achieved for glucose injection in 2 min and fructose injection in 6.5 min. Sampling time for glucose could be reduced to 37% by including fructose in excess to the sample. This also makes the regenerating step unnecessary. The specificity of this biosensor was tested for different sugars. The results showed that this biosensor system is highly specific, sensitive and suitable for dual analysis of glucose and fructose.
Glucose Fructose Fluorescence Sensor Filter Optical fiber

"Determination Of Intracellular Dehydrogenase Activities Using Flow Injection Analysis"
J. Biotechnol. 1994 Volume 33, Issue 3 Pages 221-231
Katrin Steube and Uwe Spohn*

Abstract: Reproducible rapid methods for cell disintegration in miniaturized flow cells were developed. The disintegration of yeast cells by ultrasonics and ultraturrax in a miniaturized flow-through chamber was compared with the mixer mill disintegration. The flow-through chamber was combined with a sterile online sampling device to take samples from yeast cell suspensions and with an automated flow injection analysis (FIA) set-up, to determine the released enzyme activities. The FIA set-up was tested with the sequential determination of malate dehydrogenase, formate dehydrogenase and formaldehyde dehydrogenase in mixtures. The automated FIA determination of the intracellular formate dehydrogenase of yeast cells (hansenula polymorpha) was performed.
Enzyme, formate dehydrogenase Enzyme, malate dehydrogenase Enzyme, formaldehyde dehydrogenase Cell Automation Mixing chamber

"Online Monitoring Of Monoclonal Antibody Production With Regenerable Flow Injection Immuno Systems"
J. Biotechnol. 1994 Volume 32, Issue 3 Pages 213-220
Andreas Gebbert, Manuel Alvarez-Icaza, Heinz Peters, V. J&auml;ger, Ursula Bilitewski* and Rolf D. Schmid

Abstract: In this paper two systems for the observation of the production of mouse-IgG during the cultivation of hybridoma cells in a perfusion reactor are presented. The direct immunosystem is based on the detection of changes in capacitance of a dielectric layer (tantalum oxide) on a metal surface (tantalum) when antibodies bind to immobilized anti-antibodies. The sensor consisted of a 25 nm tantalum oxide layer, electrochemically grown onto a laser patternized 1 micron thick tantalum layer. The indirect system is based on an automated fluorimetric sandwich ELISA system with β-galactosidase conjugated secondary antibodies. Two cultivations of mouse hybridoma cells in a 2-1 perfusion reactor were performed. The first cultivation was monitored with the capacitance system, the second cultivation was monitored with the fluorimetric system.
Tantalum Fermentation broth Capacitance Immunoassay Fluorescence Perfusion

"Online Immunoanalysis For Bioprocess Control"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 395-403
C. Middendorf*, B. Schulze, R. Freitag, Th Scheper, M. Howaldt and H. Hoffmann

Abstract: Immunoanalytical techniques such as ELISA are often used for the detection of proteins produced in cultivation processes. Owing to the difficulty of automating of the time-consuming traditional ELISA, there is an intense demand for a suitable online monitoring method. Combining well-known immunoassays with the FIA technique, we present the heterogeneous and the turbidimetric immuno-FIA methods. The following proteins were investigated with these FIA methods: thermostable pullulanase, IgG, antithrombin III, and recombinant tissue-type plasminogen activator. In the cases of pullulanase and monoclonal mouse IgG, the turbidimetric immuno-FIA was used for online analysis of the cultivation process. Results are presented here to demonstrate the effectiveness and application of these immunoanalysis.
Protein Enzyme, pullulanase Immunoglobulin G Antithrombin III Fermentation broth Immunoassay Turbidimetry Process monitoring

"Automated Immunochemical Binding Assay (flow-ELISA) Based On Repeated Use Of An Antibody Column Placed In A Flow Injection System"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 381-394
Mats Nilsson, Gustav Mattiasson and Bo Mattiasson**

Abstract: A computer-controlled immunological binding assay intended for monitoring concentrations of interesting metabolites during bioprocesses has been developed. The main focus has been on speed, reliability, reproducibility and operational stability of the assay. The assay is based on the repeated use of a preparation of immobilized antibodies. Stability in the regeneration step is a prerequisite and was achieved by intermittent recalibration of the system. Furthermore, before immobilizing the polyclonal antibodies, they were purified on immobilized antigen and eluted using the same procedure as used in the assay cycle. Antibody preparation could be used for more than 50 cycles and in some cases for several hundred cycles. Since the calibration curve is valid for the whole life-time of the antibody column, recalibration only involved the registration of the column capacity. Experiments have been performed concerning monitoring of chromatographic separations as well as adsorption experiments from complex mixtures.
Immunoassay Biotechnology Column

"Determination Of Minute Amounts Of ATP By Flow Injection Analysis Using Enzyme Amplification Reactions And Fluorescence Detection"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 369-380
Elo Harald Hansen, Mich&eagrave;le Gundstrup and Helle Steen Mikkelsen

Abstract: A flow injection system for assay of trace levels of ATP is described that incorporates a small column reactor containing co-immobilized hexokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase. In the presence of appropriate cofactors, ATP is by the synergistic operation of the enzymes repeatedly recycled, resulting in substrate amplification. The ultimately generated NADH is measured fluorometrically. By this approach, where the enzymatic degradation step and the detection step are completely separated, it is possible to operate them individually under optimal conditions. The amplification factor is directly proportional to the residence time of the sample zone within the enzyme reactor, which time might be manipulated by altering the flow-rate and in the extreme by performing stopped-flow experiments. Amplification factors between 15 and 1000 were obtained, but it was found that increased amplifications did not lead to significantly lower detection limits; thus, it appears that a practical lower limit of detection is of the order of 1-5 nM. An investigation of this paradoxical feature, and a possible explanation for it, is given.
Fluorescence Immobilized enzyme Stopped-flow Reactor Optimization

"Design Of Bioluminescence-based Fiber Optic Sensors For Flow Injection Analysis"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 357-368
Lo&iuml;c J. Blum, Sabine M. Gautier and Pierre R. Coulet*

Abstract: A fiber optic sensor based on enzyme-catalyzed light-emitting reactions has been developed and integrated in a flow injection analysis (FIA) system. The firefly luciferase, specific for ATP, and the bacterial oxidoreductase/luciferase system, specific for NADH, have been immobilized on preactivated polyamide membranes. ATP and NADH analysis could be performed in the range from 0.1 pmol to 3 nmol and from 0.5 pmol to 1 nmol, respectively. By co-immobilizing these two bioluminescence systems on the same membrane, a multi-function biosensor has been designed allowing the alternate determination of ATP or NADH with the same sensitivity as that obtained with the two different mono-functional biosensors. A partly self-contained biosensor has been also developed for the flow injection analysis of NADH. For this purpose, FMN (one of the substrates of the bacterial bienzymatic system) has been embedded in a synthetic matrix. Different supports have been tested for the non-covalent immobilization of this substrate and its release in the immediate vicinity of the bound enzymes. Using a photo-crosslinked poly(vinyl alcohol) support, 40 reliable assays (CV = 4.5%) could be performed without changing or reloading the matrix.
Bioluminescence Sensor Enzyme Optical fiber

"Two FIA-based Biosensor Systems Studied For Bioprocess Monitoring"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 345-346
Th. Scheper*, W. Brandes, H. Maschke, F. Pl&ouml;tz and C. M&uuml;ller

Abstract: In this paper, two different FIA-based biosensor systems are described for application to different biotechnologically relevant purposes. In the first system, single fiber optodes were used to determine the pH, urea and penicillin V concentrations. A two-channel system was developed for the simultaneous monitoring of different variables to increase the analysis accuracy. This system was used for monitoring the penicillin V concentration during a cultivation of Penicillium chrysogenum. The second system described is a calorimetric immunoassay based on the use of an enzyme thermistor. A sandwich assay with protein A immobilized on a solid support for the determination of various IgGs was established. A fusion protein of protein A and β-galactosidase obtained from a recombinant E. coli strain was used in the labelling and detection reaction. This system is designed for future application in bioprocess monitoring.
Urea pH Penicillin V Sensor Thermistor Optrode Sensor Process monitoring Multidetection

"Software FIACRE: Bioprocess Monitoring On The Basis Of Flow Injection Analysis Using Simultaneously A Urea Optode And A Glucose Luminescence Sensor"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 327-343
M. Busch, W. H&ouml;bel and J. Polster*

Abstract: Various computer programs for large-scale bioprocess control and optimization have been developed as well as software for simple laboratory routine analysis. In comparison, software can hardly be found that works on laboratory scale and provides the control of complex flow injection analysis (FIA) systems, multisubstrate determination, data evaluation as well as minimal process control abilities. The sensors applied can be of different type (luminometric or other optical as well as electrochemical biosensors). The development of such a software may be very helpful for the transfer of FIA/biosensor systems from the state of development to industrial processes. Hence, each analyzing system--even a well established biosensor--has to be individually adapted to the process, a task which is best done under laboratory conditions. Such a flexible, computer- controlled FIA system for research level based on the software FIACRE is presented. Five FIA/(bio)sensor system can be controlled simultaneously. Additionally, common temperature and pH recordings are possible. Determinations of substrate concentrations are performed by means of calibration curves which can be recorded at different times. This allows supervising the activities of the sensors during a cell cultivation and controlling the bioprocess, e.g. by adding substrate to a cell culture. The automated monitoring of the degradation of glucose and urea by two different optical sensing principles during a cell cultivation under the control of one microcomputer is presented for the first time. For this purpose, already well examined biosensors (a urease optode and a luminometric glucose sensor) were employed and their properties discussed under the aspect of working in real cultivation media. It will also be shown that substrates being of interest for bioprocess control can be detected by slight modifications of known reactions. For example, substrates of NADH-dependent enzymatic reactions can be detected by the luminol chemiluminescence system, and optodes can be employed for pH, penicillin and glucose determination.
Glucose Urea Ethanol Fermentation broth Chemiluminescence Optrode Sensor Computer Process monitoring Optimization

"Online Determination Of Ethanol In Bioprocesses Based On Sample Extraction By Continuous Pervaporation"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 317-325
I. Ogbomo*, A. Steffl, W. Schuhmann, U. Prinzing and H. -L. Schmidt

Abstract: The development of a flow injection analysis system (FIA) based on immobilized enzymes for the online determination of ethanol in biological liquids is presented. The FIA system includes prior to enzymatic analysis a continuous purification by pervaporation, which is supplemented with an electronically controlled temperature device. Fundamental experiments with the pervaporation module as well as the application of this system for the monitoring of ethanol in beer and during a bakers' yeast cultivation (measurable range 1-100 mM) are presented. The results show good reproducibility and satisfactory agreement with those of a common enzymatic test kit. Incorporation of the pervaporation module enhanced the stability of the enzyme noticeably.
Ethanol Fermentation broth Beer Sample preparation Immobilized enzyme Heated reaction Pervaporation Process monitoring Extraction

"A Reagentless Amperometric Biosensor For Alcohol Detection In Column Liquid Chromatography Based On Co-immobilized Peroxidase And Alcohol Oxidase In Carbon Paste"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 301-316
K. Johansson, G. J&ouml;nsson-Pettersson, L. Gorton*, G. Marko-Varga and E. Cs&ouml;regi

Abstract: A reagentless carbon paste electrode chemically modified with covalently bound alcohol oxidase and horse-radish peroxidase was examined as a selective sensor in flow injection and column liquid chromatography. A combination of carbodiimide, glutaraldehyde, and polyethyleneimine was used for immobilizing the enzymes in the paste. The surface of the electrodes was protected by first forming a layer of electropolymerized ortho-phenylenediamine followed by deposition of a cation exchange membrane (Eastman AQ 29D). The electrodes were used for detection of hydrogen peroxide, methanol, ethanol, propanol, isopropanol, and butanol. Preliminary investigations of the use of this sensor for bioprocess control are reported.
Alcohol Hydrogen peroxide Methanol Ethanol 1-Propanol 2-Propanol Butanol, 1- Amperometry Electrode LC Sensor

"Amperometric Biosensor For The Determination Of Phenolic Compounds Using A Tyrosinase Graphite Electrode In A Flow Injection System"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 289-300
F. Ortega*, E. Dom&iacute;nguezG. J&ouml;nsson-Pettersson and L. Gorton

Abstract: Selective and sensitive devices for the monitoring of phenol and phenolic compounds are required in clinical and environmental analysis. This paper describes a biosensor for the analysis of phenolic compounds in a flow injection system. The enzyme electrode is based on the use of immobilized tyrosinase and the amperometric detection of the enzymatic product at -50 mV vs. SCE. The enzyme is covalently immobilized on the surface of a carbodiimide-activated graphite electrode. The biosensor responds to a variety of phenolic substrates with different conversion efficiencies. The detection limit for phenol is 0.003 µM (S/N = 3), a quantification limit of 0.01 µM (rsd 3.7%), and an extended dynamic range up to 5 µM is achieved with a sample frequency of 110 samples per hour.
Phenols Environmental Amperometry Electrode Clinical analysis Sensor Immobilized enzyme

"Development Of A Process-FIA System Using Mediator-modified Enzyme Electrodes"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 277-287
B. Gr&uuml;ndig*B. Strehlitz, H. Kotte and K. Ethner

Abstract: A computer-controlled process-FIA system for monitoring industrial bioprocesses was developed using mediator-modified enzyme electrodes. The single-line FIA system was modified by replacing the mixing coil with a flexible operating sample dilution unit. By this way, the analyzer offers automatic procedures for self-calibration 'real-time' dilution and recalibration based on the current analyte concentration. In regard to the dynamic range of the sensors, the FIA system is able to self-adapting to any analyte concentration of the bioprocess. The technique was tested for control of glucose during microbial fed-batch processes of gluconic acid production. A computer-controlled process-FIA system for monitoring industrial bioprocesses was developed using mediator-modified enzyme electrodes. The single-line FIA system was modified by replacing the mixing coil with a flexible operating sample dilution unit. By this way, the analyzer offers automatic procedures for self-calibration 'real-time' dilution and recalibration based on the current analyte concentration. In regard to the dynamic range of the sensors, the FIA system is able to self-adapting to any analyte concentration of the bioprocess. The technique was tested for control of glucose during microbial fed-batch processes of gluconic acid production.
Electrode Process control Computer

"Online Determination Of Acetic Acid In A Continuous Production Of Acetobacter Aceticus"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 267-275
T. Becker*, R. Kittsteiner-Eberle, T. Luck and H. -L. Schmidt

Abstract: A flow injection system for the determination of acetic acid in the 10^-60 mM range was conceived and optimized on basis of the coupled reactions of acetate kinase/pyruvate kinase/lactate dehydrogenase. The enzymes were immobilized in series, a reagent mixture containing PEP, ATP and NADH was pulsed into the analyte stream, and the decrease of the NADH absorption was determined. One injection cycle was performed within 90 s. Two identical parallel enzyme lines were installed, one of them being in use while the other was calibrated. Automated calibration, control of sample dilution, compensation for enzyme activity loss, the switching between the two enzyme lines and the control of all instruments were performed by an appropriate computer system. The analysis of vinegar samples resulted correct and reliable results. The semi-automated control of continuous vinegar production of 60 h yielded very satisfactory results in the range of 0.5-1.5 M after 40-fold dilution and was more reliable than the reference HPLC method. The extension of the analysis system to higher concentrations ranges still demands an optimization of the dilution method. In total, the enzyme-based FIA for continuous acetic acid determination seems to be a very useful and reliable method.
Acetic acid Food Spectrophotometry Sensor Immobilized enzyme Indirect Optimization

"Comparison Of Different Biosensor Systems Suitable For Bioprocess Monitoring"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 257-266
U. Bilitewski*, W. Drewes, J. Neermann, J. Schrader, R. Surkow, R. D. Schmid and J. Bradley

Abstract: To achieve effective bioprocess monitoring, sensing systems are required which are suitable for an online determination of substrates, inhibitors, nutrients or products. Such devices may utilise biochemical principles, i.e. the specific interaction of biochemical receptors with their surroundings. They can be constructed either as in situ sensors or as flow-through sensors connected to the process via sampling devices. Hence, characteristic features of an in situ glucose electrode are described, e.g. analytical range, sensitivity and stability. The sensor was based on mediated electron transfer from the enzyme glucose oxidase to the graphite electrode, the mediators being tetrathiafulvalene (TTF) or dimethylferrocene (DMF). Additionally, various flow injection analysis (FIA) systems based on oxidases, which were immobilized either on controlled pore glass or in a membrane, were characterized with respect to analytical ranges and sensitivities and applied to glucose, lactate and glutamate determinations in off-line samples taken from an animal cell cultivation.
Glucose Lactate Glutamate Fermentation broth Sensor Electrode Electrode Method comparison Process monitoring Controlled pore glass

"Which Requirements Do Flow Injection Analyzer/biosensor Systems Have To Meet For Controlling The Bioprocess?"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 241-256
Karl Sch&uuml;gerl*

Abstract: Possible faults with flow-injection analyzers (FIA) and biosensors are identified, and the necessity of instrument adaptation and signal validation is stressed, provided these instruments are used for on-line monitoring of medium components in biotechnical processes.
Sensor

"Mediatorless Electrocatalytic Reduction Of Hydrogen Peroxide At Graphite Electrodes Chemically Modified With Peroxidases"
J. Biotechnol. 1993 Volume 30, Issue 3 Pages 315-337
E. Cs&ouml;regi, G. J&ouml;nsson-Pettersson and L. Gorton*

Abstract: The electrocatalytic reduction of H2O2 was studied for carbonaceous electrodes modified with horse-radish peroxidase (HRP), microperoxidase (MP), and lactoperoxidase (LP). The carbonaceous electrodes were of three different graphites, carbon and glassy carbon. The peroxidase modified electrode was inserted as the working electrode in a flow-through amperometric cell of the wall jet type and connected to a flow injection system. The effect of different pretreatments of the electrode surface prior to adsorption of the enzyme was investigated. Heating the electrodes in a muffle furnace at 700-degrees-C for 1.5 min was found to yield the highest currents. The electrocatalytic current for HRP-modified electrodes starts at about +600 mV vs. Ag/AgCl (pH 7.0) and reaches a maximum value at about -200 mV. For MP- and LP-modified electrodes the currents start at a lower potential (almost-equal-to 300 mV). For the best electrode material for HRP, straight calibration curves were obtained between 1 and 500 muM H2O2 at 0 mV. The mechanism for the electron transfer from the electrode to the adsorbed peroxidase is discussed. Deliberate, modification of the electrode surface with quinoid type electroactive species was found to mediate the reaction. It is proposed that spontaneously occurring electrochemically active surface groups mediate the electron transfer to the adsorbed enzyme. However, a contribution to the observed current from a direct electron transfer cannot be ruled out. [References: 31]
Hydrogen peroxide Electrode Electrode Electrode Electrode Amperometry Redox

"Control Of Microbial Activity By Flow Injection Analysis During High Cell Density Cultivation Of Escherichia Coli"
J. Biotechnol. 1993 Volume 27, Issue 2 Pages 143-157
T. Ding, U. Bilitewski*, R. D. Schmid, D. J. Korz and E. A. Sanders

Abstract: The application of an automated flow injection analysis (FIA) system for online determination of microbial activity, during high cell density cultivations of Escherichia coli is reported. Based on a bioelectrochemical principle, the FIA method used a redox mediator (potassium hexacyanoferrate(III)) to facilitate electron transfer from the microorganisms to an electrochemical detector. Assays were carried out using a new sampling device which provided aseptic operation by use of a valve and chemical sterilisation. No sample dilution or pretreatment was necessary for biomass concentrations up to approximately 40 g l-1. The sample volume was 0.5 mL and the overall analysis time was 5 min. FIA signals were found to correlate well with the oxygen uptake rate (OUR). Changes in metabolic activity due to low substrate levels or high inhibitor concentrations in the cultivation medium became obvious from the FIA signals.
Microbial activity Bacteria Fermentation broth Electrochemical analysis Process monitoring

"Electrocatalytic Oxidation Of NADH At Mediator-modified Electrode Surfaces"
J. Biotechnol. 1993 Volume 27, Issue 2 Pages 129-142
Wolfgang Schuhmanna,*, Johanna Hubera, Heidi Wohlschl&auml;gera, Beate Strehlitzb and Bernd Gr&uuml;ndigc

Abstract: The electrocatalytic oxidation of NADH at suitable electrode surfaces is of fundamental interest for the development of dehydrogenase-based enzyme electrodes. Copolymers of 3-(N-pyrrolo)-2,5,6-trichloro-1,4-benzoquinone or higher pyrrole-substituted chloranil derivatives with pyrrole and graphite/mediator electrodes with insolubilized phenazine and phenoxazine salts are investigated with respect to their electrocatalytic properties for the oxidation of NADH, their long-term operation stability, and the application in flow injection systems with enzyme columns or in enzyme electrodes with immobilized dehydrogenases.
Nicotinamide adenine dinucleotide oxidized Electrode Electrode Immobilized enzyme

"Development Of A Flow Injection Analysis Mediated Biosensor For Sulfite"
J. Biotechnol. 1993 Volume 27, Issue 2 Pages 117-127
C. A. Grooma, J. H. T. Luong*, a and C. Massona

Abstract: Among several mediators tested including N,N,N',N'-tetramethyl-1,4-phenylenediamine, 2,3,5,6-tetramethyl-1,4-phenylenediamine, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and 2,6-dichlorophenolindophenol, an organic conducting salt of tetrathiafulvalene (TTF) and tetracyanoquinodimethane (TCNQ) was selected to form a mediated biosensor for sulfite in view of its superior mediating capability to sulfite oxidase. The conducting TCNQ/TTF organic salt and sulfite oxidase were sequentially deposited onto the sensing area of a vitreous carbon electrode which was then covered by a dialysis membrane (MW cut-off 6000-8000). The mediated TCNQ/TTF working electrode was used in connection with flow injection analysis for the determination of sulfite. The linear range of the system was 0 to 5 mM with a detection limit of 5 µM. The electrode achieved 90% of maximum response in 20 s, but required 10 min to return to baseline. The system was tested for 55 repeated analyzes during 26 h of continuous operation with no loss of activity. Electrodes bearing both the conducting salt and sulfite oxidase stored in 3 M ammonium sulfate and at 4°C retained 100% of their original activity after 2 months. The biosensor system was applicable for the determination of sulfite in wine, beer and dried fruit samples and the data obtained agreed very well with the pararosaniline reference method.
Sulfite Sensor

"Flow Injection Analysis And Inline Biosensors For Bioprocess Control: A Comparison"
J. Biotechnol. 1992 Volume 25, Issue 1-2 Pages 75-80
H. L&uuml;di*, M. B. Garn, S. D. Haemmerli, A. Manz and H. M. Widmer

Abstract: The cited techniques are compared in a table in respect of ease of handling, skill requirements, ease of maintenance, need for sterility, flexibility, reliability, information content of signal, dynamic range, ease of re-calibration, and ease of multi-component analysis. It is concluded that miniaturization will lead to combinations of the two techniques. Miniaturization will unify the different approaches chosen for the application of biosensors in bioprocess control. The most versatile system, which probably is flow injection anal., will be the method of choice for the introduction of biosensors in bioprocess control. A lot of experience will be gained for the future development of miniaturized total chemical anal. systems.
Sensor Process control Method comparison Review Miniaturization

"Enzyme Sensor-FIA-system For Online Monitoring Of Glucose, Lactate And Glutamine In Animal Cell Cultures"
J. Biotechnol. 1991 Volume 21, Issue 1-2 Pages 173-185
R. Renneberg, G. Trott-Kriegeskorte, M. Lietz, V. J&auml;ger, M. Pawlowa, G. Kaiser, U. Wollenberger, F. Schubert, R. Wagner, R. D. Schmid*,* and F. W. Scheller

Abstract: Enzyme sensors for glucose, lactate and glutamine were connected via flow injection analysis (FIA) devices to two different bioprocesses. They were used for online process control of perfused bioreactor systems containing mammalian cell lines producing a monoclonal antibody and recombinant interleukin-2. The biosensor system gives direct access to important process data which can be used as control parameters for long term cell cultivation systems. Enzyme sensors for glucose (I), lactate (II) and glutamine (III) were connected via FIA to two different bioprocesses for the online monitoring of perfused bioreactor systems containing mammalian cell lines producing a monoclonal antibody and recombinant interleukin-2. The sensors comprised of glutaminase or lactate oxidase immobilized in polyurethane at pH 7 for I and II, respectively, or glutaminase and glutanate oxidase co-immobilized in gelatine at pH 5.5 for III. Resulting H2O2 was measured amperometrically at a Pt electrode at 0.6 V vs. SCE. Calibration graphs were rectilinear up to 30 mM, 20 mM and 15 mM of I, II and III, respectively, detection limits were 0.025 mM and coefficient of variation were 5%.
Glucose Glutamine Lactate Fermentation broth Amperometry Electrode Sensor Immobilized enzyme Process monitoring

"Reagentless Enzyme Electrode For Glucose 6-phosphate Using A Mediator-modified Graphite Electrode And Macromolecular NAD+"
J. Biotechnol. 1991 Volume 20, Issue 2 Pages 181-188
Mikael Skoog, Frieder Scheller, Andreas B&uuml;ckmann and Gillis Johansson*

Abstract: A graphite electrode type RW0 (Ringsdorff-Werke GmbH) was modified by adsorption of a phenoxazine derivative (prepared by reaction of Nile blue with terephthaloyl chloride). Glucose-6-phosphate dehydrogenase (0.5 mg) and polyoxyethylene glycol - NAD+ conjugate (1 mg) (cf. Bueckmann, Biocatalysis, 1987, 1, 173) was mixed with phosphate buffer solution and applied to the electrode surface. A cellulose dialysis membrane (Nephrophan, mol. wt. cut-off 12,000 to 15,000, Filmfabrik Wolfen, Germany) was tightened around the electrode to retain the macromolecules. The enzyme electrode was conditioned in buffer solution for 20 min before use. A flow injection manifold, with electrochemical cell, potentiostat and recorder was used with the prepared electrode. The calibration graph for concentration. of glucose 6-phosphate from 10 µM to 10 mM was slightly non-rectilinear. The electrode life was a few days.
Glucose 6-phosphate Electrode Electrode Buffer Dialysis Membrane

"Online Determination Of Intracellular β-galactosidase Activity In Recombinant Escherichia Coli Using Flow Injection Analysis (FIA)"
J. Biotechnol. 1991 Volume 20, Issue 1 Pages 95-104
Heinrich-Andreas Kracke-Helm*, Lutz Brandes, Bernd Hitzmann, Ursula Rinas** and Karl Sch&uuml;gerl*

Abstract: A flow injection analysis (FIA) system was developed for the determination of cytoplasmic β-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for online monitoring of β-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for online monitoring of intracellular protein formation. The method is based on that described by Miller ('Experiments in Molecular Genetics', Cold Spring Harbour Laboratory, New York, USA, 1982). Cells were grown in a Luria broth in an airlift tower loop reactor (loc. cit.). The reactor was equipped with a sterile sampling device to provide a continuous sample flow for analysis of media compounds and by-products. Data acquisition and control of the flow injection system were carried out with the CASFA process management and control system (Frueh et al., Biotech-Forum, 1986, 3, 204) and the FERAS local data management system (Wieneke, Ph.D. Thesis, Univ. Hannover, 1989).
Enzyme, galactosidase Bacteria Fermentation broth Reactor Process monitoring Computer

"Online Determination Of Glucose Concentration Throughout Animal Cell Cultures Based On Chemiluminescent Detection Of Hydrogen Peroxide Coupled With Flow Injection Analysis"
J. Biotechnol. 1991 Volume 18, Issue 1-2 Pages 161-172
Y. L. Huang*, S. Y. Li, B. A. A. Dremel, U. Bilitewski and R. D. Schmid

Abstract: A flow injection analysis (FIA) system for the online determination of glucose in animal cell cultures is described. The system is based on immobilized glucose oxidase (GOD). The hydrogen peroxide generated in the enzyme reaction is determined via a highly sensitive chemiluminescent reaction with luminol. Based on the measurement of the maximum emitted light intensity, the system was able to analyze hydrogen peroxide over the concentration range of 10^-7 to 10^-2 M. For glucose determination, the system has a linear range of 10^-5 to 5 x 10^-2 M glucose, with an RSD of 3% at the 1 mM level (5 measurements). The influence of luminol and buffer concentrations, pH and temperature on the chemiluminescent reaction were investigated. The enzyme reactor used was stable for more than 4 weeks in continuous operation, and it was possible to analyze up to 20 samples per h. The system has been successfully applied to online monitoring of glucose concentration during an animal cell culture, designed for the production of human antithrombin III factor. Results obtained with the FIA system were compared with off-line results, obtained with a Yellow Springs Instrument Company Model 27 (YSI).
Glucose Fermentation broth Chemiluminescence Buffer Immobilized enzyme pH Temperature

"Online Determination Of Glucose In Biotechnological Processes: Comparison Between FIA And An In Situ Enzyme Electrode"
J. Biotechnol. 1991 Volume 18, Issue 1-2 Pages 153-160
Claudio Filippini*, Bernhard Sonnleitner, Armin Fiechter, Joanne Bradley and Rolf Schmid

Abstract: Two different analysis techniques for online monitoring of glucose in biotechnological processes have been tested: an in situ enzyme electrode and a flow injection analysis system (FIA). The measuring ranges, detection limits, response times and the reliabilities of each system have been compared during monitoring of batch and continuous cultures of Saccharomyces cerevisiae. Two different analysis techniques for online monitoring of glucose in biotechnological processes were evaluated, viz., determination with an enzyme electrode with use of a modification of a design previously described by Bradley et al. (cf., 'Computer Applications in Fermentation Technology: Modeling and Control of Biotechnological Processes', Elsevier Science Publishers, London, 1989, p.47) and a FIA method esssentially adapted from Garn et al. (cf. Biotechnol. Bioeng., 1989, 34, 423). The measuring ranges, detection limits, response times and the reliabilities of each system were compared during monitoring of batch and continuous cultures of Saccharomyces cerevisiae. Results are tabulated.
Glucose Fermentation broth Electrode Biotechnology Enzyme Method comparison Process monitoring

"Development Of A Gas Diffusion FIA System For Online Monitoring Of Ethanol"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 127-140
Wolfgang K&uuml;nnecke and Rolf D. Schmid*

Abstract: A flow injection analysis (FIA) system for online monitoring of ethanol in cultivation media was developed, which combines the selectivity of a gas diffusion membrane with the substrate specificity of immobilized alcohol oxidase (AOD). The optimization of membrane material and immobilized enzyme was performed using different FIA modes such as dual detection and dual injection. A simple modification of a polypropylene membrane with silicone enabled a very flexible adjustment of the linear range for alcohol detection between 0.0006 and 60% (v v-1). The ethanol content of cultivation media could be determined continuously with a frequency of 120-180 samples per hour with an excellent correlation to gas chromatographic analysis (r = 0.9996). The relative standard deviation for 10 successive injections was lower than 0.5%. Samples (10 µL) were injected into a carrier stream and passed into a perspex gas diffusion unit (52 mm x 1.4 mm x 0.5 mm) containing a composite polypropylene - silicone membrane and maintained at 30°C. The vapor diffused across the membrane into the acceptor stream (1.5 mL min-1) then into the enzyme reactor (2 cm x 2 mm) that contained alcohol oxidase immobilized (by the glutaraldehyde method) on pore glass. Electrochemical detection of the resulting H2O2 was with a platinum electrode at 700 mV vs. Ag - AgCl. The calibration graph was rectilinear from 0.0006 to 60% of ethanol and coefficient of variation (n = 10) was 0.5%. Sample throughput was 180 h-1.
Ethanol Fermentation broth Electrode Electrode Electrode Gas diffusion Selectivity Silicone membrane Immobilized enzyme Dual detection Linear dynamic range Glass Calibration Optimization

"Simultaneous Determination Of Glucose, Ethanol And Lactate In Alcoholic Beverages And Serum By Amperometric Flow Injection Analysis With Immobilized Enzyme Reactors"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 115-126
Kiyoshi Matsumoto*, Hiroaki Matsubara, Masashi Hamada, Hiroyuki Ukeda and Yutaka Osajima

Abstract: Glucose, ethanol and lactate were determined simultaneously in a flow injection system by using a parallel configuration of immobilized enzyme reactors. Hydrogen peroxide produced was monitored amperometrically at the potential of +0.65 V vs. Ag/AgCl. Linear relations between sensor responses and each species were observed in the ranges of 0.02-10 mM (glucose), 5 x 10^-4-0.1% (v/v) (ethanol) and 0.005-1 mM (lactate) with correlation coefficients larger than 0.999 for each species. The relative standard deviations for 10 successive injections were 1.4, 0.5 and 1.1% for glucose (1 mM), ethanol (5 x 10^-3% (v/v] and lactate (0.05 mM), respectively. Analysis of serum samples was performed with urate-eliminating reactors which were set just before each immobilized enzyme reactor. Interference of ascorbate in a serum sample was completely eliminated by using an ascorbate-eliminating reactor which was set before the sample injection valve. Application of the system to alcoholic beverages and control serum was described and the results were compared with those of free enzymatic, spectrophotometric analysis (F-kit or C-test method). Glucose, ethanol and lactate were determined simultaneously in reactor vessels containing immobilized glucose oxidase, alcohol oxidase and lactate oxidase, respectively in a flow injection analysis (FIA) system. Samples were injected through a 16-way switching valve into the reactor vessels and the resulting H2O2 was measured amperometrically at +0.65 V vs. Ag - AgCl. Urate and ascorbate were removed from serum prior to its analysis using online eliminating-reactors containing uricase - catalase and ascorbate oxidase, respectively. Calibration graphs were rectilinear from 0.02 to 10 mM, 5 x 10^-4 to 0.1%, 5 µM to 1 mM of glucose, ethanol and lactate, respectively with corresponding coefficient of variation (n = 10) of 1.4. 0.5 and 1.1%.
Lactate Ethanol Glucose Beverage Blood Serum Amperometry Immobilized enzyme Simultaneous analysis Interferences Valve Calibration

"Monitoring Of Enzymes During Chromatographic Separations"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 99-114
Wolfgang W. Stamm and Maria-Regina Kula*

Abstract: An online enzyme assay is presented based on flow injection techniques combined with fluorimetric detection. It allows to monitor NAD-dependent oxidoreductases during the purification of microbial crude extracts or partially purified enzymes by fast protein liquid chromatography (FPLC) in a near real-time mode. The arrangement is simple and can be easily integrated in the chromatographic system avoiding dead volumes. A high measuring frequency (up to 180 samples h-1) and a short response time (10-30 s) are achieved. The method has a low limit of detection (approximately 0.01 U mL-1), and a good reproducibility (1-4%), the injected sample volume is only 2 µL. Microbial alanine dehydrogenase, phenylalanine dehydrogenase and formate dehydrogenase were purified using fast protein liquid chromatography (Pharmacia LKB). Eluates passed through a UV detector then flow injection analysis (FIA) valve (Cheminert U Auto CSV-2) before collecting as fractions. At timed intervals 2 µL of sample was injected into the reagent mixture composed of buffer solution, substrate, and NAD coenzyme. The resulting NADH was estimated in the flow cell of an HPLC fluorescence detector at 460 nm (excitation at 340 nm). Sample throughput was 180 h-1, the limit of detection was 0.007 to 0.1 U mL-1 and the coefficient of variation (n = 13) was 1.0 to 1.4%.
Enzymes HPLC Fluorescence Enzyme Detection limit Detector Flowcell

"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. 1990 Volume 14, Issue 1 Pages 89-97
Toshio Yao*, Yoshihiro Matsumoto and Tamotsu Wasa

Abstract: A sensitive and highly selective method for the simultaneous determination of purine bases and their nucleosides is proposed. An amperometric flow injection system with the two immobilized enzyme reactors (guanase immobilized reactor and purine nucleoside phosphorylase/xanthine oxidase co-immobilized reactor) is used as the specific post-column detection system of HPLC, to convert compounds separated by a reversed-phase. HPLC column to electroactive species (hydrogen peroxide and uric acid) which can be detected at a flow-through platinum electrode. The proposed detection system is specific for a group of purine bases and purine nucleosides and does not respond for purine nucleotides and pyrimidine bases. The linear determination ranges are from 10 pmol to 5 nmol for four purine bases (hypoxanthine, xanthine, guanine, and adenine) and four purine nucleosides (inosine, xanthosine, guanosine, and adenosine). The detection limits are 1.2-5.5 pmol. Samples were separated by HPLC on a column (25 cm x 4.6 mm) of ODS-A operated at 40°C with a mobile phase (1 mL min-1) of 0.05 M ammonium phosphate buffer solution (pH 2.5). The eluate was mixed with 0.3 M Na2PO4 buffer solution of pH 7.5 (0.5 mL min-) and the mixture was passed through a reactor containing co-immobilized nucleoside phosphorylase - xanthine oxidase. The resulting H2O2 was monitored amperometrically at 0.5 V vs. Ag - AgCl. Calibration graphs were rectilinear form 10 pmol to 5 nmol for the purine bases and purine nucleosides studied and the detection limits were 1.2 to 5.5 pmol. Coefficients of variation (n = 5) were 2.1 to 6.8% for 0.5 nmol of base or nucleoside.
Hypoxanthine Xanthine Guanine Adenine Inosine Xanthosine Guanosine Adenosine HPLC Amperometry Electrode Electrode Column Immobilized enzyme Selectivity Sensitivity Reverse Linear dynamic range Detection limit Buffer pH Calibration Post-column derivatization

"An Automated Spectrophotometric Flow Injection Procedure For The Determination Of Cellulase Activity In Bioreactor Preparations"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 81-87
P. J. Worsfold*,*, I. R. C. Whiteside, H. F. Pfeiffer and H. Waldhoff

Abstract: Sample (0.6 mL min-1) was injected through a rotary valve into a carrier stream of 0.05 M acetate buffer solution of pH 5 (0.2 mL min-1) until the sample loop (3900 µL) was filled. The loop contained two knitted reactor coils of 200 cm and 300 cm maintained at 50°C. Substrate solution (920 µL ) of 4% Na carboxymethyl cellulose in acetate buffer solution was injected through a second valve, 16 s after which the sample pump was turned off. After incubation the reducing sugar product was reacted with aqueous 5% p-amino-benzoylhydrazide in another reactor coil at 76°C. and the absorbance of the derivative was measured at 410 nm. The calibration graphs were rectilinear up to 0.56 U mL-1 of cellulase with a detection limit of 0.1 U mL-1. Sample throughput was 5 h-1. The method is applicable to other carbohydrases acting on soluble substrates and producing reducing sugars, e.g. amylase, dextranase, xylanase, glucanase, polygalacturonase. A spectrophotometric procedure for the determination of cellulase activity in precipitated bioreactor preparations and culture filtrates is described. It is based on the determination of reducing sugar produced by the action of the enzyme on carboxymethylcellulose. The reducing sugar is derivatized with p-aminobenzoyl-hydrazide and permits a limit of detection of 0.1 U mL-1 cellulase in the presence of background sugar, with a sampling rate of 5 h-1. The method can readily be applied to the determination of any carbohydrase acting on soluble substrates and producing reducing sugars, e.g. amylase, dextranase, xylanase, glucanase and polygalacturonase.
Enzyme, cellulase Spectrophotometry Automation Activity Enzyme Valve Buffer Calibration Detection limit Knotted reactor Heated reaction

"Flow Injection Analysis And Biosensors: Applications For Biotechnology And Environmental Control"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 71-79
H. L&uuml;di*, M. B. Garn, P. Bataillard and H. M. Widmer

Abstract: Our experience in industrial bioprocess monitoring and environmental control let us develop a concept for biosensor research which distinguishes itself from other, more popular, approaches. Biosensors must improve and/or simplify existing state-of-the-art analysis systems. Only the parallel development of biosensors and their complementary metrology leads to industrially sound solutions. The combination of flow injection analysis with immobilized enzymes in the form of enzyme columns is already used today for the solution of online analytical problems in bioprocesses and environmental control. Combinations of flow injection analysis (FIA) and biosensors for monitoring of bioprocesses are discussed. For example, the uptake of glucose by cultures of S. Cervisiae was monitored with an online sensor consisting of glucose oxidase deposited on a microelectrode operated at +400 mV vs. Ag - AgCl to detect H2O2. Another manifold system was described for monitoring downstream processing of NN-dimethylformamidase (details given). Strategies for using immobilized enzymes in FIA - biosensor systems for environmental analytical chemistry are also discussed.
Environmental Sensor Biotechnology Immobilized enzyme Enzyme Column

"Prerequisites For The Online Control Of Microbial Processes By Flow Injection Analysis"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 63-70
I. Ogbomo, U. Prinzing* and H. -L. Schmidt

Abstract: Problems associated with the use of biosensors in process control, e.g. difficulties of sterilization and sensor fouling, are shortly displayed, and possibilities to overcome them are outlined. The advantages of flow injection analysis (FIA) are demonstrated and examples for efficient sampling systems connected with this method are reviewed. Special emphasis is given to problem-orientated sample pretreatments, preventing fast inactivation of immobilized enzymes in the analysis system. Examples of problem-orientated sample pretreatment units are given. A proposal for a computer-controlled self-calibrating FIA system is given. A review is presented, with 32 references, on sampling methods for flow injection analysis (FIA) of substances from bioreactors. Emphasis is given to problem-orientated sample pre-treatments, preventing fast activation of immobilized enzymes in the analysis system. Examples of sample pre-treatment units are given and a proposal for a computer-controlled self-calibrating FIA system is given.
Fermentation broth Sensor Process control Immobilized enzyme Computer Calibration Review

"Fluorometric Determination Of Urea By Flow Injection Analysis"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 53-61
M. S. Abdel-Latif and G. G. Guilbault*

Abstract: Urea was determined using fluorometry with flow injection analysis. O-phthalaldehyde (OPA) reacts with enzymatically generated ammonia and sulfite in alkaline medium to give a highly fluorescent compound that has an excitation wavelength of 372 nm and an emission wavelength of about 430 nm. The method is more selective to ammonia than the one which uses mercaptoethanol in place of the sulfite. Urease was immobilized to a Pall Immunodyne membrane which is commercially available. The immobilization occurs through covalent bonding which results in a highly stable enzyme preparation. The enzymatic membrane was fitted in a 5 cm long, 0.125 inch o.d. Teflon tubing which served as the enzymatic reactor. The system is difficult to use for the analysis of urea in serum because some compounds normally present in serum fluoresce at the same wavelength. This results in higher values for urea. If the reaction system is to be used for the evaluation of urea in serum, a blank should be run so that urea concentration can be calculated by difference. Sample solution was injected into a carrier stream (1 mL min-1) of 0.05 M phosphate buffer solution into a reactor containing urease immobilized to a Pall Immunodyne membrane. The NH3 produced was reacted with o-phthalaldehyde and Na2SO3 in 0.5 M NaOH (1 mL min-1) and the fluoresence of the product was measured at 430 nm (excitation at 372 nm). The calibration graph was rectilinear from 1 to 60 mM urea and coefficient of variation (n = 10) was 2%. Sample throughput was 40 h-.
Urea Blood Serum Fluorescence Immobilized enzyme

"Determination Of Alanine Aminotransferase In Human Serum In An Open-closed Flow Injection Configuration"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 43-52
J. M. Fern&aacute;ndez-Romero, M. D. Luque de Castro and M. Valc&aacute;rcel*

Abstract: Open-closed flow injection systems allow the development of methods based on multidetection by using conventional detectors. The methods proposed here for the determination of alanine aminotransferase (ALT) utilize a photometric detector included in the closed circuit, which allows one to perform fixed-time and reaction-rate measurements whose sensitivity can be increased by using the sum of the analytical signals. The methods, with determination ranges between 1 and 500 U L-1 of ALT, feature sampling frequencies up to 60 h-1. Their application to serum samples provided excellent results, with recoveries between 95 and 106% and averaging at 99.9%. The determination of alanine aminotransferase (I) is based on the transfer of the amino group from L-alanine to 2-oxoglutarate, yielding glutamate and pyruvate within a reactor. A selector valve switched the flow into a closed circuit where, in another reactor, pyruvate was reduced with lactate dehydrogenase and NADH. This indicator reaction was subsequently monitored by the decrease in absorbance at 340 nm in a spectrophotometer flow cell. Repeated passage of the reactive plug through the closed circuit allowed an increase in the sensitivity of the measurements. Recoveries were quantitative for 1 to 500 U L-1 of I. Sample throughput was up to 60 h-1.
Alanine aminotransferase Serum Human Spectrophotometry Multidetection Detector Sensitivity Flowcell Closed loop

"A Flow Injection Analysis System Involving Immobilized NADH Oxidase In Column Form For Clinical Analysis"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 33-41
Takashi Murachi*, Masayuki Totani, Masaki Ikemoto and Masayoshi Tabata

Abstract: A highly sensitive FIA system for chemiluminometric determination of reduced coenzyme, NADH, was developed, using immobilized NADH oxidase from Brevibacterium ammoniagenes. The enzyme catalyzed the oxidation of NADH generating hydrogen peroxide which emitted chemiluminescence when mixed with luminol and potassium ferricyanide. The immobilized enzyme reactor was a mini-column, measuring 1 or 2 mm in inner diameter and 20 mm in length, and the sample volume was only 1 µL per assay, with a feeding speed of one sample per min and a lowest detection limit of 10 pmol NADH. A FIA system was also developed for the determination of magnesium in human serum, using an enzyme column reactor with simultaneously coimmobilized hexokinase, D-glucose-6-phosphate dehydrogenase, and NADH oxidase. The performance of the system was as satisfactory as a routine colorimetric assay, but with much higher sensitivity. A flow injection analysis (FIA) system is described for the determination of NADH with a detection limit of 10 pmol. Samples (1 µL) were passed through a mini-column (2 cm x 2 mm) containing immobilized NADH oxidase and the H2O2 generated was determined by a chemiluminometric method using luminol and potassium ferricyanide. A FIA system was also developed for the determination of Mg in human serum using a reactor with co-immobilized hexokinase, D-glucose-6-phosphate dehydrogenase and NADH oxidase. The sensitivity was higher than that for the colorimetric assay using Xylidyl Blue.
Magnesium Nicotinamide adenine dinucleotide oxidized Serum Human Chemiluminescence Clinical analysis Immobilized enzyme Enzyme Column Catalysis Detection limit Sensitivity Method comparison

"Flow Injection Analysis Based On Enzymes Or Antibodies-applications In The Life Sciences"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 3-31
Rolf D. Schmid* and Wolfgang K&uuml;nnecke

Abstract: A review is presented, with 172 references, on flow injection analysis (FIA). Procedures discussed are: measurement of enzyme activity, use of enzymes in FIA assays and flow injection immuno analysis (FIA).
Enzyme, activity Fermentation broth Biochemical analysis Immunoassay Enzyme Review

"Application Of FIA In The Life Sciences - Dedication "
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 1-2
Prof. Dr. Rolf D. Schmid

Abstract: The international scientific community has honoured Dr. Fiechter on many occasions. If now the accomplishments and contributions of Dr. Fiechter to science, science policy and education are acknowledged again in symposia and commemorative volumes such as this, it is meant to honour the distinguished scientist, the successful science publisher, the inspired teacher of more than 50 Ph.D. students, but in particular a most gentleman-like and friendly person.
Biological