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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Fermentation broth

Classification: Fermentation broth

Citations 207

"New Ways Of Enzymatic Two-substrate Determinations In Flow Injection Systems"
Anal. Chim. Acta 2000 Volume 421, Issue 1 Pages 7-18
Thomas Matthias Becker and Hanns-Ludwig Schmidt

Abstract: This contribution presents two new conceptional strategies for the simultaneous determination of two substrates in flow injection systems. In most biotechnological processes, the substrate consumed as well as the product produced are of special interest for supervision and control. In most cases, substrate and product are interrelated via a redox reaction chain or the law of mass action, respectively. Their enzymatic analysis using dehydrogenases needs often in most cases the use of the cosubstrate in opposite redox states (NAD(+) or NADH). The consequence in a simultaneous two-substrate analysis is a more complex experimental setup. This paper describes two principal new concepts, using only one cosubstrate solution and in situ generation of the cosubstrate in the opposite redox state or a procedure using equilibrium constants, respectively. So opposite configuration requirements, or two substrates which are interrelated as substrate and product of the enzymatic analysis can be determined simultaneously, keeping the analysis system minimised. The determination of ethanol/acetate and the analysis of glucose/fructose serve as models for the proposed methods. A comparison of the results with established standard analysis kits is given.
Ethanol Acetate ion Glucose Fructose Sensor Process control Method comparison Enzyme Simultaneous analysis Multicomponent

"Application Of Convective Interaction Media Disks With Immobilised Glucose Oxidase For On-line Glucose Measurements"
Anal. Chim. Acta 2000 Volume 407, Issue 1-2 Pages 105-110
M. Vodopivec, M. Berovič, J. Jančar, A. Podgornik and A. Štrancar

Abstract: A new polymeric macroporous material, Convective Interaction Media (CIM) was applied as a support for glucose oxidase (GOD) immobilization. CIM epoxy disks with the immobilized enzyme were integrated as an enzyme reactor in a flow injection analysis (FIA) system and applied to on-line monitoring of glucose during cultivation of Saccharomyces cerevisiae and citric acid production by Aspergillus niger. The developed CIM GOD disk-FIA system exhibited good signal reproducibility and satisfactory long-term stability with a linear response in the range 10^-200 mg 1-1. The CIM disk applied as an enzyme reactor proved to be a successful replacement for conventionally used packed-bed columns and as such it was well suited for on-line monitoring of bioprocesses.
Glucose Spectrophotometry Immobilized enzyme Process monitoring

"Spectrophotometric Determination Of Phosphate And Silicate By Sequential Injection Using Molybdenum Blue Chemistry"
Anal. Chim. Acta 2000 Volume 417, Issue 2 Pages 191-200
Cristiane X. Galhardo and Jorge C. Masini

Abstract: This paper presents a spectrophotometric sequential injection (SI) determination of phosphate and silicate in environmental samples and cell cultivation medium using the molybdenum blue reaction. The interference of silicate in the determination of phosphate was eliminated by using a reagent composed of 5 mmol L-1 ammonium molybdate in 0.2 mol L-1 nitric acid, containing 0.25% (w/v) oxalic acid to avoid the formation of molybdosilicic acid. The interference of phosphate in the determination of silicate was avoided adding a 10% (w/v) oxalic acid solution to the reaction zone where the molybdophosphoric and molybdosilicic acids were previously formed, in order to destroy the molybdophosphoric acid. To perform this task in the single Line SI system, obtaining a total sample and reagent zones penetration, it was used as a combination of sandwiching the sample zone between reagent zones and flow reversal through an auxiliary reaction coil. The method has a phosphate sampling frequency of 75 hr-1, with a linear dynamic range between 0.2 and 7 mg L-1 and a detection limit of 0.1 mg L-1 PO43 = P. For silicate, the sampling frequency is 40 h-1 with a linear dynamic range between 5 and 50 mg L-1 and a Limit of detection of 1 mg L-1 SiO32 = Si.
Phosphate Silicate Spectrophotometry Sequential injection Interferences Flow reversal

"Determination Of Fungal α-amylase By Flow Injection Analysis"
Anal. Chim. Acta 1984 Volume 158, Issue 2 Pages 375-377
Preben W. Hansen

Abstract: A manual iodine - starch method has been adapted for use in the analysis of broths from Aspergillus oryzae fermentations. The absorbance of the residual starch - iodine complex was measured at 570 nm following degradation of starch by the enzyme at 37°C and pH 4.7. Rectilinear calibrations were obtained over the range 0.01 to 0.1 amylase units mL-1. Results agreed well for the determination of α-amylase in diluted culture broth by flow injection analysis and by the manual method, but the flow injection method affords a sampling rate of 80 h-1, ~5 times that possible by the manual method.
α-Amylase Biochemical analysis Spectrophotometry Method comparison Indirect Complexation

"Computer Monitoring Of Sugars, Acids And Volatile Compounds In Fermentations"
Anal. Chim. Acta 1984 Volume 163, Issue 1 Pages 275-280
J. C. Motte, X. Monseur, M. Termonia, M. Hofman, G. Alaerts, A. De meyer, P. Dourte and J. Walravens

Abstract: A brief account is given of the use of head-space g.c. - computer-assisted m.s. to monitor the formation of ethanol in fermentation media, HPLC with computerized integration to monitor the disappearance of sugars and the formation of acids during fermentation, and flow injection analysis to monitor feed streams of low sugar content.
Sucrose Computer

"Evaluation Of A Dialysis Probe For Continuous Sampling In Fermentors And In Complex Media"
Anal. Chim. Acta 1984 Volume 163, Issue 1 Pages 135-141
C. F. Mandenius, B. Danielsson and B. Mattiasson

Abstract: The steam-sterilizable probe described yields samples suitable for, e.g., HPLC, flow injection analysis or enzyme calorimetry. The analyte is transferred from the sample to a flowing stream by passage through a dialysis membrane that is protected from fouling by the sample mixture by means of a magnetic stirring-bar rotating close to the membrane surface.
Dialysis Apparatus Membrane

"Measurement Of Biological Parameters During Fermentation Processes"
Anal. Chim. Acta 1984 Volume 163, Issue 1 Pages 111-118
T. Scheper, A. Gebauer, A. Sauerbrei, A. Niehoff and K. Schügerl

Abstract: Three techniques were tested. Firstly, the NADH-dependent fluorescence of micro-organisms was measured in batch and continuous cultures under aerobic and anaerobic conditions to provide information on the metabolic status of the cells. The effects of cell concentration. and of glucose, ethanol and O were studied. Secondly, various substrates were determined calorimetrically with an enzyme - thermistor device. Penicillin was determined selectively in a fermentation broth with use of immobilized penicillin amidase or penicillinase, and penicillin amidase activity was also measured. Thirdly, laser flow cytometry, with use of a commercial double-beam flow system, was used to determine the protein, DNA and RNA contents of single cells. The results obtained by this technique are discussed.
Penicillin Calorimetry Thermistor Flow cytometry Immobilized enzyme

"Determination Of Penicillin-V In Fermentation Samples By Flow Injection Analysis"
Anal. Chim. Acta 1984 Volume 166, Issue 1 Pages 293-295
Ib Schneider

Abstract: The flow injection procedure involves three reagent streams, each at 0.92 mL min-1 and maintained at 45°C. Sample solution (3 µL) is injected into a penicillinase solution stream (in citrate - phosphate buffer solution of pH 6.2) and pumped through a 20-cm mixing coil to allow enzymatic hydrolysis of the penicillin to penicilloic acid. The stream is then mixed with aqueous HgCl2 and aqueous molybdoarsenic acid and passed through a 50-cm mixing coil. The 'molybdenum blue' formed is measured at 670 nm. The calibration graph was rectilinear for 100 to 1000 iu mL-1 of phenoxymethylpenicillin and sample throughput was 90 h-1. Results obtained by this procedure were compared with those obtained with an AutoAnalyzer (r = 0.954).
Phenoxymethylpenicillin Penicillin V Spectrophotometry Heated reaction Method comparison Small sample

"Online Flow Injection Monitoring Of The Enzyme Inductor L-phenylaniline In The Continuous Cultivation Of Rhodococcus Sp. M4"
Anal. Chim. Acta 1988 Volume 213, Issue 1-2 Pages 55-60
U. Nalbach, H. Schiemenz, W. W. Stamm, W. Hummel and M. -R. Kula

Abstract: A flow injection system with a dual-channel manifold is described in which 2 µL injections of sample could be analyzed at 90 to 100 h-1 by the reaction of L-phenylalanine(I) with phthalaldehyde(II) and spectrofluorimetric monitoring at 455 nm (excitation at 336 nm). The carrier solution (1.2 mL min-1) was 1 M borate - NaOH buffer of pH 10.4, which, after sample injection, was blended with reagent solution (0.4 mL min-1) containing 2 mg mL-1 of II in the cited buffer - water - methanol - 3-mercaptopropionic acid (20:24:4:1) before reaction in a PTFE coil (75 cm x 0.5 mm). Rectilinear calibration graphs were obtained for 0.1 to 1.2 mM or for 1 to 30 mM I, according to fluorimeter setting, with coefficient of variation of 1.6% at 0.1 mM (n = 10). Other amino-acids interfere and only small amounts of other amino-group donors can be tolerated.
l-Phenylalanine Fluorescence Interferences Tecator Process monitoring

"Continuous, Reliable Online Analysis Of Fermentation Media By Simple Enzymatic/spectrophotometric Assays"
Anal. Chim. Acta 1988 Volume 213, Issue 1 Pages 61-68
T. D. Gibson and J. R. Woodward

Abstract: On-line analysis of fermentation media is often necessary for control of the fermentation process. A simple reliable semi-automatic analytical procedure with a dialysis probe is described. Ethanol and glucose were determined in a model fermentation system by using this probe with enzymatic spectrophotometric and direct peroxide detectors. The results obtained by using the dialysis probe in a semi-automated system compare favourably with those from samples withdrawn directly from the fermenter.
Spectrophotometry Enzyme

"Inline Flow Injection Analysis For Monitoring Lactic Acid Fermentation's"
Anal. Chim. Acta 1988 Volume 214, Issue 1-2 Pages 137-145
K. Nikolajsen, J. Nielsen and J. Villadsen

Abstract: A flow injection system is described for the monitoring of lactic acid fermentation products, i.e., lactic acid, glucose and proteins, plus cell density, under personal-computer control. Glucose and lactic acid are determined with use of selective enzymatic (oxidase) oxidation and chemiluminescence detection of the products via the luminol - H2O2 reaction. Proteins are determined via reaction with biuret in a 2-m reaction coil and spectrophotometric detection at 565 nm. The absorbance at 565 nm is also used to measure cell density. Detection limits are ~5 mg L-1 for glucose, 50 mg L-1 for lactic acid and 0.2 g L-1 for proteins (rectilinear calibration ranges 5 mg L-1 to 2 g l-1, 0.1 to 2 g L-1 and 0.2 to 8 g l-1, respectively). The apparatus is particularly suitable for studies of fermentation kinetics.
Glucose Lactic acid Proteins Chemiluminescence Spectrophotometry Computer Immobilized enzyme Kinetic

"In-situ Ethanol Probe Based On Sample Dilution In A Double Membrane System"
Anal. Chim. Acta 1988 Volume 215, Issue 1 Pages 71-77
Carl Fredrik Mandenius

Abstract: An in-situ probe suitable for monitoring ethanol in fermenters or other bioreactors is described. It is constructed with an ethanol-permeable double membrane covering a solid-state tin(IV) oxide sensor for gas detection. A stream of nitrogen is passed between the two membranes in order to dilute the ethanol vapor from the fermenter that has passed through the first membrane, before it reaches the second membrane covering the detector. A 100-fold dilution was obtained at a flow rate of 30 mL min-1 of the diluent gas. The delay time was less than 5 min to obtain 80% of maximum response.
Ethanol Biotechnology Conductometry Electrode Dilution Gas diffusion Membrane

"High Reliability And Stability Of Enzyme Cartridges In Flow Injection Analysis"
Anal. Chim. Acta 1989 Volume 225, Issue 2 Pages 253-262
T. Dullau, B. Reinhardt and K. Schügerl

Abstract: A flow injection system consisting of a membrane pump, a sampling valve, two or more enzyme cartridges and an amperometric oxygen electrode detector was used to determine glucose, lactate and maltose in fermentation broths during cell cultivation and product formation. Selectivity was achieved by immobilizing glucose oxidase, lactate oxidase and α-glucosidase on polymeric carrier materials. Calibration graphs for glucose and lactate were rectilinear up to 6 and 8 g l-1, respectively. The cartridges were easily prepared, reliable and stable for 6 months. Concentrations of different substrates could be measured in a single analysis by switching from one cartridge to another. The system could be used for online measurement and control of biotechnological production processes.
Glucose Lactose Maltose Sensor Amperometry Electrode Immobilized enzyme Valve Detector Selectivity Calibration

"Application Of Flow Injection Analysis In The Online Monitoring Of Sugars, Lactic Acid, Protein And Biomass During Lactic Acid Fermentations"
Anal. Chim. Acta 1990 Volume 237, Issue 1 Pages 165-175
Jens Nielsen, Karin Nikolajsen, Stig Benthin and John Villadsen

Abstract: A sample from the fermenter is cooled to 3°C, and a 100 µL portion is injected into analyzer.s for glucose, lactose, galactose, lactic acid and protein. A continuous-flow analyzer. is used to measure biomass concentration. Oxidase-catalyzed reactions are used for the sugars, the H2O2 produced being measured by chemiluminescence (cf. Biotechnol. Bioeng., 1989, 33, 1127); the required enzyme is preferably immobilized on the inner surface of nylon tubing. Lactic acid is determined by enzymatic oxidation to pyruvate in the presence of lactate oxidase, and the Biuret reaction is used for protein. Biomass is determined by spectrophotometry at 565 nm. The various reactions and interference are discussed and details of the reagents are given. Data are presented to demonstrate the value of flow injection analysis for laboratory or industrial-scale online monitoring of dynamic fermentation processes.
Sugars Lactic acid Protein Glucose Galactose Lactose Chemiluminescence Spectrophotometry Enzyme Immobilized enzyme Nylon Interferences Process monitoring

"Immunological Online Detection Of Specific Proteins During Fermentation Processes"
Anal. Chim. Acta 1991 Volume 249, Issue 1 Pages 113-122
Ruth Freitag, Christel Fenge, Thomas Scheper, Karl Schügerl, Andreas Spreinat, Garo Antranikian and Elisabeth Fraune

Abstract: A merging zones stopped-flow flow injection system is described (with diagram) for online immunoturbidimetric determination of proteins in fermentation processes. Sample and reagent solution are injected into separate buffer streams, the streams are merged and the flow is stopped to allow incubation at 37°C. After incubation, turbidity is measured at 340 nm vs. a sample blank. For determination of monoclonal IgG, the reagent solution (0.1 mL min-1) was anti-mouse IgG (1.8 mg mL-1), the buffer solution (1 mL minmin1) was 0.01 M sodium phosphate buffer solution (pH 7.2) containing PEG 6000 (30 g l-1) and NaCl (4.5 g l-1) and the incubation time was 90 s. For assay of α-dextrin endo-1,6-α-glucosidase (I), the reagent was rabbit anti-I antibody solution, the buffer solution was the same as that for IgG, but with 40 g L-1 of PEG 6000, and the incubation time was 120 s. From 10 to 1000 iu L-1 of I and 1 to 1000 mg L-1 of IgG could be determined.
Proteins Turbidimetry Buffer Heated reaction Merging zones Stopped-flow Turbulent flow

"Novel Flow Injection Analysis Method For Bioprocess Monitoring"
Anal. Chim. Acta 1991 Volume 249, Issue 1 Pages 77-85
Suzanne Chung, Xiaowen Wen, Kirsten Vilholm, Michael De Bang, Gary Christian and Jaromir Ruzicka

Abstract: A single-stream system is described for online double-injection stopped-flow flow injection analysis of fermentation broths. The broth is sampled and filtered by using the system described by Garn et al. (Biotechnol. Bioeng., 1989, 34, 423). The filtrate (150 µL) is injected simultaneously with reagent solution (250 µL) into the carrier solution, the stream is passed through a mixing coil and into the dual-wavelength diode array detector. The system was applied in the determination of glucose, NH4+ and total protein in fermentation broth from a fed-batch fermentation.
Ammonium Glucose Protein, total Spectrophotometry Stopped-flow Dual injection

"Monitoring Of Substrates And Products During Fed-batch Penicillin Fermentations On Complex Media"
Anal. Chim. Acta 1991 Volume 249, Issue 1 Pages 123-136
Lars H. Christensen, Jens Nielsen and John Villadsen

Abstract: An automated monitoring system is described (with diagrams) comprising a membrane sampling module for fermentation media, flow injection analysis (FIA) systems for ammonium and glucose determination and exhaust gas analysis system for monitoring O and CO2 and a microcomputer for data acquisition. The O is determined paramagnetically and CO2 determined by IR absorbance. The FIA system for glucose contains a mixing chamber for dilution of the sample and a column of immobilized glucose oxidase. Detection is by chemiluminescence. The RIA system for NH4+ contains a mixing chamber and a gas diffusion device. Cresol red is used as pH indicator and spectrophotometric detection is at 565 nm. The monitoring system was applied to penicillin V fermentation broth.
Penicillin V Glucose Chemiluminescence Spectrophotometry Automation Dilution Gas diffusion Immobilized enzyme Computer Process monitoring

"Online Monitoring And Control Of Fermentation Processes By Flow Injection Analysis"
Anal. Chim. Acta 1991 Volume 249, Issue 1 Pages 101-111
Lawrence W. Forman, Benjamin D. Thomas and Fredric S. Jacobson

Abstract: An online tangential-flow filtration device is described (with photographs) for particle-free sampling of fermentation media. The device contains a Durapore 0.2 µm membrane filter disc sandwiched between two stainless-steel plates. The plates have grooves in them which form process flow and filtrate channels. The device was applied in conjunction with flow injection systems for online determination of acetate (I) and phosphate (II) in fermentation media. The I system involved the use of a CO2 stripper and a gas diffusion module and detection of I at 500 nm with phenol red as indicator. The II system involved the reaction of II with molybdic and ascorbic acids and detection at 800 nm. Calibration graphs were rectilinear up to 80 mM I and from 0 to 1 and 1 to 20 mM II. Online flow injection determination of I gave results slightly higher than those obtained by ion chromatography due to the presence of formate. The online flow injection method for II was preferred over an ion-chromatographic method.
Acetate ion Phosphate Spectrophotometry Filtration Gas diffusion

"Fermentation Monitoring And Control By Online Flow Injection And Liquid Chromatography"
Anal. Chim. Acta 1991 Volume 249, Issue 1 Pages 87-100
K. Schügerl, L. Brandes, T. Dullau, K. Holzhauer-Rieger, S. Hotop, U. Hübner, X. Wu and W. Zhou

Abstract: Flow injection manifolds are described (with diagrams) for the determination of mono- and di-saccharides, lactate, amino-acids and glutamine in fermentation broths from the production of alkaline protease and penicillin. All manifolds contain immobilized enzyme cartidges and detection is by O-electrode. HPLC conditions for online and offline monitoring are also tabulated for determination of cephalosporins, penicillins, carbohydrates, amino-acids and inorganic anions.
Amino Acids Anions, inorganic Carbohydrates Cephalosporin C Disaccharides Glutamine Lactate Monosaccharides Penicillin HPLC Immobilized enzyme

"Inline Tubular Ion-exchanger To Enhance Selectivity In Enzyme-based Flow Injection Potentiometry: Application To Determination Of L-glutamine In Bioreactor Media"
Anal. Chim. Acta 1992 Volume 258, Issue 2 Pages 281-287
Sara A. Rosario and Mark E. Meyerhoff, Marek Trojanowicz

Abstract: The samples in 0.5 M Li acetate - acetic acid buffer of pH 4.9 were passed down a tube (300 cm x 0.625 mm) of Nafion at 41°C and 2 mL min-1, wherein NH4+, Na+ and K+ were exchanged for, e.g., Li+ before reaction with glutaminase immobilized on glass beads. The generated NH4+ was determined with an NH4+-selective electrode. The calibration graph was rectilinear from 0.2 to 4.0 mM with a slope of 50 to 55 mV per decade. A new approach for reducingpositive errors caused by endogenous cationic interferences when using ammonium ion-selective electrodes as detectors in immobilized enzyme-based flow injection analysis systems is described. The method involves the use of an inline tubular cation-exchange unit (e.g., Nafion) placed between the injection valve and the downstream immobilized enzyme reactor/electrode detector portion of the system. Interferent cation species within the sample slug are exchanged for other cations (replacement ions; e.g., Li+) contained within a reservoir solution surrounding the ion-exchange tubing. The membrane electrode exhibits much less response toward the replacement cations; consequently, the detected concentration. of ammonium ions generated downstream within the enzyme reactor is directly proportional to the level of analyte substrate present in the sample. The influence of various experimental parameters on the efficiency of the inline exchanger as well as the general advantages and limitations of this approach are examined The anal. utility of the concept is demonstrated by the rapid and accurate determination of L-glutamine in bioreactor media via the use of immobilized glutaminase enzyme.
l-Glutamate Ion exchange Potentiometry Selectivity Interferences Immobilized enzyme Glass beads Nafion membrane

"Flow Injection Analysis Of Micromolar Concentrations Of Glucose And Lactate In Fermentation Media"
Anal. Chim. Acta 1992 Volume 261, Issue 1-2 Pages 145-153
Stig Benthin*, Jens Nielsen and John Villadsen

Abstract: Samples (8 ml) of fermentation liquor are mixed with 0.8 mL of 6.6 M HClO4 and cooled in ice for 15 min to quench metabolic activity, then adjusted to pH 6.3 with 6.6 M KOH and diluted to 12 mL. The ppt. is centrifuged off and 500 µL of supernatant solution is injected into 10 mM phosphate buffer of pH 6.3. The stream is passed through a reactor containing lactate oxidase or glucose oxidase immobilized on a nylon tube, and is then merged with luminol - Fe(III) reagent for spectrophotometric determination of the H2O2 produced. A reactor in which the nylon tube was knitted in a figure-of-eight shape around two glass spatulas (diameter 4 mm) gave better conversion than one with the tube coiled around a cylinder (diameter 10 mm). With use of the purer (USB 16135; 300 iu mg-1) of two glucose oxidase preparations, the calibration graph (based on peak heights) was rectilinear up to almost 200 mg l-1. The upper rectilinear limit for lactic acid was ~50 mg l-1. Limits of determination were 50 and 150 µg L-1 for lactic acid and glucose, respectively. The technique was used to study bacterial and yeast fermentations, especially the product formation pattern at very low glucose concentration. and the microbial preference for α- or β-glucose. Flow injection analyzers for glucose and lactate were optimized for the measurement of very low concentrations. in complex fermentation media. The analytes were enzymatically oxidized and the H2O2 formed was detected by chemiluminescence. The enzymes were immobilized on a nylon tube and, by measuring the flow-rate dependence of the conversion of H2O2 by catalase, the performance of differently combined enzyme reactors was evaluated. In the resulting glucose and lactate analyzers, the limits of determination in aqueous solutions were 50 µg lactic acid/L and 150 µg glucose/L. When measuring glucose concentrations. in the range 0.5-10 mg/L, the purity of the enzyme used in the analyzer must be high. The analyzers were used to study bacterial and yeast fermentations, in particular the product formation pattern at very low glucose concentrations. and the microbial preference for α- and β-glucose.
Glucose Lactate Chemiluminescence Immobilized enzyme Nylon Knotted reactor Optimization

"Determination Of Penicillin-V In Standard Solution And In Fermentation Broth By Flow Injection Analysis Using Fast-responding Enzyme Glass Electrodes In Different Detection Cells"
Anal. Chim. Acta 1992 Volume 264, Issue 1 Pages 13-22
Helmut Meier* and Canh Tran-Minh

Abstract: A flow-through, a wall-jet and a magnetically stirred flow cell were evaluated in batch tests and in a flow injection system (illustrated) for the determination of phenoxymethylpenicillin (I) with use of a glass membrane pH electrode (Ingold LoT 403-M8-S7 or Schott H 8281) coated with glutaraldehyde-immobilized penicillinase (cf. Ibid., 1991, 249, 405). Potentiometric measurements were made with a Radiometer pHM-64 research pH meter. With use of a carrier stream (4 mL min-1) of 10 or 5 mM phosphate buffer (pH 6.5) containing 0.1 M NaCl and an Ingold (flat-ended) electrode in the stirred flow cell (the system with the best long-term stability), I could be determined in fermentation broths. For I in prepared solution, response with the wall-jet cell and the Schott electrode was rectilinear up to 20 mM. An enzyme immobilization technique to measure penicillin concentration. was developed. Penicillinase is immobilized by crosslinking with a very fine film of glutaraldehyde, which is sprayed on to the sensitive ends of two different pH glass electrodes. An extremely short response time of less than 10 s is guaranteed with electrodes made by this technique when measurements of penicillin are conducted in batch. The electrodes show good stability for 12 days when the sensor remains at room temperature As the response time of the sensor is very short, an immediate determination of penicillin-V is possible by incorporating this enzyme electrode in a continuous-flow system, thereby dispensing with the need for an online enzyme reactor. The penicillin concentration. is calculated as the difference in the potentiometric signal obtained as a peak when the sample flows past the sensor. Sample throughputs up to a max. of 200 H-1 can been achieved. Samples are injected into a carrier stream consisting of a buffer of constant pH. Three different configurations of detection cells were designed and tested with the penicillinase electrode. First the performance of a magnetically stirred flow cell was studied. Subsequently a modified flow-through cell was tested, where the sensor was placed perpendicular to the direction of flow. A wall-jet cell was used to test the electrode where the flow direction coincided with the electrode axis. Finally, the magnetically stirred flow cell was used to detect penicillin-V in fermentation broth samples.
Penicillin V Electrode Electrode Electrode Potentiometry Immobilized enzyme

"Characteristics And Readout Correlation Of Flow Injection Analysis For Penicillin"
Anal. Chim. Acta 1992 Volume 269, Issue 1 Pages 35-40
Xiang-Ming Li*, Man-Xiang Chen, Fu-Chang Ruan, Wing-Yan Ng

Abstract: A flow injection system is described for the determination of penicillin free from CaCl2 and MgSO4 which are commonly found present in fermentation broths. The system consists of a reaction column containing silica gel beads with immobilized penicillinase (60 mg) which hydrolyses the amide bond in the β-lactam ring and the resulting concentration of penicilloic acid is measured by a pH electrode sensor. A carrier solution consists of 1 mM phosphate buffer (pH 7) with 0.1 M NaCl. The enzymatic reactor has a lifetime of 600 h. A flow injection analysis (FIA) system based on the enzymatic reactor with immobilized penicillinase on silica gel is developed for the measurement of penicillin. The basic principle of the system is to convert penicillin in the injected sample into penicilloic acid through the enzymatic reactor and to detect the penicilloic acid with a pH sensor in a flow cell following the reactor. A model based on theoretical analysis is developed to correlate the output signal of the pH sensor with the sample concentrations, which shows superior linearity compared to that correlating the peak height of the pH sensor output with the sample concentrations The life time of the enzymatic reactor is about 600 h. The reactor can be easily replaced after the enzyme activity has declined to a level where the penicillin in the passing carrier stream cannot reach hydrolysis equilibrium. The signal output of the pH sensor is processed online by an interfaced microcomputer using an algorithm of the model.
Penicillin Electrode Immobilized enzyme Silica gel Computer

"Flow Injection Analysis For The Measurement Of Penicillin V In Fermentation Media"
Anal. Chim. Acta 1993 Volume 274, Issue 1 Pages 117-123
Morten Carlsen, Lars H. Christensen and Jens Nielsen*

Abstract: Iodimetric, potentiometric and molybdenum blue flow injection analysis systems for the determination of penicillin (I) in fermentation media were evaluated. All three methods were based on the enzymatic hydrolysis of I to penicilloic acid (II) using β-lactamase, followed by the detection of II. β-Lactamase was immobilized on aminopropyl controlled-pore glass beads (120 to 200 mesh) and the beads were packed in a column and used as a packed-bed reactor. Conversion of I to II was 100%, therefore total I and II present in the sample was determined. The iodometric method was the best for measurements in fermentation media. By replacing the enzyme reactor with an enzyme-free dummy reactor, II alone was determined, and hence the concentration. of I was calculated. The limit of detection was 2.5 mg L-1 and calibration graphs were rectilinear from 2.5 to 150 mg l-1. There was no interference from other components in the fermentation medium. The iodometric analyzer. was applied to the monitoring of a fed-batch I fermentation; results correlated well with those obtained by LC.
Penicillin V Potentiometry Spectrophotometry Method comparison Immobilized enzyme Controlled pore glass Interferences

"Monitoring And Control Of Recombinant Protein Production"
Anal. Chim. Acta 1993 Volume 279, Issue 1 Pages 3-16
Karl Schügerl*, Lutz Brandes, Xiaoan Wu, Jens Bode, Jong Il Ree, Jens Brandt and Bernd Hitzmann

Abstract: Fermentation liquors for endonuclease production were tested for cell concentration, total cell count, cell viability and the number of colony-forming units by established methods. The plasmid copy number and stability and the product concentration. were determined by gel electrophoresis. The concentration. of the substrate, glucose and the metabolites pyruvate, succinate, lactate, glycerol, acetate, methanol and ethanol were determined online or offline by HPLC on an IONpak column (25 cm x 8 mm) at 60°C, with a mobile phase of 0.9 mM H2SO4 and a refractive index detector. Sugar components were also analyzed on a Nucleosil 100-5 NH2 column (25 cm x 4.6 mm) at 28°C, with a mobile phase (1 ml/min) of acetonitrile-phosphate buffer (31:9), containing 4 mM tetrabutylammonium hydrogen sulfate. The analysis was also performed by FIA in combination with enzyme cartridges. Examples of the monitoring of a 60 l batch process are illustrated.
Proteins Glucose Pyruvate Succinate Lactate Glycerol Acetate ion Methanol Ethanol Immobilized enzyme

"Automated Monitoring Of Biotechnological Processes Using Online Ultrafiltration And Column Liquid Chromatography"
Anal. Chim. Acta 1993 Volume 279, Issue 1 Pages 39-50
N. C. van de Merbel*, H. Lingeman and U. A. Th Brinkman, A. Kolhorn, L. C. de Rijke

Abstract: The efficient control of fermentation processes requires reliable monitoring systems. This paper describes a fully automated monitoring system based on the online combination of ultrafiltration and column liquid chromatography. An evaluation of the performance of a hollow-fiber ultrafiltration module is provided and a method for the determination of sugars, alcohols and organic acids during a gluconic acid and a beer fermentation is presented. The system can be used for at least 250 h with complex fermentation broths under both aerobic and anaerobic conditions without any detrimental effect on either the fermentation or the chromatographic part. Depending on the application 3-5 analyzes can be performed every hour, using only 3.0 µL of sample per analysis. Using refractometric or direct UV detection the linear dynamic range is from 0.2 to 150 g L-1 for most of the compounds, while with a simple post-column derivatization procedure sugars can be determined in the low mg L-1 range. [References: 21]
Sugars Alcohols Acids, organic LC Filtration Linear dynamic range

"Online Monitoring Of Penicillin V During Penicillin Fermentations: A Comparison Of Two Different Methods Based On Flow Injection Analysis"
Anal. Chim. Acta 1993 Volume 279, Issue 1 Pages 51-58
Morten Carlsen, Claus Johansen, Rong Wei Min and Jens Nielsen*, Helmut Meier and Francois Lantreibecq

Abstract: Two methods were compared for the cited analysis. In method (i), cell-free sample from the reactor (0.78 ml/min) was injected into a carrier stream of 10 mM phosphate buffer of pH 6.5 (1.1 ml/min) and mixed in the stirred 2 mL detection cell. The cell contained a rapid response glass electrode, on which β-lactamase had been immobilized, which measured the pH change caused by the hydrolysis of phenoxymethylpenicillin (I) to penicilloic acid. The β-lactamase reaction was also used in method (ii), but the penicilloic acid was detected by its bleaching of starch-iodine at pH 6. Using a second injector valve, the enzyme reactor was bypassed and the pre-existing penicilloic acid could also be measured. The first method was simpler, requiring no pre-dilution of the sample and could be used for routine determinations of 35 g/l of I. The second method required three dilution ranges to analyze 0.1-30 g/l of I, smaller samples (10 µL) and more frequent calibration. Both methods were used for continuous monitoring of 200 h production runs.
Penicillin V Electrode Method comparison Process monitoring

"Flow Injection Determination Of Tylosin In Fermentation Broth"
Anal. Chim. Acta 1993 Volume 281, Issue 2 Pages 243-247
Frank L. Neely*

Abstract: Fermentation broth was extracted with isopropyl acetate. A portion (5 µL) of the solution was injected into the reagent stream (3 ml/min) of cis-aconitic anhydride in acetic anhydride/isopropyl acetate (1:1). The reaction took place at 45°C in a stainless-steel tube reactor and the absorbance of the red salt formed was measured at 525 nm. The calibration graph was linear for 0.11-2.2 mM tylosin (I). The linear range was extended to 11 mM I, by changing the wavelength to 560 nm. Recovery was 96.6-106.1% for fermentation broth spiked with a concentration. aqueous solution of I over the domain 50-500% of initial I concentration. The method may be applied in the determination of a variety of antibiotics possessing a tertiary amine moiety.
Tylosin Antibiotics Spectrophotometry Sample preparation Extraction Reactor

"Industrial Online Monitoring Of Penicillin V, Glucose And Ethanol Using A Split-flow Modified Thermal Biosensor"
Anal. Chim. Acta 1993 Volume 281, Issue 3 Pages 521-526
M. Rank, J. Gram, B. Danielsson

Abstract: Bioreactor fermentation broth was sampled online with a hydrophilized polypropylene filtration probe (Advanced Biotechnology, Puchheim, Germany) and pumped into an enzyme thermistor with a split-flow biosensor comprising a column of β-lactamase, penicillin V acylase, glucose oxidase/catalase or alcohol oxidase immobilized onto controlled-pore glass using γ-aminopropyltriethoxysilane and glutaraldehyde (details given), and a parallel reference column without enzyme. Columns were eluted (0.9 ml/min) with 0.1 M phosphate buffer of pH 7 containing 4 mM NaN3 or 5 g/l of benzoic acid (for the glucose oxidase/catalase column). The difference between the enthalpy changes on the two columns was monitored, amplified and processed by computer. The calibration graph for the penicillin V acylase column was linear from 0.5-150 mM penicillin V (20-500 µL samples); the determinations of penicillin V, penicilloic acid and p-hydroxypenicillin V agreed well with those obtained by LC. The β-lactamase column values were 10% higher than those obtained by LC. Results for ethanol (alcohol oxidase column) agreed with those obtained by GC.
Penicillin V Glucose Ethanol Enthalpimetry Sensor Thermistor Biotechnology Controlled pore glass Immobilized enzyme

"Flow Injection Analysis - Wall-jet Electrode System For Monitoring Glucose And Lactate In Fermentation Broths"
Anal. Chim. Acta 1993 Volume 283, Issue 2 Pages 763-771
Yu Liang Huang, Soo Beng Khoo, Miranda G. S. Yap

Abstract: The flow injection manifold consisted of two peristaltic pumps, an injection valve with a 20 µL sample loop, an enzyme reactor column (10 x 1 mm i.d.) containing either glucose oxidase or lactate oxidase immobilized onto controlled-pore glass beads, and a large volume wall-jet detector cell. The detector contained a Pt disc working electrode modified with Fe(II), a Ag/AgCl reference electrode and a vitreous C counter electrode. Fermentation broth was centrifuged and the supernatant was diluted prior to analysis. The resulting solution was injected into the 0.1 M potassium phosphate buffer of pH 7 carrier stream (0.5 ml/min). The H2O2 produced by the enzymatic reaction of both glucose (I) and lactose (II) was detected at 0.65 V vs. Ag/AgCl. The calibration graphs were linear up to 0.5 and 0.04 g/l of I and II, respectively. The RSD (n = 54) was 3% over a 10 h measurement period. The procedure was used to monitor I and II concentrations in various fermentation broths and the results agreed well with those obtained using a commercial I-II analyzer..
Glucose Lactate Electrode Method comparison Controlled pore glass Immobilized enzyme

"Ion Chromatography For Monitoring Biotechnological Processes. 2. Applications For Industrial Cultivation Media"
Anal. Chim. Acta 1994 Volume 286, Issue 2 Pages 179-187
Ute Scheller, Detlef Siedenberg, Uwe Hübner, Michael Siebold, Gerlinde Kretzmer and Karl Schügerl*

Abstract: The production of alkaline protease by Bacillus licheniformis, and the cultivation of Cephalosporium acremonium was monitored by the ion-chromatography of chloride, phosphate and sulfate anions in the broth. Sample proteins were filtered, and filtrates analyzed on a Hamilton PRP-X 100 ion-exchange column with a Hamilton PRX- x 100 pre-column and RI detection (no other details given). Lactic acid, acetic acid, citric acid and glucose, present during the production of lactic acid by Lactobacillus salivarius subsp. salivarius were also monitored by ion-exclusion chromatography (no details given). Glucose and lactic acid results compared favourably with those obtained by an enzyme FIA method using immobilized glucose oxidase and lactate oxidase amperometric detectors (no details given). The problems involved in sampling, sample pre-treatment, the optimization of the chromatographic analysis and the enrichment of the analytes after dialysis are discussed.
Lactic acid Acetic acid Citric acid Glucose HPIC Biotechnology Dialysis Optimization

"Online Monitoring Of Intracellular Enzyme Activities With Flow Injection Analysis"
Anal. Chim. Acta 1994 Volume 287, Issue 3 Pages 235-246
K. Steube and U. Spohn*

Abstract: A FIA method was developed for the determination of enzyme activities in yeast cells during fermentation. The FIA system (diagram given) comprised an online sampling device, a cell disintegration chamber with a glass micro-ball mixer mill, an enzyme membrane reactor and a fluorescence detector; ultrasonic and ultraturrax disintegration chambers were less effective. Samples (20 µL) were injected simultaneously with 100 µL of 30 mM NAD+ solution and mixed with appropriate buffer and substrate solution to enable up to six different enzymes to be assayed. A thin-layer dialysis membrane device was used to separate the enzymatically-produced NADH from the cell debris and high mol. wt. substances, allowing it to diffuse into an acceptor solution which flowed to the detector for determination at 340 nm. The calibration graphs for three dehydrogenases were linear from 0.001-2 iu/ml with RSD of 1%; the recoveries from disintegrated cell suspensions were 95-102%. The intracellular formate dehydrogenase activities determined during a yeast fermentation process using the system agreed well with those determined off-line.
Enzyme, activity Spectrophotometry Dialysis

"Application Of Enzyme Field-effect Transistor Sensor Arrays As Detectors In A Flow Injection System For Simultaneous Monitoring Of Medium Components. 1. Preparation And Calibration"
Anal. Chim. Acta 1994 Volume 296, Issue 3 Pages 263-269
T. Kullick, M. Beyer, J. Henning, T. Lerch, R. Quack, A. Zeitz, B. Hitzmann, T. Scheper and K. Schügerl*

Abstract: Enzymes were co-immobilized on the pH-sensitive gates of an 8-channel array of field-effect transistors (FETs). Glucose was determined with a glucose dehydrogenase (GDH) FET, maltose with a co-immobilized maltase (MAL)/GDH FET, sucrose with a co-immobilized invertase (INV)/GDH FET, lactose with a β-galactosidase/galactose dehydrogenase (β-GAL/GALDH)-fusion protein FET and ethanol with a co-immobilized alcohol dehydrogenase/aldehyde dehydrogenase (ADH/ALDH) FET. These EnFETs were integrated into FIA systems, and were calibrated and characterized with respect to pH, buffer capacity, stirrer speed, NAD concentration and cross sensitivity. Because the signals of the EnFETs were sensitive to pH and buffer capacity, the pH was monitored with a pH-FET in the array, and the buffer capacity and substrate concentration were calculated from the shape of the signal of the FIA system. The EnFETs were stored at 4°C for many months without activity loss. They have satisfactory activity for performing a large number of analyzes. However, glucose reversibly inhibited the sucrose signal and added to the maltose signal. Hence the sucrose monitor can only be used when glucose is practically absent, and the maltose sensor requires simultaneous determination of glucose.
Lactose Ethanol Sucrose Glucose Maltose Field effect transistor Sensor Simultaneous analysis pH Buffer Optimization

"Application Of Enzyme Field-effect Transistor Sensor Arrays As Detectors In A Flow Injection System For Simultaneous Monitoring Of Medium Components. 2. Monitoring Of Cultivation Processes"
Anal. Chim. Acta 1995 Volume 300, Issue 1-3 Pages 25-31
T. Kullicka, U. Bocka, J. Schuberta, T. Scheperb and K. Schügerla,*

Abstract: A biosensor array consisting of a single pH-FET and seven enzyme FET channels integrated into a single flow injection system was used for the online monitoring of pH, glucose, maltose, sucrose, lactose, ethanol and urea during the cultivation of Escherichia coli and Saccharomyces cerevisiae in synthetic media. The components were monitored using glucose dehydrogenase, maltase-glucose dehydrogenase, invertase-glucose dehydrogenase, β-galactosidase-galactose dehydrogenase, alcohol dehydrogenase-aldehyde dehydrogenase and urease (co)immobilized on the pH sensitive gates of the FET. The array was integrated in a commercial FIA system. The results agreed with those obtained by offline concentration measurements.
Ethanol Glucose Lactose Maltose Sucrose Urea Field effect transistor Field effect transistor Sensor Method comparison

"Online Monitoring Of D-lactic Acid During A Fermentation Process Using Immobilized D-lactate Dehydrogenase In A Sequential Injection Analysis System"
Anal. Chim. Acta 1995 Volume 300, Issue 1-3 Pages 277-285
Hun-Chi Shu, Håkan Håkanson and Bo Mattiasson*

Abstract: A PC-automated sequential injection analysis (SIA) system was used to monitor the production of D-lactic acid during the batch fermentation of Lactobacillus delbrueckii ATCC 9649. Sampling was carried out from the flux of a cellulose acetate membrane filtration unit through which the broth was continuously circulated. Cell-free permeate was diluted with 0.1 M phosphate buffer of pH 7 and passed through rotary and three-way valves to enzyme and blank reactors. The enzyme reactor contained D-lactate dehydrogenase co-immobilized with L-alanine aminotransferase on porous glass. The measuring conditions were: 0.1 M glycylglycine buffer including 70 mM glutamic acid as carrier buffer of pH 10, 4 mM NAD+ cofactor in glycylglycine buffer solution and an incubation time of 90 s. Detection was by spectrophotometric absorbance measurement (340 nm for NADH). The total analysis time was 182 s including the incubation time while the flow was stopped. The calibration graph was linear in the range 2-25 mM D-lactic acid and the detection limit was 1 mM. The results from SIA were comparable with those obtained by the offline Boehringer enzymatic kit method.
d-Lactic acid Spectrophotometry Sequential injection Stopped-flow Automation Computer Method comparison Immobilized enzyme Glass

"Aldose Dehydrogenase-modified Carbon Paste Electrodes As Amperometric Aldose Sensors"
Anal. Chim. Acta 1995 Volume 302, Issue 2-3 Pages 233-240
Maria Smolander*, György Marko-Varga and Lo Gorton

Abstract: A solution of aldose 1-dehydrogenase in 10 mM sodium acetate of pH 5 containing 0.1% of Triton X-100 was applied to the polished tip (diameter 1.7 mm) of a carbon paste electrode; results were less satisfactory when the enzyme was first mixed with the carbon paste. Incorporation of 2% (relative to graphite) of dimethylferrocene in the paste led to high catalytic response. The prepared electrode was coated with Eastman AQ-29D from aqueous 0.5% solution; this improved stability and minimized non-specific responses. Amperometric measurements with the electrode were made at +200 mV vs. Ag/AgCl (Pt counter electrode) in a flow injection system with 50 mM sodium phosphate buffer of pH 6.5 as carrier at 0.7 ml/min. Solutions of glucose and xylose standards and aldose samples were prepared in the same buffer. Xylose from a yeast fermenter was separated by LC on an Aminex HPX-87P column (30 cm x 7.8 mm i.d.) in Pb(II) form. The enzyme was active mainly towards the β-anomer of glucose. Response was linear up to 100 mM xylose and 10 mM glucose.
Aldoses Glucose Xylose Sensor Amperometry Electrode Electrode Apparatus Triton X Surfactant

"Development Of Enzyme-cartridge Flow Injection Analysis For Industrial Process Monitoring. 2. Application For Monitoring Of Micro-organism Cultivations"
Anal. Chim. Acta 1995 Volume 302, Issue 2-3 Pages 289-296
H. Jürgens, L. Brandes, R. Joppien, M. Sieboldb J. Schubert, X. Wu, G. Kretzmer and K. Schügerl*

Abstract: Cartridges (1 ml) were filled with 200 mg of VA-Epoxy-Biosynthe E3 (Riedel-de Haen) on which glucose oxidase, alcohol oxidase, pyranose oxidase, L-amino acid oxidase or L-lactate monooxidase had been immobilized from phosphate buffer medium. Up to four enzyme cartridges were used in series or parallel for FIA of glucose, ethanol, L-amino-acids and lactate on Eppendorf instruments. Conditions are given for determining glucose and ethanol in Saccharomyces cerevisiae cultures, amino-acids during the production of fusion protein by recombinant Escherichia coli, and L-lactate during the production of lactic acid by Lactobacillus salivarius. The cartridges showed good long-term stability and response times of a few minutes.
Ethanol Glucose Amino acids, L Lactate Immobilized enzyme Process monitoring

"Sequential Enzymic Monitoring Of Glucose, Ethanol And Glutamate In Bioreactor Fermentation Broth Containing A High Salt Concentration By A Multi-channel Flow Injection Analysis Method"
Anal. Chim. Acta 1995 Volume 308, Issue 1-3 Pages 145-151
Richie L. C. Chen and Kiyoshi Matsumoto*

Abstract: An automated glucose analyzer. was modified to form a multichannel FIA system for the sequential determination of glucose, ethanol and glutamate. The modifications consisted of inserting a computer controlled column switching valve downstream from the injector. Each channel was equipped with a mini-reactor column containing immobilized glucose oxidase, alcohol oxidase or glutamate oxidase. The product from the enzyme reactions, H2O2, was detected electrochemically. The carrier solution (3 ml/min) was 0.1 M phosphate buffer of pH 6.8 and the injection volume was 2 µL for each determination. The apparatus was evaluated by monitoring the fermentation of a fish sauce with Torulopsis versatiles immobilized beads. Due to the high salt concentration in the fermentation broth it was necessary to insert a Dowex 1-X8 anion-exchange column between the injector and the column switching valve to eliminate the interference of NaCl. Linear calibration graphs were obtained for 0.5-5% (w/v) glucose and ethanol and 0.1-2% (w/v) glutamate. The results were confirmed by analysis with assay kits.
Ethanol Glucose Glutamate Electrochemical analysis Column Computer Dowex Immobilized enzyme Interferences Method comparison Multichannel

"Immunobased Elution Assay For Process Control"
Anal. Chim. Acta 1995 Volume 309, Issue 1-3 Pages 301-305
Kerstin Beyer, Martin Reinecke, Wolfgang Noe and Thomas Scheper*

Abstract: Portions of 200 µg of two different monoclonal and one polyclonal antibody of recombinant tissue-type activator plasminogen (I) and the antibody of recombinant antithrombin III (II) were separately dissolved in 800 µL 100 mM phosphate buffer of pH 7.4 and the antibodies immobilized onto 270 mg cyanogen bromide-activated Sepharose-4B, and packed into a small cartridge (no dimensions given). Portions of 138 µL I and II were injected into a carrier stream (1 ml/min) of buffer, bound on-cartridge and eluted with 100 mM K3PO4 buffer of pH 12.3 with fluorimetric detection at 340 nm (excitation at 273 nm). Calibration graphs were linear for 2-500 µg/ml of I. The binding strength of I was maximum with the immobilized polyclonal antibodies. The immobilized protein cartridges maintained satisfactory performance for at least two weeks.
Antithrombin III Fluorescence Immobilized antibody Process control

"Development Of An Extremely Flexible Automatic Analyser With Integrated Biosensors For Online Control Of Fermentation Processes"
Anal. Chim. Acta 1995 Volume 315, Issue 1-2 Pages 113-122
Wolfgang Schuhmann*, Heidi Wohlschläger, Johanna Huber, Hanns-Ludwig Schmidt and Herbert Stadler

Abstract: A fully automatic sequential injection system, named OLGA, for the online control of fermentation processes is described. It consisted of a sampling and injection section with a high-precision, stepping motor-driven micropump, magnetic snap valves and two manifolds manufactured in polyacryl and a detection section equipped with a low-volume, four-electrode flow-through detection cell. The detection cell was fitted with two Pt working electrodes (0.5 mm diameter) to monitor H2O2 at 650 mV vs. Ag/AgCl and Ag pseudo-reference electrodes. The flow system was controlled by a computer program written in Microsoft Visual Basic 3.1 Professional and operated within the Windows 3.1 environment. The software package allow automatic calibration and recalibration with up to five standard solutions, calculation of the analyte concentration using the most recent calibration data and the injection of samples from up to 4 sampling sites. The system was evaluated for the determination of glucose and the simultaneous determination of glucose and lactate. Enzyme membranes containing immobilized glucose oxidase or lactate oxidase were mounted over the working electrodes. A carrier stream of 0.1 M sodium phosphate buffer of pH 7.4 was used with a injection volume of 3-5 µL. The OLGA system was also applied to monitor glucose concentration in a baker`s yeast fermentation for up to 35 h.
Glucose Lactate Electrode Sensor Sequential injection Computer Simultaneous analysis Immobilized enzyme

"Online Monitoring With Feedback Control Of Bioreactors Using A High Ethanol Tolerance Yeast By Membrane Introduction Mass Spectrometry"
Anal. Chim. Acta 1995 Volume 316, Issue 2 Pages 269-276
N. Srinivasan, N. Kasthurikrishnan, R. G. Cooks*, M. S. Krishnan and G. T. Tsao

Abstract: We have investigated the fermentations of glucose with a genetically engineered yeast, strain 1400, which has a tolerance to high ethanol concentration. The concentration of the major product in the liquid phase, ethanol, was monitored online as a function of time using membrane introduction mass spectrometry (MIMS). Minor products produced during the fermentation and identified by MIMS include lactic acid and glycerol. The concentration of the substrate, glucose, was monitored off-line using a glucose analyzer. A flow injection analysis sampling system was used to inject, in sequence, the sample, the standard, and the flush solution (deionized water). Microfiltered broth plugs were introduced into the mass spectrometer, through a direct insertion membrane probe which uses a hydrophobic silicone membrane as the interface between the aqueous sample and the mass spectrometer. Isobutane chemical ionization was used to produce the protonated molecular ion of ethanol (m/z 47) in experiments with a quadrupole mass spectrometer and water chemical ionization was used in comparative experiments with an ion trap. All operations such as sampling, scanning, data acquisition, control of the FIA, calibration, and feedback control were carried out automatically, with the help of a control program written in C. The feedback control system described in this paper was employed to automate substrate addition. This allowed the inhibition of ethanol formation due to high substrate (glucose) concentration to be avoided. Batch fermentations with initial concentrations of glucose up to 150 g/l were carried out in this mode and an ethanol concentration of about 50 g/l was achieved after monitoring for 8 h. Fed-batch fermentations were also carried out, to reduce the inhibitory effect of the substrate. In a typical fed-batch fermentation, a final ethanol concentration of about 120 g/l was achieved, and the bioreactor was monitored for 50 h. (27 references)
Ethanol Mass spectrometry Silicone membrane Process control

"Characterization And Application Of An Online Flow Injection Analysis/wall-jet Electrode System For Glucose Monitoring During Fermentation"
Anal. Chim. Acta 1995 Volume 317, Issue 1-3 Pages 223-232
Yu Liang Huanga, Titus J. Foellmera, Koon Chye Anga, Soo Beng Khoob,* and Miranda G. S. Yapa

Abstract: An online system for glucose monitoring during fermentation was developed. The system employed flow injection analysis coupled with a wall-jet electrode. Glucose was converted enzymatically in a tubular reactor packed with controlled pore glass beads containing immobilized glucose oxidase. The hydrogen peroxide produced was detected amperometrically. A filtration/sampling module operated by a peristaltic pump enabled sample withdrawal from the fermenter. The system was interfaced to a programmable language controller which was in turn linked to a microcomputer. The dual injection valve of the flow system was operated in a novel configuration to achieve sample dilution, by dispersion, and standardization simultaneously. The developed system was characterized for dilution factor, stability and linearity of response. After conditioning of the reactor, sample peak heights were found to have a precision of 1.07% (RSD, 25 determinations) when the glucose concentration was 1.32 g l-1. Under the conditions studied, linear response to glucose concentration from 0.062 to 4.0 g L-1 was obtained. Sample throughput was about 7 per hour. Interferences from the fermentation media were negligible after filtration through the sampling module and dilution. The online system was applied to glucose monitoring during E. coli fermentation. Good results were obtained as shown by the high cell density (OD6-00 nm = 168 in 9 h fermentation), constant specific growth rate (µ= 0.66) and lower acetate accumulation (less than 2 g L-1 when OD-600 nm was over 50). (18 References)
Glucose Electrode Controlled pore glass Immobilized enzyme Interferences Apparatus Detector Computer Process monitoring

"Online And Off-line Monitoring Of The Production Of Alkaline Serine-protease By Bacillus-licheniformis"
Anal. Chim. Acta 1995 Volume 317, Issue 1-3 Pages 247-258
A. B. Van Puttena, F. Spitzenbergerb, G. Kretzmerb, B. Hitzmannb and K. Schügerlb,*

Abstract: Subtilisin Carlsberg alkaline serine protease was produced by Bacillus licheniformis on complex medium in a stirred tank reactor and the turbidity and culture fluorescence were monitored in situ, absorbance online and off-line, (dry) sediment, (dry) cell mass and DNA concentrations off-line. These data were correlated with each other. The cultivation medium composition: starch, maltose, glucose, urea, ammonia, phosphate concentrations and protease activity were monitored by a six-channel flow injection analysis system online and off-line. Protein concentration, α-amylase activity and intracellularly phosphate concentration were evaluated off-line. The online and off-line data agreed well. Differences in the online and off-line monitored protease activities are caused by the membrane fouling of the sampling system. (18 references)
Ammonia Glucose Maltose Phosphate Starch Urea Turbidimetry Process monitoring Method comparison Multichannel

"Online Monitoring Of Glucose And Penicillin By Sequential Injection Analysis"
Anal. Chim. Acta 1996 Volume 320, Issue 2-3 Pages 199-205
Rong Wei Min, Jens Nielsen* and John Villadsen

Abstract: In the sequential injection flow analysis (SFIA) system, sample was injected via a six-position valve into a bioreactor. The enzyme reactors were prepared by immobilizing glucose oxidase and penicillinase on a piece of nylon tubing. Glucose was converted to gluconolactone and the H2O2 formed was detected by a chemiluminescence reaction involving luminol. The penicillin was converted to penicilloic acid, which was detected by its quenching effect on the chemiluminescence signal obtained when luminol and iodine reacted. It was also detected spectrophotometrically by the decrease in absorbance of an iodine-starch complex. Online and offline measurements showed good agreement. The SFIA system was simpler than conventional FIA and running costs were lower. The system was used to analyze Penicillium cultivations.
Glucose Penicillin Chemiluminescence Spectrophotometry Sequential injection Immobilized enzyme Method comparison Nylon Quenching

"Sequential Injection Determination Of D-glucose By Chemiluminescence Using An Open Tubular Immobilized Enzyme Reactor"
Anal. Chim. Acta 1996 Volume 326, Issue 1-3 Pages 1-12
Xuezhu Liu and Elo Harald Hansen

Abstract: The method was based on the luminol chemiluminescence detection of H2O2 formed by glucose oxidase catalyzed oxidation of D-glucose. The manifold was equipped with an enzyme reactor prepared by immobilizing glucose oxidase onto a nylon tube (1 m x 1 mm i.d.) and a photodiode detector. The analysis was performed by aspirating at 75 µL/s 225 µL of sample solution into the holding coil (160 cm x 0.8 mm i.d, 800 µL) via the enzyme reactor and adding 75 µL 1.5 mM luminol in 0.1 M K2CO3 of pH 11.2 and 37.5 µL 30 mM potassium hexacyanoferrate(III). The contents of the holding coil was aspirated through the detector. The calibration graph was linear for 30-600 µM-D-glucose, the detection limit was 15 µM and the RSD (n = 6) for 50 µM-glucose was 2.7%. The recoveries of 42.3-332 µM-D-glucose from spiked samples of fermentation broth were >97%. The sampling frequency was 54 samples/h.
d-Glucose Chemiluminescence Immobilized enzyme Reactor Sequential injection Photodiode Nylon

"Characterization Of A Membrane Interface Designed For Analytical Scale Sample Introduction Into A Mass-spectrometer"
Anal. Chim. Acta 1996 Volume 327, Issue 1 Pages 1-16
Ashok R. Dongré and Mark J. Hayward*

Abstract: A membrane interface has been constructed for analytical scale sample introduction into a mass spectrometer. The interface was designed to be easy to use while achieving the following two goals: (i) to improve the mass transport efficiency of the analyte from the sample solution to the mass spectrometer ion source and (ii) to improve performance while reducing the sample sizes utilized for routine direct analyzes with membrane introduction mass spectrometry (MI/MS). In order to systematically characterize this interface, a variety of key mass transport parameters including membrane thickness, temperature, analyte flow rate, and pneumatic assist carrier gas flow rate have been examined. Interface characterization studies have focused primarily on two analytes, benzene and ethanol. These two analytes were chosen to compare the relative importance of each of the key mass transport parameters for the two most common applications of MI/MS, environmental analyzes and fermentation monitoring. The results of these studies suggest that mass transport of the analyte from solution phase to the membrane (including the use of the thin layer approach), membrane dimensions and membrane temperature are among the more important factors affecting analyte response and limits of quantification. Other significant results suggest that, when given a warm unrestricted path from the membrane to the ion source, the permeation (flux) of water from the aqueous matrix may be a major factor affecting the mass transport of analyte from the membrane to the ion source of the mass spectrometer. The comparison of the effects of the key mass transport parameters for environmental analyzes and fermentation monitoring shows that each of these two applications depend on a markedly different set of parameters for achieving optimal performance.
Organic compounds Mass spectrometry Interface Membrane Optimization Apparatus

"Pervaporation: An Interface Between Fermentors And Monitoring"
Anal. Chim. Acta 1996 Volume 330, Issue 2-3 Pages 265-272
I. Papaefstathioua, U. Bilitewskib and M. D. Luque de Castroa,*

Abstract: An enzymatic spectrophotometric method is described for determining acetaldehyde in fermentation broths. A 1 mL sample of the fermentation broth is injected into a carrier stream of water and propelled at 0.49 ml/ml through a pervaporation module (at 60°C) fitted with a PTFE membrane (1.5 mm thickness, 5 µm pore size). The vaporized acetaldehyde diffuses through the membrane and is collected in an acceptor stream (0.49 ml/min) containing 1 mM NAD+, 0.2 M KCl and 6 mM 2-mercaptoethanol in 0.1 M Na2HPO4/NaH2PO4 buffer at pH 8. The acceptor stream passes through an enzyme reactor (at 30°C) containing aldehyde dehydrogenase immobilized on controlled-pore glass beads and a spectrophotometric detector equipped with a 1 cm detector cell; the NADH formed by the enzymatic oxidation of acetaldehyde is detected at 340 nm. The calibration graph was linear for 1-30 µg/ml of acetaldehyde, the detection liwas 0.9 µg/ml and the RSD (n = 8) at 20 µg/ml was 2.7%. The sampling frequency was 8 per h. The recoveries of 6.5 µg/ml of acetaldehyde added to fermentation broths containing 2-15 µg/ml of acetaldehyde were >92.7%.
Acetaldehyde Spectrophotometry Controlled pore glass Pervaporation Teflon membrane Heated reaction

"Membrane Introduction Mass-spectrometry"
Anal. Chim. Acta 1997 Volume 350, Issue 3 Pages 257-271
N. Srinivasan, R. C. Johnson, N. Kasthurikrishnan, P. Wong and R. G. Cooks*

Abstract: An overview of membrane introduction mass spectrometry (MIMS) is presented and comparisons are made with other direct sample introduction techniques. Special attention is given to the unique advantages and the limitations of newer variants on the MIMS technique, including affinity MIMS, reverse-phase and trap MIMS. The salient features of the interfaces used in MIMS are summarized and the various membrane materials commonly used are delineated. The applicability of MIMS is illustrated via discussion of (i) bioreactor monitoring (represented by yeast fermentation), (ii) environmental monitoring (illustrated by analysis of contaminated ground water samples) and (iii) online chemical reaction monitoring (exemplified by the photolysis of aryl esters). The applicability of MIMS to the analysis of environmental samples, including complex mixtures in water, air and soil, is noted. 77 References
Mass spectrometry Membrane Process monitoring Photochemistry Review

"Electrocatalytic Oxidation Of H2O2 At An Oxycobalt Film-modified Glassy-carbon Electrode For Fermentation Monitoring"
Anal. Chim. Acta 1997 Volume 351, Issue 1-3 Pages 133-142
Soo Beng Khooa,*, M. G. S. Yapc, b, Yu Liang Huangb and Sixuan Guoa

Abstract: Glassy carbon electrodes coated with thin oxymetallic films of Co(II), Fe(II), Ni(II), Pb(II), Ce(III), Cr(III), Tl(I) and Mn(II) were investigated for the presence of electrocatalytic effect towards H2O2 oxidation. Of these, Co(II), Ni(II), Pb(II), Ce(III) and Mn(II) were found to exhibit such an effect in alkaline medium, with Co(II) showing the strongest effect. Further studies with the Co(II) modified glassy carbon electrode as amperometric detector, in the wall-jet configuration, for flow injection analysis (FIA) demonstrated that only a low operating potential of +0.175 V (vs. Ag/AgCl, saturated KCl) was needed for good sensitivity. For flow injection analysis, linearity of H2O2 calibration was found from 5.00 x 10^-8 to 0.10 M and the detection limit was 5.0 x 10^-9 M (S/N=3). The electrode showed good stability (at least two weeks operation) and can be usefully applied to glucose determination in the fermentation broths without interferences. 30 References
Glucose Hydrogen peroxide Electrode Electrode Interferences Sensitivity Process monitoring

"A Flow Injection Mediated Biosensor For Online Monitoring Of Lactate In Mammalian Cell Culture"
Anal. Chim. Acta 1997 Volume 351, Issue 1-3 Pages 159-167
Keith B. Male, P. Octavian Gartu, A. Amine Kamen and John H. T. Luong*

Abstract: A flow-injection (FI) biosensor system has been developed for on-line monitoring of lactate formation during mammalian cell cultivation. The culture sample was peristaltically withdrawn from the bioreactor and, after cell separation by a steam sterilizable ceramic microfilter, the cell-free filtrate was fed to the FI system. Lactate oxidase was covalently immobilized onto a preactivated nylon membrane and attached to the sensing area of a platinum working electrode. The enzyme reaction was coupled with a water-soluble mediator 1,1'-dimethylferricinium (DMFe+)-cyclodextrin inclusion complex to recycle the reduced lactate oxidase to its original active state. 1.1'-Dimethylferrocene (DMFe) was then reoxidized to DMFe+ at the surface of the platinum electrode poised at +0.15 V vs. silver/silver chloride. The FI biosensor was linear up to 3 mM lactate with a detection limit of 0.05 mM, and possessed ±1.5% reproducibility over 154 repeated analyzes during a 77 h continuous operation. Due to a significant accumulation of lactate during fed-batch cultivation of 293S mammalian cells, a dialysis membrane with 1000 molecular weight cut-off was placed over the lactate oxidase membrane to extend the linear detection range up to 40 mM. Excellent reproducibility (±1.3%) was observed for 96 repeated hourly analyzes with culture medium containing 15 mM lactate. When applied to (5-10)-day fed-batch cultivation of 293S mammalian cells, the results obtained from the biosensor system compared well with HPLC and spectrophotometric assay data.
Lactate Sensor Process monitoring

"Simultaneous Determination Of α-amylase And Amyloglucosidase Activities Using Flow Injection Analysis With Fourier Transform Infrared Spectroscopic Detection And Partial Least-squares Data Treatment"
Anal. Chim. Acta 1998 Volume 366, Issue 1-3 Pages 35-43
R. Schindler, B. Lendl* and R. Kellner

Abstract: A flow injection analysis (FIA) system with fourier transform IR (FTIR) spectroscopic detection for the simultaneous determination of amyloglucosidase (exo-glucan 1,4-α-glucohydrolase, EC 3.2.1.3) and α-amylase (endo-glucan 1,4-α-glucanohydrolase, EC 3.2.1.1) activities in aqueous solution is proposed. Starch hydrolysis catalyzed by both enzymes was monitored in the mid-IR range by recording IR-spectra of the assay solutions before and after hydrolysis. The intensities of the resulting difference spectra were directly related to the enzyme activities. Furthermore, the different reaction pathways of the two enzymes under investigation produced distinct spectral changes resulting in difference spectra characteristic for each enzyme. Using partial least squares (PLS) regression it was possible to simultaneously determine both enzyme activities covering a range of 38-150 U/l (630-2500 nkat/l) for amyloglucosidase and a range of 500-3750 U/l (8300-62000 nkat/l) for α-amylase. Absorption due to the sample matrix was successfully eliminated by calculation of the difference spectra. The developed method was also successfully applied to the anal. of spiked fermentation broth samples yielding mean deviations of 2.6% and 10.8% for amyloglucosidase and α-amylase, respectively.
α-Amylase Amyloglucosidase Spectrophotometry Simultaneous analysis Catalysis Interferences Partial least squares

"Simultaneous Monitoring Of Glucose And L-lactic Acid During A Fermentation Process In An Aqueous Two-phase System By Online FIA With Microdialysis Sampling And Dual Biosensor Detection"
Anal. Chim. Acta 1998 Volume 366, Issue 1-3 Pages 127-135
Rong Wei Mina, Vijay Rajendrana, Niklas Larssona, Lo Gortona,*, Jordi Planasb and Bärbel Hahn-Hägerdalb

Abstract: The prodn. of L-lactic acid by Lactococcus lactis (ATCC 19435) during fermentation in an aqueous two-phase system (ATPS) was monitored online using microdialysis sampling coupled to a dual flow-through electrochemical cell housing glucose and lactate biosensors, which enabled the simultaneous and selective monitoring of both glucose and L-lactic acid. The amperometric biosensors were based on the co-immobilization of glucose oxidase (GOD) for the glucose sensor and L-lactate oxidase (LOD) for the lactate sensor, respectively, with horseradish peroxidase (HRP) in a carbon paste matrix. The sensors characterized regarding the sensitivities, pH optima and operational stabilities were found to be satisfactory within the required range and time of measurements. The online setup was found to be a flexible system for the monitoring of both glucose and L-lactic acid simultaneously, allowing a sampling frequency of 15 h-1 and with a delay between sampling and detection of less than 3 min. Comparison of the online measurements with a standard off-line anal. using HPLC agreed well suggesting the suitability of the system for application in very complex matrixes.
Glucose l-Lactic acid Sensor Amperometry Electrode Electrode Optimization Simultaneous analysis Method comparison Dialysis

"Potentiometric Biosensor For Control Of Biotechnological Production Of Penicillin G"
Anal. Chim. Acta 1998 Volume 368, Issue 3 Pages 205-210
Ewa Leszczyskaa, Stanis

Abstract: A pH-membrane electrode with tridodecylamine as a hydrogen ion-selective ionophore was used for the construction of a biosensor for the benzylpenicillin determination The electrode was enzymatically modified by covalent binding of penicillinase directly to the membrane surface. The high sensitivity and short response time of the biosensor allows its application in flow injection analysis (FIA). Up to 30 samples per h can be analyzed in the flow system. The FIA system with the biosensor was successfully used for the determination of penicillin G in fermentation broth. An optimized anal. procedure results in minimization of interferences from pH and buffer capacity of the real samples. No interferences from inorganic, organic and bioorganic components present in the fermentation medium were observed An important advantage of the presented anal. system is the long-term stability due to the long lifetime of the biosensor. Consequently fermentation bioprocesses can be controlled using the same biosensor for about two months. Due to the high sensitivity and selectivity, the presented method for the penicillin determination in real biotechnology samples gives analytical results comparable to those obtained using methods recommended by pharmacopoeias.
Benzylpenicillin Penicillin G Electrode Electrode Electrode Potentiometry Sensor Optimization Process monitoring Method comparison Interferences

"Development Of A Spectrophotometric Determination Of Siderophores Using Flow Injection Analysis"
Talanta 1995 Volume 42, Issue 6 Pages 837-843
R. Trsková, P. Rychlovský, I. Nmcová and A. Jegorov

Abstract: A method is presented for the spectrophotometric determination of siderophores in culture fluids of fungi using FIA (schematic diagram given) based on the reaction of siderophores with the ternary complex Eriochrome Cyanine R-Fe(III)-cetyltrimethylammonium bromide. The reagent (0.4 ml/min) was prepared by mixing 12.5 mL 2 mM Eriochrome Cyanine R (Mordant Blue 3) with 2.5 mL 1 mM FeCl3 and allowing reaction for 3 min, followed by addition of 12.5 mL 50 mM cetyltrimethylammonium bromide mixed with 75 mL of 1.67 M piperazine buffer of pH 7. The volume was made up to 250 mL with water. The carrier stream (1 ml/min) was water and detection was at 650 nm. The sample injection volume was 100 µL. The calibration graph for tolypocine was linear from 2.6 µM (determination limit) to 150 µM. RSD (n = 5) were 1.2-3.5%. The method was applied to the determination of the complexation ability of siderophores produced by some entomopathogenic fungi. Samples could be analyzed at the rate of 30/h.
Tolypocine 2,3-Dihydroxybenzoic acid Spectrophotometry Chelation

"Inverse Spectrophotometric Detection In Flow Injection Analysis. Determination Of Nitrite Using Cerium(IV) As The Chromophore"
Analyst 1988 Volume 113, Issue 10 Pages 1597-1599
Thomas P. Lynch

Abstract: In the flow injection system a sample was injected into water (2 mL min-1), which was mixed with cerium(IV) ammonium sulfate (20 g l-1; 0.6 mL min-1) in 0.5 M H2SO4 and passed through a mixing coil (2.3 m). The absorbance of the solution was monitored at 407 nm. The calibration graph of decrease in absorbance vs. concentration. of NO2- was not rectilinear from 200 to 1000 mg l-1, but the fourth-degree polynomial regression curve showed r >0.9999. The range of application could be extended from 10 to 9000 mg L-1 by using reagent solution of varying concentration. The method was applied in the determination of NO2- in culture media.
Nitrite Spectrophotometry Calibration

"Simultaneous Determination Of Ammonia Nitrogen And L-glutamine In Bioreactor Media Using Flow Injection"
Analyst 1993 Volume 118, Issue 11 Pages 1361-1365
Bernhard O. Palsson, Bing Q. Shen, Mark E. Meyerhoff and Marek Trojanowicz

Abstract: Cell culture medium from reactors with growing retroviral producer cells was injected into a carrier stream of water (1 ml/min). The stream was split in half. One stream was treated with 10 µM-NH4Cl (1 ml/min) followed by 0.1 M NaOH containing 0.01 M EDTA (1.5 ml/min) at 20°C and NH3-N was determined using an NH3 gas-sensing electrode fitted with a wall-jet cap. The other stream was mixed with 0.5 M acetate buffer of pH 4.9 (1.5 ml/min) before passing through a Nafion ion-exchange coil (2 m) at 40°C, followed by a glass bead reactor containing immobilized glutaminase. The ammonium ions liberated by hydrolysis were detected potentiometrically by an ISE using absolute peak potentials. The maximum sampling rates were 40 and 35 samples/h for NH3-N and L-glutamine, respectively. In simultaneous determination of the analytes, NH3-N levels of up to 10 mmol/l could be tolerated without affecting L-glutamine measurement. The system was calibrated with Iscove's Modified Dulbecco's Medium. Results agreed with those obtained using commercial, manual enzymatic-spectrophotometric kits. A novel split stream flow injection (FI) system suitable for the simultaneous determination of L-glutamine and ammonia nitrogen (ammonia- N) in cell culture media is described. Potentiometric detection of ammonia-N in one portion of the manifold is achieved using a commercial ammonia gas-sensing electrode fitted with a wall-jet cap. L-Glutamine is quantified in the other part of the split sample by potentiometric detection of ammonium ions (by an ammonium-selective polymer membrane electrode), liberated from the hydrolysis of glutamine after the sample flows through a glass bead reactor containing immobilized glutaminase. Endogenous ammonia-N and potassium ions that would normally interfere with the glutamine measurement are removed upstream using a unique tubular cation-exchange unit. Using 50 µL sample volumes and mixed solutions of ammonium chloride and L-glutamine in Iscove's Modified Dulbecco's Medium to calibrate the FI measuring system, values for ammonia-N and L-glutamine determined for 22 media samples obtained from a bioreactor growing retroviral producer cells correlate well with those measured with commercial, manual enzymatic-spectrophotometric assay kits.
Ammonia, nitrogen l-Glutamine Potentiometry Electrode Sensor Nafion membrane Immobilized enzyme Glass beads Heated reaction Method comparison Interferences

"Rapid Determination Of Total Biomass From A Yeast Fermentation Using Sequential Injection"
Analyst 1994 Volume 119, Issue 8 Pages 1807-1812
P. J. Baxter, G. D. Christian and J. Ruzicka

Abstract: Samples were obtained directly from a feed-batch aerobic fermentation of Saccharomyces cerevisiae and were analyzed without pretreatment. A 38 µL sample was pulled into a holding coil via a multiport valve by means of a peristaltic pump. The sample was delivered to a stirred flow cell (volume 1.7 ml) in a water carrier stream (2.2 ml/min) containing 2 ppm of Brijj-35. The signal was recorded by measuring either total transmitted light or light scattered at 90°C. Diagrams of the sequential-injection manifold and flow cell used are given. The sample range for the assay was from 0.2 to at 800 g/l of biomass. RSD (n not given) were 3%. The total assay time was 4 min.
Biomass Nephelometry Turbidimetry Sequential injection Process monitoring Surfactant Valve

"Fermentation Monitoring Using A Glucose Biosensor Based On An Electrocatalytically Bulk-modified Epoxy-graphite Biocomposite Integrated In A Flow System"
Analyst 1995 Volume 120, Issue 8 Pages 2255-2258
F. Céspedes, F. Valero, E. Martínez-Fàbregas, J. Bartrolí and S. Alegret

Abstract: A graphite-epoxy-Au-Pd-glucose oxidase biocomposite, prepared as described previously (Cespedes et al., Anal. Chim. Acta., 1993, 273, 409 and 1993, 284, 21), was used to construct a glucose biosensor, which was integrated in a flow system and used to monitor glucose consumption during a fermentation process. The sample (5 ml) was filtered and the filtrate was mixed successively with two diluting streams of 0.1 M phosphate buffer of pH 7/0.1 M KCl (solution A). A portion (350 µL) of the diluted sample was then injected into a carrier stream (0.6 ml/min) of solution A and glucose was detected amperometrically at the biosensor by applying a potential of +900 mV vs. Ag/AgCl and measuring the current produced. A diagram of the manifold used is given. The calibration graph was linear from 0.01-10 mM of glucose. The RSD (n = 3) was 5%. The throughput was 20 samples/h. The results obtained agreed with those obtained with a commercial glucose analyzer. and by HPLC.
Glucose Amperometry Electrode Electrode Sensor Method comparison

"Automated Determination Of Microbial Peroxidase Activity In Fermentation Samples Using Hydrogen Peroxide As The Substrate And 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) As The Electron Donor In A Flow Injection System"
Analyst 1995 Volume 120, Issue 8 Pages 2101-2105
Kaj André Holm

Abstract: The sample was diluted with 0.1 M phosphate buffer of pH 7 containing 0.1 M ammonium sulfate and 1.5 g/l of Triton X-405. A portion (40 µL) of the solution was injected into a carrier stream (1.2 mL/min) of 0.2 M phosphate buffer of pH 7 containing 0.1 M (NH4)2SO4 and 1.5 g/l of Triton X-405. Reagent streams (both 0.8 mL/min) of 7.1 mM H2O2 and 1.6 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) were mixed in a 30 cm coil before merging with the carrier stream. The mixture was passed through a 30 cm coil before being incubated at 40°C in a 200 cm coil. The solution was then passed through a 60 cm coil and the absorbance was measured at 418 nm. A diagram of the manifold used is given. Detection and determination limits were 0.005 and 0.05 iu/ml of peroxidase, respectively. Inter- and intra-day RSD (n = 63 and 14, respectively) were 1.1 and 4.8%, respectively. The throughput was ~e;100 samples/h. An automated flow injection method has been developed for the determination of microbial peroxidase activity. The substrate used was hydrogen peroxide and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) was used as the electron donor. In the presence of hydrogen peroxide, peroxidase catalyses the dehydrogenation of ABTS, resulting in the formation of a resonance-stabilized radical cation of ABTS. The green-blue color formed, recorded at 418 nm, is taken as a measure of the peroxidase activity. The general technical conditions and the general enzymatic kinetics have been optimized. Conditions for activation and stabilization of the enzyme were found, e.g., ammonium sulfate acts as a peroxidase activator. The resulting method has a good precision, sensitivity and speed.
Enzyme, peroxidase Spectrophotometry Heated reaction Immobilized enzyme Triton X Surfactant

"Online Spectrophotometric Determination Of Phosphate In Bioprocesses By Sequential Injection"
Analyst 1995 Volume 120, Issue 5 Pages 1583-1587
Jorge C. Masini, Pamela J. Baxter, Kandace R. Detwiler and Gary D. Christian

Abstract: Automation of a reduced molybdophosphoric acid method by sequential injection (SI) for the on-line monitoring of phosphate concentrations is presented. Simplicity, robustness and computer compatibility of the SI system has made possible the monitoring of a wide range of phosphate concentrations (0.1 to 30 mmol l-1) without the necessity of mechanical reconfiguration of the manifold. Dilution, when required, was performed in a mixing chamber connected to the selector valve. Decisions about the necessity of dilution were made through software. Real-time data may be provided to process controllers. Results obtained on-line agreed with those obtained off-line. A sampling frequency of 25 per hour is possible. The method was demonstrated in a study of the influence of phosphate concentration on the production of biomass in aerobic fermentations of Saccharomyces cerevisae. The addition of phosphate to the fermentation before its depletion from the medium enhanced the production of biomass by 25 to 35%.
Phosphate Spectrophotometry Automation Sequential injection

"Development Of Ultraviolet-polymerizable Enzyme Pastes: Bioprocess Applications Of Screen-printed L-lactate Sensors"
Analyst 1996 Volume 121, Issue 6 Pages 877-881
Ingrid Rohm, Meike Genrich, Wendy Collier and Ursula Bilitewski

Abstract: Amperometric enzyme electrodes for L-lactate were prepared by screen-printing a mixture of UV-polymerizable, water-soluble paste, lactate oxidase, DEAE-dextran (1%) and lactitol (5%) on to the surface of one of two Pt working electrodes of a screen-printed thick-film transducer. The second Pt electrode was untreated. Polymerization was performed by UV irradiation of the enzyme paste. The enzyme electrodes were incorporated in a flow injection system and used for online monitoring of L-lactate in cultivations of Geotrichum candidum. The sample was filtered in situ and then injected into a carrier stream of Clark and Lubs buffer of pH 8 (buffer A). The mixture was passed through a dialysis module and the L-lactate that diffused into the acceptor stream (buffer A) was detected amperometrically at the Pt enzyme paste electrode at 0.6 V vs. Ag/Pd. The calibration graph was linear for 0.55-50 mM L-lactate. The results obtained agreed with those obtained by a photometric assay.
Lactate Sensor Dialysis

"Determination Of Amyloglucosidase Activity Using Flow Injection Analysis With Fourier Transform Infrared Spectrometric Detection"
Analyst 1997 Volume 122, Issue 6 Pages 531-534
R. Schindler, B. Lendl and R. Kellner

Abstract: Samples (250 µL each) and a 55 g/l starch solution in 0.1 M acetate buffer of pH 4.3 were simultaneously injected into two aqueous carrier streams (both at a flow rate of 0.88 ml/min) and merged in a reaction coil (250 cm x 0.5 mm i.d.) maintained at 54°C. When the reaction plug had filled the reaction coil, the flow was stopped for 5 min after which the FTIR spectrum was recorded from 950-1300 cm-1. The amyloglucosidase (I) activity was calculated from the difference in the absorbances at 1078 and 1020 cm-1 compared with a reference spectrum of unreacted starch. The calibration graph was linear from 50-2000 U/l I. The method was applied to fermentation broths: recoveries were 98-102% of added I.
Amyloglucosidase Spectrophotometry Stopped-flow Heated reaction

"Microbial Detection By A Glucose Biosensor Coupled To A Microdialysis Fibre"
Analyst 1997 Volume 122, Issue 10 Pages 1125-1128
F. Palmisano, A. De Santis, G. Tantillo, T. Volpicella and P. G. Zambonin

Abstract: The use of a glucose biosensor coupled to microdialysis sampling in a flow injection analysis system is described to follow the growth of Escherichia coli in a glucose-containing liquid culture medium. The experimental set-up permitted a throughput rate of 25 samples h-1. Growth curves were modelled by a modified Gompertz equation, which permitted the determination of lag time and maximum specific growth rate. The time required to produce an appreciable variation in the biosensor response (minimum detection time, MDT) was determined. A plot of MDT versus microbial concentration was found to be linear in the range 10(6)-10(10) colony forming units (cfu) mL-1. A microbial concentration of 10(6) cfu mL-1 can be detected after about 5 h.
Glucose Sensor Dialysis Process monitoring

"Flow Injection Determination Of Penicillins Using Immobilized Penicillinase In A Single Bead String Reactor"
Anal. Chem. 1985 Volume 57, Issue 6 Pages 1005-1009
R. Gnanasekaran and Horacio A. Mottola

Abstract: Procedures for etching the glass beads with NH4HF2, silylation with (aminophenyl)trimethoxysilane, activation with glutaraldehyde and immobilization of penicillinase on the glass beads are described in detail. With use of optimum reaction conditions established for the flow injection determination of penicillins (at pH 6.4, 25°C and a flow rate of 4 mL min-1), benzylpenicillin was determined in tablets and injection solution and phenoxymethylpenicillin was determined in fermentation broths. Rectilinear calibration graphs were obtained for concentration. up to 5 mM of each drug.
Benzylpenicillin Phenoxymethylpenicillin Penicillins Potentiometry Glass beads Immobilized enzyme Optimization Single bead string reactor

"Online Monitoring Of Bioreactions Of Bacillus Polymyxa And Klebsiella Oxytoca By Membrane Introduction Tandem Mass Spectrometry With Flow Injection Analysis Sampling"
Anal. Chem. 1990 Volume 62, Issue 17 Pages 1798-1804
Mark J. Hayward, Tapio Kotiaho, Anita K. Lister, R. Graham Cooks, Glen D. Austin, Ramani Narayan, and George T. Tsao

Abstract: The monitoring of the fermentation of the cited organisms to produce butan-2,3-diol (I) was studied. A flow injection sampling system is described, which was used to deliver rapidly fermentation broth or an external standard to the mass spectrometer. Analyte introduction was via a direct insertion membrane probe in which the aqueous solution flowed past a membrane within the ion source. The concentration. of the liquid-phase products (acetic acid, 3-hydroxybutan-2-one, I and ethanol) were monitored as a function of time after permeation through the membrane and chemical ionization. MS - MS confirmed that these measurements were made without interference. The results agreed with those using off-line GC. Dissolved O in the fermentation broth and O and CO2 in the off-gases were also measured. The results agreed with those from common measurement techniques. The use of MS - MS also allowed the detection of trace metabolites.
Carbon dioxide Oxygen Acetic acid 2,3-hydroxybutanone Mass spectrometry Membrane Interferences Process monitoring

"Improved Fiber Optic Chemical Sensor For Penicillin"
Anal. Chem. 1995 Volume 67, Issue 24 Pages 4471-4476
Brian G. Healey and David R. Walt

Abstract: An optical penicillin biosensor is described, based on the enzyme penicillinase. The sensor is fabricated by selective photodeposition of analyte-sensitive polymer matrices on optical imaging fibers. The penicillin-sensitive matrices are fabricated by immobilizing the enzyme as micrometer-sized particles in a polymer hydrogel with a covalently bound pH indicator. An array of penicillin-sensitive and pH-sensitive matrices are fabricated on the same fiber. This array allows for the simultaneous, independent measurement of pH and penicillin. Independent measurement of the two analytes allows penicillin to be quantitated in the presence of a concurrent pH change. An analysis was conducted of enzyme kinetic parameters in order to model the penicillin response of the sensor at all pH values. This analysis accounts for the varying activity of the immobilized penicillinase at different pH values. The sensor detects penicillin in the range 0.25-10.0 mM in the pH range 6.2-7.5. The sensor was used to quantify penicillin concentration produced during a Penicillium chrysogenum fermentation. (27 References)
Penicillin pH Sensor Sensor Immobilized enzyme Optical fiber Simultaneous analysis

"Online Determination Of Enzymes In Bioprocessing With The Emphasis In Flow Injection Analysis And Continuous Sampling"
Fresenius J. Anal. Chem. 1988 Volume 329, Issue 6 Pages 718-725
K. H. Kroner

Abstract: A review is presented, with 19 references, of the current status of online assay of enzymes with use of flow injection techniques. Continuous sampling is possible with a module based on membrane filtration, the process fluid being pumped through the module and back to the reactor. Examples are given of determinations of enzymes, e.g., β-galactosidase, in fermentation and in downstream processing.
Enzymes Spectrophotometry Optimization Process control Tecator Review

"Determination Of Total Ammonium-nitrogen And Free Ammonia In A Fermentation Medium By Sequential Injection Analysis"
Fresenius J. Anal. Chem. 1993 Volume 346, Issue 6-9 Pages 813-818
Ingrid Lukkari, Jaromir Ruzicka and Gary D. Christian

Abstract: Samples (0.2 ml) were mixed with carrier (H2O or 1 M NaOH) and transported into a diffusion cell incorporating a Celgard 2400 hydrophobic membrane. The receptor side of the membrane contained either indicators or Berthelot reagents, depending on the NH3 concentration. expected. Optical fibers were fixed to the acceptor side of the flow cell for absorbance measurement at 580 or 620 nm. When the absorption peak max. reached the detector, the flow was stopped for 10 s and the rate was measured. For 0.05-0.5 mM NH3, the detector reagent was aqueous 0.1 mM bromothymol blue (pH 6.5). For 0.7-10 mM NH3 the reagent was a mixture of 0.02% of cresol red, 0.04% of bromothymol blue and 0.08% of bromocresol purple in 1 mM KH2PO4/1 mM Tris (pH 6.5). When the concentration. of the buffer components were increased to 50 mM each, the rectilinear range was 7-125 mM (or up to 350 mM with a second-order polynomial). With the Berthelot reaction, the rectilinear range was 1-100 mM (or up to 250 mM with a second-order polynomial). The method was suitable for online monitoring if the membrane was washed with a proteinase after every 10 samples.
Ammonia Ammonia, nitrogen Spectrophotometry Sequential injection Hydrophobic membrane Celgard

"Amperometric Penicillin Biosensor And Its Application In Flow Injection Analysis System For Determination Of Penicillin In Broth"
Microchem. J. 1995 Volume 52, Issue 2 Pages 166-173
Li Q. S., Zhang S. L. and Yu J. T.

Abstract: Penicillinase was immobilized by crosslinking with glutaraldehyde and BSA onto a Pt disc electrode which was then covered by a dialysis membrane. The electrode was incorporated into a flow-through cell with a Pt counter electrode and SCE reference electrode. Sample (30 µL) was injected into 0.1 M phosphate buffer of pH 7.5 containing 5-10 mM I2 and 10^-20 mM KI (2 ml/min). Excess I2 not consumed by the penicilloic acid produced was determined at +100 mV. Calibration graphs for penicillin (I) obtained at 5-10 mM I2 were linear (graphs shown). No RSD or detection limit is given. Sample throughput was 80/h. Ascorbic acid interfered. The biosensor was stable for 2 weeks (>1000 assays). It was applied to the determination of I in fermentation liquor; the results agreed well with those obtained by the conventional I2 titration method.
Penicillins Amperometry Electrode Electrode Electrode Sensor Method comparison Interferences Dialysis

"Determination Of Trehalose By Flow Injection Analysis Using Immobilized Trehalase"
Anal. Biochem. 1997 Volume 253, Issue 1 Pages 8-12
H. -D. Meyer zu Düttingdorfa, B. Bachmannb, M. Buchholzc and W. Leuchtenbergerb

Abstract: A new method for the determination of trehalose by flow injection analysis (FIA) is described. The basic principle is the hydrolysis of the disaccharide trehalose into its monomer D-glucose by trehalase, a periplasmic enzyme of Escherichia coli. D-glucose is quantified spectrophotometrically after reaction with hexokinase and glucose-6-phosphate dehydrogenase. Trehalase is prepared by osmotic shock from a recombinant E, coli strain and precipitated with ammonium sulfate. The enzyme is immobilized on VA-Epoxy Biosynth from Riedel-de-Haen, The immobilization rate is about 60%. The FlA signals show a nonlinear dependence on the trehalose concentration. The resulting curve corresponds to a second-order polynomial that serves as a calibration function for test samples. Immobilized trehalase was used during a period of 4 months without any loss of suitability. Several samples of fermentation broth were tested. The results are verified by HPLC. Within an interval of 2 to 10 g/L trehalose the recovery is about 100-120% with a precision of 7% (coefficient of variation). (C) 1997 Academic Press. 10 References
Trehalose Spectrophotometry Immobilized enzyme Kinetic Method comparison

"Entirely Automated Glucose Monitoring System Based On A Flow Injection Analysis Apparatus"
Anal. Lett. 1994 Volume 27, Issue 5 Pages 833-848
Pilloton, R.;Mignogna, G.;Fortunato, A.

Abstract: A polysulfone hollow fiber membrane (HFM) was used to immobilize a β-galactosidase enzyme by pumping 500 mL of β-galactosidase solution (1 g/l) with 0.1% glutaraldehyde, BSA (0.5 g/l) in 25 mM citrate buffer of pH 4.5, into the HFM module (1 ml/min) using a back-flushing configuration for 6 h. This was followed by a washing solution comprising 30 mM glycine/25 mM citrate buffer of pH 4.5. A glucose biosensor was constructed by coupling an amperometric H2O2 sensor with glucose oxidase enzyme. A 10 µL sample loop was used to inject the sample into the carrier stream of 0.1 M phosphate buffer of pH 7, with 0.01% NaN3 and amperometric detection at 0.65 V vs. Ag/AgCl. To control data acquisition, sampling and calibration, a GWBASIC program was developed. The method improved the upper limit of detection 30-fold up to 150 mM compared to that possible with the conventional amperometric biosensor and the RSD was ±2%. The system could be expanded and used to control fermentation processes; it may also be possible to extend the work to lactose and lactic acid.
Glucose Lactose Lactic acid Amperometry Sensor Electrode

"Sequential Injection Analysis Of Ethanol Using Immobilized Alcohol Dehydrogenase"
Anal. Lett. 1996 Volume 29, Issue 7 Pages 1109-1124
Hedenfalk, M.;Mattiasson, B.

Abstract: A sequential injection analysis (SIA) system (schematic of manifold is given) was developed for the determination of ethanol in fermentation broth. The system was optimized in terms of pH, cofactor concentration in the reagent segments and stopping time using the aspiration sequence: 1-6 mL wash/carrier buffer, NAD+ in buffer (0.15 ml), sample or standard (15 ml) NAD+ in buffer (0.15 ml) and wash/carrier buffer (0.14 ml). The optimum pH was 8.8 but the type of buffer, i.e. Tris hydrochloride or glycine/NaOH was unimportant. The stopping time had little effect reaching a plateau after 30 s. The optimum concentration of cofactor was 0.5 µmol cofactor/assay. The assay cycle was complete within 140 s. The SIA system was based on the use of immobilized NAD+ dependent alcohol dehydrogenase and the calibration range (non-linear) extended from 0.25-100 mM ethanol. The RSD (n = 50) was 1.4% at the 2 mM ethanol level.
Ethanol Sequential injection Immobilized enzyme

"Flow Injection Piezoelectric Determination Of Brix In Sugar Cane Juice And In The Alcoholic Fermentation Process"
Anal. Lett. 1996 Volume 29, Issue 13 Pages 2411-2419
Adriana Magna; A. Fernando de Oliveira; Orlando Fatibello-Filho

Abstract: A schematic of the piezoelectric FIA system is given. Sample (500 µL) was aspirated from the fermentation reactor at 1 ml/min and transported by the water carrier to the piezoelectric cell. The flow of solution was directed to the centre of one side of the 10 MHz AT-cut piezoelectric crystal coated with Au on both sides. The calibration graph was linear from 0.5-20% brix (total dissolved solids) with a detection limit of 0.3%. The RSD (n = 15) was ?. The results were comparable with those obtained from refractometric and densitometric measurements.
Brix Sensor Piezoelectric crystal Method comparison

"A Bioelectrochemical Method For The Determination Of Acetate With Immobilized Acetate Kinase"
Anal. Lett. 1997 Volume 30, Issue 14 Pages 2469-2483
Xiao-Jing Tang; Gillis Johansson

Abstract: sample solution containing acetate was injected into a flow (0.1 ml/min) of water, which merged with a flow (0.1 ml/min) of 0.1 M imidazole buffer of pH 7.6 containing 0.1 M KNO3, 0.015 M magnesium nitrate, 6 mM ATP, 2 mM phosphoenolpyruvate and 0.25 mM NADH and then passed through an enzyme reactor containing acetate kinase, pyruvate kinase and lactate dehydrogenase. The stream then merged with a flow (0.1 ml/min) of 0.1 M sodium acetate buffer of pH 4.5 and passed through a flow cell equipped with a Meldola Blue-modified graphite working electrode, a Pt auxiliary electrode and a SCE reference electrode for amperometric detection at an applied voltage of 0 V. The calibration graph was linear from 0.3-2 mM acetate, the detection limit was 0.2 mM acetate and the RSD was 3.4%. The method was suited to the determination of acetate in fermentation liquors where acetate is the carbon source.
Acetate ion Electrochemical analysis Immobilized enzyme

"Column Liquid Chromatography In Combination With Immobilized Enzymes And Electrochemical Detection And Its Applications In Some Industrial Processes"
Electroanalysis 1992 Volume 4, Issue 4 Pages 403-427
György A. Marko-Varga

Abstract: A review, with 206 references. The first part of the paper provides a review of the pre- and post-column derivatization systems used in column liquid chromatography (CLC) in combination with immobilized enzyme reactors (IMERs) and electrochemical detection (EC). In the second part, an outline of important factors to consider in the optimization of CLC-IMER-EC systems are presented. Three industrial applications are described utilizing enzyme-based detection or enzymes for sample handling purposes. In two of the cases, CLC-IMER were used in combination with amperometric detection using chemical modified electrodes (CME). These applications were performed in the author's lab., and the high selectivity and sensitivity of these systems as well as the problems encountered in these complex samples will be discussed.
HPLC Electrochemical analysis Review Post-column derivatization Pre-column derivatization Immobilized enzyme Optimization

"Ferrocene-containing Polymers As Electron Transfer Mediators In Carbon Paste Electrodes Modified With PQQ-dependent Aldose Dehydrogenase"
Electroanalysis 1995 Volume 7, Issue 10 Pages 941-946
Maria Smolander, Lo Gorton, Hung Sui Lee, Terje Skotheim, Hsing-Lin Lan

Abstract: Four different polymer-bound ferrocene derivatives were evaluated as electron transfer mediators in aldose biosensors based on aldose dehydrogenase with pyrroloquinoline quinone (PQQ) as co-enzyme. The polymeric ferrocene derivatives (syntheses described) were mixed with C paste and packed into a plastic syringe holder that had been pre-packed with unmodified C paste. A solution of aldose dehydrogenase in 10 mM sodium acetate of pH 5 containing 0.1% Triton X-100 was applied to the electrode surface and allowed to dry. The electrodes were evaluated in a flow injection system equipped with a wall-jet amperometric cell using 50 mM sodium phosphate buffer of pH 6.5 as carrier (0.7 ml/min). Maximum response was obtained at +300 mV vs. Ag/AgCl but measurements were normally made at +200 mV to minimize interference from non-specific oxidation processes. The electrodes were more stable than those modified with monomeric dimethylferrocene. The best response was obtained with polymethyl(11-ferrocenyl-4,7,10-trioxa-undecanyl)methyl (12-amino-4,7,10-tioxa-dodecyl)siloxane (1:1) and electrodes prepared from this derivative was applied to the determination of xylose in fermentation mixtures; the results agreed with those obtained by LC.
Aldoses Xylose Sensor Electrode Electrode Amperometry Electrode Interferences Method comparison Triton X Surfactant

"Determination Of Sterigmatocystin In Fermentation Broths By Reversed-phase High Performance Liquid Chromatography Using Post-column Fluorescence Enhancement"
J. Chromatogr. A 1990 Volume 523, Issue 1 Pages 305-311
Frank L. Neely* and Curt S. Emerson

Abstract: Sterigmatocystin (I, fungal toxin often found in food) was separated from the broth by homogenization with methanol, filtration, extraction of I from the filter-cake with methanol and 0.45 µm re-filtration of the supernatant solution A portion of solution was subjected to HPLC in a column (25 cm x 4.6 mm) of Beckman Ultrasphere C18 (5 µm) with use of a guard column (1.5 cm x 3.2 mm) of Brownlee Newguard RP-18 (7 µm), a mobile phase (0.5 mL min-1) of aqueous 88% methanol, post-column derivatization (reaction coil diameter 0.01 in.) at 35°C with aqueous 5% AlCl3 (added at 0.5 mL min-1) and fluorimetric detection at 455 nm (excitation at 254 nm). The limit of detection was 0.09 ppm, with rectilinear calibration range from 0.1 to 15 ppm and coefficient of variation (n = 12) of 0.2% at 0.82 ppm. A TLC separation on Whatman LK-5D silica with a mobile phase of 1% acetic acid in CCl4 - CHCl3 (1:1) and fluorimetric detection of the same derivative is summarized.
Sterigmatocystin HPLC Fluorescence Sample preparation Column Filtration Extraction C18 Post-column derivatization Detection limit Calibration Silica

"Sampling And Analytical Strategies In Online Bioprocess Monitoring And Control"
J. Chromatogr. A 1996 Volume 725, Issue 1 Pages 13-27
N. C. van de Merbel, H. Lingeman and U. A. Th. Brinkman*

Abstract: A review is presented of the uses of online systems for monitoring small organic molecules in biotechnological processes are also discussed. Sampling techniques used including non-membrane, dialysis and ultrafiltration techniques are discussed. Analysis by FIA is fast, simple and inexpensive, but relatively non-selective compared to LC, which allows determination of more than one component per run. (37 references).
Review Dialysis

"Fluorimetric Determination Of Rat Growth Hormone In Pituitary Cell Culture By High Performance Liquid Chromatography With Post-column Derivatization"
J. Chromatogr. B 1987 Volume 417, Issue 1 Pages 409-413
Hiroshi Nakazawa

Abstract: Rat pituitary cell culture mixture was treated with human growth-hormone-releasing factor and then analyzed for growth hormone by HPLC on a Bio-Rad RP-304 column (25 cm x 4.6 mm) with gradient elution (details given) by using aqueous acetonitrile containing 0.1% of trifluoroacetic acid at 1 mL min-1. The eluate was passed through a reaction coil and the growth hormone was derivatized with ethanolic 0.1% phthalaldehyde containing 0.107% of 2-mercaptoethanol; fluorimetric detection was at 440 nm (with excitation at 340 nm). The detection limit was 60 ppb; calibration graphs were rectilinear from 0.4 to 10 ppm. The coefficient of variation was 2.7%; results correlated well (r = 0.922) with those obtained by RIA.
Hormone HPLC Fluorescence Method comparison Post-column derivatization Biorad

"Covalent Immobilization Of Glucose Oxidase On Silanized Platinum Microelectrode For The Monitoring Of Glucose"
Sens. Actuat. B 1993 Volume 15, Issue 1-3 Pages 127-134
A. Nyamsi Hendji, P. Bataillard and N. Jaffrezic-Renault

Abstract: Glucose oxidase was covalently immobilized on an aminosilanized Pt micro-electrode and used in batch and flow injection systems. The best sensitivity was obtained by coupling the enzyme through benzene-1,2,4,5-tetracarboxylic dianhydride (I) or succinic anhydride to the aminosilanized electrode. This gave a response range up to 30 mM under batch conditions. Cross-linking of the enzyme with BSA with use of glutaraldehyde gave a 200-fold better sensitivity, but a dynamic range limited to 2 mM. With coupling through I the sensitivity after storage for 2 months was 50% of the initial value. The use of glucose oxidase (GOD) in electrochemical sensors for glucose detection is reported. The aim of this study was to show the feasibility and the performances of enzymatic platinum electrodes prepared by direct covalent coupling of GOD. The glucose oxidase enzyme electrode is used in a batch and flow injection analysis (FIA) system. It has been demonstrated that oxidized Pt can be silanized with surface densities comparable to those of silica; the grafting densities do not depend on the preparation method of Pt oxide, but on the nature of the monofunctional silane used. The best sensitivities for glucose detection are obtained using the coupling of 1,2,4,5-benzene tetracarboxylic acid dianhydride (BTCAD) and succinic anhydride with the aminosilanized electrodes. This coupling procedure leads to a dynamic range of response up to 30 mM in batch conditions because of no limitation by diffusion. In comparison, the BSA crosslinking technique results in a much higher sensitivity (about 200-fold) but also in the dynamic range limited to 2 mM. The sensitivity after a two-month storage remains 50% of its initial level for BTCAD coupling. The sensitivity can be amplified by performing an additional coupling process using the CDI reagent on a BTCAD-coupled enzyme electrode, whereas the opposite effect is obtained with GA. Nevertheless, the succinic anhydride-coupled electrode exhibits high non-specific signals in yeast fermentation broth samples because of the presence of electroactive interferents. [References: 14]
Glucose Electrode Electrochemical analysis Immobilized enzyme Interferences

"Application Of Optrodes In FIA-based Fermentation Process Control Using The Software Package FIACRE"
Sens. Actuat. B 1993 Volume 11, Issue 1-3 Pages 407-412
M. Busch, F. Gutberlet, W. Höbel, J. Polster*, H. -L. Schmidt and M. Schwenk

Abstract: A description is given of biosensors for ethanol and urea that can be used in fermentation processes. The ethanol sensor measures NADH fluorescence during an enzymatic reaction involving alcohol dehydrogenase and aldehyde dehydrogenase. The urea sensor is based on measuring a change in the fluorescence of fluorescein. Both sensors can be used for controlling fermentation processes involving the production or consumption of ethanol and urea. A description is given of an Enterobacteriaceae Proteus vulgaris fermentation that is monitored by a urea optode controlled by a FIACRE (Flow injection Analysis: Computer Regulated and Evaluated) software package. The lifetime of the urease optrode can be long.
Ethanol Urea Fluorescence Sensor Optrode Computer Process control

"Bio Field-effect Transistors For Process Control In Biotechnology"
Sens. Actuat. B 1991 Volume 4, Issue 3-4 Pages 315-318
U. Brand, B. Reinhardt, F. Rüther, T. Scheper and K. Schügerl

Abstract: The application of enzyme-modified FET in biotechnology is discussed. The sensors are applied in flow injection systems for determination of penicillin, glucose and urea in cultivation media and for online biotechnological process control. A sample rate of 15 to 20 per h was achieved with use of small-volume flow cells.
Glucose Penicillin Urea Field effect transistor Biotechnology Process control

"Fermentation Control With Biosensors In Flow Injection Systems - Problems And Progress"
Sens. Actuat. B 1990 Volume 1, Issue 1-6 Pages 542-545
U. Prinzing, I. Ogbomo, C. Lehn and H. -L. Schmidt

Abstract: The inclusion of a pervaporation stage is recommended in order to protect the biosensor from being fouled by macromolecular components of the fermentation broth, and a laboratory-built pervaporation module is described and illustrated. This consists of an upper and a lower compartment separated by a horizontal semipermeable membrane; the carrier stream enters the lower compartment, but is separated from the membrane by an air gap. The carrier stream is heated, and volatile analytes diffuse across the air gap and pass through the membrane to an acceptor stream, which carries them to the biosensor. As examples are described the determination of ethanol and biacetyl in simulated brewing mixtures.
Ethanol Biacetyl Sensor Membrane Diffusion Pervaporation

"Improved Micro Enzyme Sensor For Bioprocess Monitoring By Flow Injection Analysis"
Sens. Actuat. B 1992 Volume 7, Issue 1 Pages 404-407
S. Haemmerli, A. Schaeffler, A. Manz and H. M. Widmer

Abstract: The cited sensor, based on an integrated microelectrode, was used for the online determination of glucose. After cleaning for 5 min in an ultrasonic bath, the sensor was coated in a solution of glucose oxidase in a poly-(4-vinylpyridine) matrix doped with ferrocene monocarboxylic acid as mediator and allowed to dry at room temperature The mediated sensor was tested as a detector for the monitoring of yeast cultivation by flow injection analysis (cf. Ibid., 1991, 4, 309). Sensitivity towards interferents was 2.5-fold lower than than the unmediated sensor, making it more suitable for monitoring cultivations at low glucose concentration. However, the measured glucose concentration was lower than the actual value obtained colorimetrically, so the unmediated enzyme electrode was preferred for the determination of glucose at levels of 5 to 200 mM. An improved µenzyme electrode is presented for online determination of glucose during bioprocesses. The disposable sensor was constructed on the basis of an integrated microelectrode (from the Institute of Microtechnique, University of Neuchatel) and used as detector in a flow injection analysis (FIA) system. Glucose oxidase was incorporated in a polyvinyl pyridine matrix doped with mediator molecules and deposited on the electrode. The performance of this sensor was tested during the cultivation of yeast. The obtained results were compared with the data from a different flow injection method, using enzyme columns and colorimetric detection.
Glucose Electrode Electrode Sensor Process monitoring Method comparison Interferences

"Automated Glucose Measurement With Micro-structured Thin-layer Biosensors For The Control Of Fermentation Processes"
Sens. Actuat. B 1994 Volume 19, Issue 1-3 Pages 603-606
A. P. Loibner, O. Doblhoff-Dier, N. Zach, K. Bayer and H. Katinger, Ch. Lobmaier, Th. Schalkhammer and F. Pittner

Abstract: A FIA system for the online determination of glucose in animal cell fermentation broth is described. It comprised an aseptic sampling system (details given) and a micro-structured thin-film amperometric biosensor (diagram given) based on glucose oxidase cross-linked with an amine - aldehyde-based polymer (cf. Schallkammer et al., Ibid., 1992, 7, 356) with a Ag/AgCl reference electrode. Fermentation broth (30 µL) was carried in a stream (3 ml/min) of phosphate buffer of pH 7.4 (1 g/l of NaH2PO4.H2O and 6.8 g/l of Na2HPO4) containing 0.5 g/l of Titriplex III.H2O, 2.75 g/l of NaCl and 5 mg/l of gentamycin sulfate. The computer control of the system is described. The system was calibrated with 50-500 mg/l glucose solution Data obtained from the analysis of a fermentation over 33 h, with one calibration per run, are shown. The sensor performance remained satisfactory over 150 days.
Glucose Electrode Sensor

"Microfabricated Glucose, Lactate, Glutamate And Glutamine Thin-film Biosensors"
Sens. Actuat. B 1994 Volume 19, Issue 1-3 Pages 587-591
Th. Schalkhammer, Ch. Lobmaier, B. Ecker, W. Wakolbinger, E. Kynclova, G. Hawa and F. Pittner

Abstract: Glutaminase, glucose, lactate and glutamate oxidase-bonded sensors were fabricated on Al2O3 ceramics by immobilization in a polyimine gel or co-crosslinking with polyvinylpyrrolidone and nitrene (details given). The sensors contained integrated Pt and Ag/AgCl counter and reference electrodes. Sensors were mounted in a flow cell and test solution were pumped through the cell at 3 ml/min. Cyclovoltammetry was performed by scanning from -500 to +700 mV at 50 mV/s. Calibration graphs were linear for 0-20 mM glucose and 0-120 mM lactate, glutamate and glutamine. After use, the cell assembly was washed with 0.12 M PBS of pH 7. Applications to the FIA analysis of glucose, lactate, glutamate and glutamine in plasma, blood and fermentation broths are discussed.
Glucose Lactate Glutamate Glutamine Voltammetry Electrode Electrode Sensor

"Flow Injection Determination Of Hydrogen Peroxide By Bis-(2,4,6-trichlorophenyl)oxalate Chemiluminescence In O/W Emulsion"
Anal. Sci. 1996 Volume 12, Issue 6 Pages 881-885
T. HASEBE, E. HASEGAWA and T. KAWASHIMA

Abstract: A schematic of the FIA manifold is presented. Sample (60 µL) was injected into the water carrier stream (0.6 ml/min) and merged with 0.6 M Tris buffer of pH 9 (0.6 ml/min). The mixture was transferred to a heating coil (3 m length) in a water bath at 37±0.1°C. A 1.5 wt.% Tween 20 solution and a mixture of 0.1 mM bis-(2,4,6-trichlorophenyl)oxalate and 1 mM perylene in ethyl acetate were mixed (0.6 ml/min) to form an oil/water (O/W) type emulsion. The mixture was then pumped (1.2 ml/min) and merged with the carrier stream in front of the flow cell. The light emitted from the chemiluminescence reaction was measured over the range 400-600 nm and was proportional to the H2O2 concentration. The calibration graph was linear from 0.1-1 µM-H2O2. The method was applied to the determination of L-glutamic acid in culture medium as H2O2. Results are discussed.
Hydrogen peroxide Chemiluminescence Heated reaction

"A New Version Of An In-situ Sampling System For Bioprocess Analysis"
Acta Biotechnol. 1996 Volume 16, Issue 2-3 Pages 185-192
J.-M. Hilmer, Th. Scheper

Abstract: In this article, attention is focused on an online sampling device for bioreactors and its characterization regarding sterility and response time. The integration of this device into analytical systems for biotechnology (FIA with biosensors) resulted in online analysis systems for bioprocess control. The new ESIP version of an in situ sampling system for bioprocess analysis produced by the EPPENDORF-NETHELER-HINZ GmbH, Germany, had a short response time of 8 min (99%), which was determined by means of conductivity measurement after an increase of the medium conductivity due to the gradual addition of KCl. Effectiveness and reliability of the module were tested by bubble point measurement resulting in a bubble point pressure of 2.1 bars. The sampling probe was tested successfully for use in a broad variety of microorganisms and cultivations.
Microbial activity Glucose Maltose Sucrose Sensor Biotechnology Process control Review

"Glucose Monitoring Of Fermentation With An FIA Wall-jet Electrode System"
Am. Biotechnol. Lab. 1993 Volume 11, Issue 5 Pages 18-20
Huang YL, Khoo SB, Yap MG

Abstract: The wall-jet electrode (WJE) has been used as an electrochemical detector in many flow applications. It offers some useful features such as well-defined hydrodynamic properties, low dead volume, good sensitivity, fast response, and ease of operation. As evaluated by the authors, WJE also demonstrated good performance when it was used as the detector for flow-injection analysis (FIA). Based on a previous work, a simple method for glucose monitoring was developed. The main objective is to demonstrate the applicability of the FIA/WJE system together with an immobilized glucose oxidase reactor for monitoring glucose concentration in fermentation broths. On the basis of conditions used, several samples from different sources (with high glucose contents [15-20 g/L] casein hydrolyzate, tryptose, or horse serum) were tested and the results showed good agreement between the data produced by the FIA/WJE system and those determined by the commercial analyzer.
Glucose Electrode Process monitoring

"Computer-aided Detection Of Failures In Flow Injection Analysis Systems"
Am. Biotechnol. Lab. 1993 Volume 11, Issue 5 Pages 78-80
Brandt, J.;Hitzmann, B.

Abstract: The capabilities of an expert system to detect and diagnose a failure in a flow-injection anal. system are illustrated by the enzymatic determination of glucose measured during cultivation of Penicillium chrysogenum.
Glucose Computer Process monitoring

"Oxidation Of Yeast Iso-1 Ferrocytochrome C By Yeast Cytochrome C Peroxidase Compounds I And II. Dependence Upon Ionic Strength"
Biochemistry 1995 Volume 34, Issue 31 Pages 9991-9999
Andrea L. Matthis, Lidia B. Vitello, and James E. Erman

Abstract: The reduction of cytochrome c peroxidase compound I by excess yeast iso- 1 ferrocytochrome c is biphasic. Two pseudo-first-order rate constants can be measured by stopped-flow techniques. The fastest rate process is the reduction of cytochrome c peroxidase compound I to compound II, and the slower process is the reduction of II to the native enzyme. The yeast iso-1 ferrocytochrome c concentration dependence of the reduction of cytochrome c peroxidase compound I to compound II is consistent with a mechanism involving two binding sites for cytochrome c on cytochrome c peroxidase. Electron transfer from cytochrome c bound at the high- affinity binding site to the Fe(IV) site in cytochrome c peroxidase compound I is dependent upon ionic strength, increasing from 15±6 to 2000±100 s-1 over the ionic strength range 0.01-0.20 M. The reduction rate of the Fe(IV) site in the 2:1 yeast iso-1 ferrocytochrome c/cytochrome c peroxidase compound I complex is essentially independent of ionic strength with a value of 3800±300 s-1. The Fe(IV) site in cytochrome c peroxidase compound I is preferentially reduced by yeast ferrocytochrome c between 0.01 and 0.20 M ionic strength while the Trp-191 radical is preferentially reduced above 0.30 M ionic strength. The association rate constant for the binding of yeast iso-1 ferrocytochrome c to cytochrome c peroxidase compound I can be evaluated and varies from a remarkable 1 x 10(10) M-1 s-1 at 0.01 M ionic strength to 1.2 x 10(5) M-1 s-1 at 1.0 M ionic strength. Between 0.01 and 0.20 M ionic strength, the reduction of cytochrome c peroxidase compound II to the native enzyme is anomalous. The reaction is independent of the cytochrome c concentration and directly proportional to the initial cytochrome c peroxidase compound I concentration.
Enzyme, cytochrome c peroxidase Rate constants Stopped-flow Ionic strength

"Evidence For A Glutathionyl-enzyme Intermediate In The Amidase Activity Of The Bifunctional Glutathionylspermidine Synthetase/amidase From Escherichia Coli"
Biochemistry 1997 Volume 36, Issue 48 Pages 14930-14938
Chun-Hung Lin, David S. Kwon, J. Martin Bollinger, Jr., and Christopher T. Walsh

Abstract: Glutathionylspermidine (Gsp) is a metabolite common to Escherichia coli and protozoal parasites of the Trypanosoma family. Though its role in E. coli is unknown, Gsp is known to be an intermediate in the biosynthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), a metabolite unique to trypanosomatids that may allow the parasites to overcome oxidative stresses induced by host defense mechanisms. The bifunctional Gsp-synthetase/amidase from E. coli catalyzes both amide bond formation and breakdown between the N1-amine of spermidine [N-(3-aminopropyl)-1,4-diaminobutane] and the glycine carboxylate of glutathione (γ-Glu-Cys-Gly), with net hydrolysis of ATP [Bollinger et al. (1995) J. Biol. Chem. 270 (23), 14031-14041]. Synthetase and amidase activities reside in separate domains of the protein, and liberation of the amidase domain from the synthetase domain activates the amidase activity as much as 70-fold in kcat/K(m) for a chromogenic substrate γ-Glu-Ala-Gly-pNA [Kwon et al., (1997) J. Biol. Chem. 272 (4), 2429-2436]. When substrates for the Gsp-synthetase activity are present (GSH, ATP-Mg2+), Gsp-amidase is highly activated (15-fold). We provide kinetic and mutagenesis evidence suggesting that the amidase operates by a nucleophilic attack mechanism involving cysteine as the catalytic nucleophile. Stopped-flow studies on the 25 kDa Gsp-amidase fragment and the 70 kDa full-length Gsp-synthetase/amidase with γ- Glu-Ala-Gly-ONp demonstrate burst kinetics characteristic of a covalent acyl-enzyme intermediate. Studies using various group-specific protease inhibitors, such as iodoacetamide, suggest an active-site cysteine or histidine as being relevant to amidase activity, and site-directed mutagenesis indicates that Cys-59 is essential for amidase activity.
Enzyme, activity Bacteria Stopped-flow Kinetic

"FIA For Down-stream Processing-determination Of β-galactosidase In Viscous Extraction Media"
Bioprocess Eng. 1991 Volume 7, Issue 1-2 Pages 47-52
C. Silfwerbrand-Lindh, L. Nord, L. Häggström and F. Ingman

Abstract: A flow injection analysis method for determination of the enzyme β-galactosidase produced in a fermentation process has been developed. The analytical range was 10^-1000 U/mL and the sample throughput was 40 h-1. The FIA set up was connected directly to the down-stream process and the enzyme activity in the extraction step was measured online. The viscosity of the samples from the process is high causing certain physical effects on the FIA system.
Enzyme, galactosidase Viscosity

"Flow Injection Analysis In Bioprocess Control"
Bioprocess. Technol. 1990 Volume 6, Issue 1 Pages 221-241
Reed AL

Abstract: A review with 17 references. Topics include: principles of operation; practical applications in bioprocesses; and connecting flow injection analysis to a process.
Catalysis Review

"Development Of Analytical Method For Selective Detection Of Cardenolides By High Performance Liquid Chromatography"
Biosci. Biotechnol. Biochem. 1992 Volume 56, Issue 6 Pages 967-967
Baku Maekawaa & Kazuo Morimoto

Abstract: Cardenolides were separated on a column (25 cm x 4.6 mm) of TSK-gel ODS 120T operated at 25°C, with gradient elution (1 mL min-1) with aqueous methanol (details given). The eluate was mixed with Kedde reagent and passed through a reaction coil (5 m x 0.33 mm) followed by detection at 540 nm. The method compares well with the method using UV detection at 220 nm. The method is suitable for the analysis of cardenolides biotransformed by plant cell cultures.
Cardenolides HPLC Spectrophotometry Post-column derivatization

"Flow-through Enzyme Analyser For Determination Of L-lysine Concentration"
Biosens. Bioelectron. 1991 Volume 6, Issue 2 Pages 93-99
A. L. Simonian, G. E. Khachatrian, S. Sh. Tatikian, Ts. M. Avakian and I. E. Badalian

Abstract: The flow-through analyzer was based on an oxygen electrode and L-lysine-2-monooxygenase immobilized on silica gel. A schematic diagram is given of the analyzer. and a cross-section of the measurement cell shows a special construction allowing the buffer and its waste to be placed proportionally under the O electrode. The buffer solution was pumped through a pulse damper into the column containing immobilized enzyme. The sample was dosed by a device which injected 70 µL of sample into the column without interruption of the buffer flow. Oxygen concentration. was measured by means of a Clark membrane electrode (0.5 M KCl solution as the electrolyte, Pt cathode and Ag anode, pulse height -0.65 V). Calibration graphs were rectilinear from 5.5 to 55 mM (1 to 10 g l-1) of L-lysine I at pH 8.2. The analyzer. showed high specificity for I. The response time was 15-30 s, with a total analysis time about 3 min. The immobilized enzyme retained at least 50% of its activity for 2 months. Measurement have been made in the fermentation broth of fermenters and in fodder L-lysine concentrate. The analyzer. can be used autonomously and also with a microcomputer or microprocessor.
l-Lysine Electrode Buffer Column Computer Immobilized enzyme Silica gel

"Online Determination Of Glucose And Lactate Concentrations In Animal Cell Culture Based On Fibre Optic Detection Of Oxygen In Flow Injection Analysis"
Biosens. Bioelectron. 1992 Volume 7, Issue 2 Pages 133-139
B. A. A. Dremel, S. -Y. Li and R. D. Schmid

Abstract: A flow injection (FIA) system based on fiber-optic detection of O consumption using immobilized glucose oxidase and lactate oxidase (Boehringer, Mannheim, Germany) from Pediococcus sp. is described. The enzymes were immobilized on controlled-pore glass in enzyme reactors linked to a specially designed fiber-optic flow-through cell covering the optrode. The enzyme reactors were stable for >1 month in continuous operation and it was possible to analyze 20 samples h-1. The system was applied to the online monitoring of glucose (I) and lactate (II) concentration. of an animal cell culture designed for the production of recombinant antithrombine III. The calibration graphs were rectilinear for 0 to 30 mM I or II and the coefficient of variation (n = 5) were 5%. A flow injection analysis (FIA) system based on fiber optic detection of oxygen consumption using immobilized glucose oxidase (GOD) and lactate oxidase (LOD) is described for the online monitoring of glucose and alctate concentrations. in animal cell cultures. The consumption of oxygen was determined via dynamic quenching by mol. oxygen of the fluorescence of an indicator. GOD and LOD were immobilized on controlled pore glass (CPG) in enzyme reactors which were directly linked to a specially designed fiber optic flow-through cell covering the oxygen optrode. The system is linear for 0-30 mM glucose, with an RSD of 5% at 30 mM (5 measurements) and for 0-30 mM lactate, with an RSD of 5% at 30 mM (5 measurements). The enzyme reactors used were stable for more than 4 wk in continuous operation, and it was possible to analyze up to 20 samples per h. The system has been successfully applied to the online monitoring of glucose and lactate concentrations. of an animal cell culture designed for the prodn. of recombinant human antithrombin III (AT-III). Results of the online measurement obtained by the FIA system were compared with the off-line results obtained by a glucose and lactate analyzer from Yellow Springs Instrument Company (YSI).
Glucose Lactate Fluorescence Optrode Immobilized enzyme Optical fiber Indirect Quenching Method comparison Process monitoring Controlled pore glass

"A Chemiluminescence Fiber-optic Biosensor System For The Determination Of Glutamine In Mammalian Cell Cultures"
Biosens. Bioelectron. 1992 Volume 7, Issue 8 Pages 569-574
M. V. Cattaneo, K. B. Male and J. H. T. Luong*

Abstract: A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens. Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column. Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate. In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system. In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal. There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10^-6 to 10^-3 M. A complete analysis could be performed in 2 min including sampling and washing. Each immobilized enzyme column was stable for at least 300 repeated analyzes without any loss of activity. When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high performance liquid chromatography, thus validating the applicability of the CL fiber-optic system.
Glutamine Chemiluminescence Sensor Biotechnology Optical fiber Immobilized enzyme Porous glass beads Method comparison

"Studying The Bienzyme Reaction With Amperometric Detection For Measuring Maltose"
Biosens. Bioelectron. 1993 Volume 8, Issue 6 Pages 339-345
M. Váradi* and N. Adányi, G. Nagy, J. Rezessy-Szabó

Abstract: A sensor for monitoring maltose concentration (0.2-4 mM, during e.g. yeast culture and fermentation) is described and tests on optimization of its use are reported. It was based on a thin-layer reactor in which glucose oxidase and amyloglucosidase (preferred over α-glucosidase) had been immobilized on a protein membrane (e.g. pig small intestine). In a flow injection analysis system, this was followed by an amperometric measuring cell having Pt anode, Ag/AgCl reference cathode and Pt auxiliary electrode, operated with recording of the anodic current at bias +600 mV. Some interference from higher oligosaccharides, dextrins and starch occurred.
Maltose Amperometry Optimization Interferences

"Online Monitoring Of Glucose, Glutamate And Glutamine During Mammalian Cell Cultivations"
Biosens. Bioelectron. 1995 Volume 10, Issue 6-7 Pages 543-551
S. F. White and A. P. F. Turner, U. Biltewski, J. Bradley and R. D. Schmid

Abstract: Glucose (I), glutamine (II) and glutamate (III) amperometric biosensors were prepared based on rhodinized C electrodes (details given). The sensors were used in a parallel three cell FIA system with Ag/AgCl and stainless-steel tube, reference and counter electrodes, respectively, to detect I, II, and III, simultaneously, in perfusion cultures. Samples were pumped through the system every 30 min following calibration and the biosensors were changed as necessary. The results compared well with those obtained by an off-line FIA method (validated by HPLC). Amperometric biosensors (based on rhodinised carbon electrodes) for glucose, glutamine and glutamate were constructed. The sensors were incorporated into a three cell parallel FIA system and used to monitor the three analytes online during two mammalian cell perfusion cultures. All measurements were made simultaneously from undiluted media sample. Use of the FIA system enabled easy and rapid exchange of the sensors, during cultivation. The inclusion of a calibration step, regularly for all sensors, helped to maintain the accuracy of all measurements. Comparison with off-line measurements indicated that all three biosensors operated successfully, providing accurate information.
Glucose Glutamate Glutamine Sensor Amperometry Electrode Biotechnology Simultaneous analysis Multichannel Method comparison Process monitoring Perfusion

"Determination Of Glutamine And Glutamic Acid In Mammalian Cell Cultures Using Tetrathiafulvalene Modified Enzyme Electrodes"
Biosens. Bioelectron. 1996 Volume 11, Issue 3 Pages 271-280
Ashok Mulchandani* and Amarjeet S. Bassi

Abstract: A tetrathiafulvalene-carbon paste electrode was fabricated (details given) and the electrode tip was immersed in 0.1% Triton-X for 30 min. A solution of L-glutamate oxidase or L-glutamate oxidase and glutaminase were applied to the surface and allowed to absorb for 1.5 h. The electrode was used as the working electrode in a three electrode cell with a Ag/AgCl reference electrode and a Pt wire counter electrode. The cell contained 1.5 mM resorcinol and 1.5 M 1,3-phenylenediamine in 50 mM phosphate buffer of pH 6.5 and the working electrode potential was cycled between 0 and 0.6 V at 20 mV/s for 8 cycles to deposit a polymer film to entrap the enzyme. The biosensors were used in a FIA system with a carrier buffer of 0.1 M acetate buffer of pH 5.5 containing 0.12 M NaCl. Sample of mammalian cell culture was injected into the carrier buffer which passed to the glutamate oxidase biosensor, the flow was then switched to the glutamate oxidase and glutaminase biosensor and a second sample was injected into the carrier stream. The current responses were recorded. Calibration graphs were linear for 0-0.2 mM glutamic acid and 0-3 mM glutamine with detection limits of 0.01 and 0.04 mM, respectively.
Glutamine Glutamic acid Sensor Electrode Electrode Buffer Triton X Surfactant

"Application Of Biosensor For Monitoring Galactose Content"
Biosens. Bioelectron. 1996 Volume 11, Issue 10 Pages 1051-1058
E. E. Szabó, N. Adányi and M. Váradi

Abstract: A modified thin-layer enzyme cell (Varadi et al., Ibid., 1993, 8, 339) connected to an electrochemical detector cell was used to prepare an online monitoring system for determining galactose in fermentation broths. The enzyme galactose oxidase was immobilized on to a Teflon sheet; this oxidized galactose and H2O2 was generated which was detected with an amperometric detector. The detector contained a Pt wire (0.4 x 5 mm) as a working electrode and a Ag/AgCl reference electrode. For the enzyme system the optimal flow rate was 0.31 ml/min; optimum conditions were from 32-45°C at pH 6.6. Results compared well with those obtained using a standard UV method.
Galactose Sensor

"Reversible Coupling Of Glucoenzymes On Fluoride-sensitive FET Biosensors Based On Lectin-glucoprotein Binding"
Biosens. Bioelectron. 1996 Volume 11, Issue 12 Pages 1229-1236
R. Köneke, C. Menzel, R. Ulber, K. Schügerl and T. Scheper*, M. Saleemuddin

Abstract: A reloadable affinity biosensor is described in which concanavalin A was immobilized on the surface of a n-Si/SiO2/Si3N4/LaF3 fluoride ISFET and in a second step glycoproteins, namely glucose oxidase and peroxidase, were attached. The enzymes were reversibly coupled and removal was readily effected with acidic acetate solution of pH 1; further enzymes could then be coupled. The biosensor was applied to online monitoring of glucose in a Saccharomyces cerevisiae culture by FIA.
Glucose Sensor Field effect transistor Process monitoring Immobilized enzyme

"A Tryptophan-2-monooxygenase Based Amperometric Biosensor For L- Tryptophan Determination: Use Of A Competitive Inhibitor As A Tool For Selectivity Increase"
Biosens. Bioelectron. 1997 Volume 12, Issue 5 Pages 363-371
A. L. Simoniana, b,*, E. I. Raininaa, P. F. Fitzpatricka and J. R. Wilda

Abstract: A new flow injection amperometric biosensor based on immobilized tryptophan-2-monooxygenase (TMO) has been developed for reagentless L- tryptophan determination. Concentrations of L-tryptophan between 0.1 and 50 mM could be measured with the linear part of the calibration curve between 0.1 and 2 mM. The response time was 30 s and the total analysis time was less than 3 min. The biosensor retained activity for greater than 4 months, when operated daily at 25°C and stored at 8°C. The biosensor was characterized by a relatively high sensitivity to phenylalanine (54% that of L-tryptophan), a modest response to L-methionine (less than 6%) and virtually no response to other amino acids. However, the biosensor selectivity to L-tryptophan could be dramatically increased when indoleacetamide (IA), a competitive inhibitor of TMO, was introduced. In the presence of 10 µM IA, the biosensor response to L-phenylalanine decreased to 7-4% of the unaffected rate for L-tryptophan. In the absence of L-tryptophan and IA the biosensor could be used for L-phenylalanine determination in the concentration range from 1 to 50 mM. The biosensor was successfully used for L-tryptophan determination in nutritional broth.
l-Tryptophan Amperometry Sensor Selectivity

"Multianalyte Biosensors On Optical Imaging Bundles"
Biosens. Bioelectron. 1997 Volume 12, Issue 6 Pages 521-529
Brian G. Healey, Lin Li and David R. Walt*

Abstract: We present an optical biosensor design that expands the utility of enzyme biosensors. These biosensors are fabricated by site-selective photodeposition of analyte-sensitive polymer matrices on optical imaging fibers. These dual-analyte arrays allow for the simultaneous, independent measurement of the analyte of interest and the transducing analyte. The first integrated optical-biosensors using this design have been prepared that allow both the dependent and independent analytes to be measured simultaneously, for example penicillin. and pH (Healey and Walt, 1995) or glucose and O-2 (Li and Walt, 1995). Independent measurement of the transducing analyte allows penicillin or glucose to be quantitated in the presence of a concurrent pH or O-2 change, respectively. Penicillin can be measured in the range 0.25-10.0 mM in the pH range 6.2-7.5 . Glucose can be measured in the range 0.6-20.0 mM in the O-2 range 20-100%. The utility of the sensor design was demonstrated by using the penicillin-dual-analyte biosensor to quantitate penicillin produced during a Penicillium chrysogenum fermentation. (C) 1997 Published by Elsevier Science Limited. 25 References
Glucose Penicillin pH Fluorescence Sensor Sensor Multicomponent Enzyme

"Multichannel Flow Injection Analysis Biosensor System For Online Monitoring Of Glucose, Lactate, Glutamine, Glutamate And Ammonia In Animal Cell Culture"
Biotechnol. Appl. Biochem. 1994 Volume 20, Issue 2 Pages 291-307
Blankenstein G, Spohn U, Preuschoff F, Thommes J, Kula MR

Abstract: The application of a chemiluminometric method for the online monitoring of a hybridoma cell culture is described. Enzyme sensors for glucose, lactate, glutamine, glutamate and ammonia, based on oxidase- catalyzed reactions, were developed and connected to a flow injection analysis (f.i.a.) biosensor. H2O2 produced by the oxidase-catalyzed enzyme reaction was detected by luminol chemiluminescence with a fiber- optic H2O2 biosensor. The system has been used to monitor animal cell cultures. A continuous hybridoma cell cultivation for the production of monoclonal antibodies is presented as an example. It was possible to monitor the bioprocess over a period of 15 days. A complete analysis of all five components could be performed within 42 min. The enzyme sensors were stable during the whole cultivation time without significant loss of activity. The computer-controlled biosensor f.i.a. works with good reliability. The precision for all five components ranged between 2.2 and 4.5%. It was possible to determine glutamine in one step using an anti-interference enzyme reactor. Endogenous glutamate was completely removed up to a level of 0.5 mM.
Glucose Lactate Glutamate Ammonia Glutamine Chemiluminescence Sensor Process monitoring Interferences Multichannel

"Enzyme Electrode For Specific Determination Of L-lysine"
Biotechnol. Bioeng. 1983 Volume 25, Issue 11 Pages 2557-2566
J. L. Romette, J. S. Yang, H. Kusakabe, D. Thomas

Abstract: L-Lysine α-oxidase from Trichoderma viride Y244-2 was immobilized in gelatin and fixed on a pO sensor. The resulting electrode was used for continuous-flow measurement of L-lysine in a fermenter. Dependence of the signal on the O content of the sample was minimized by the enzyme support properties. The measuring range was 0.2 to 4 mM lysine, and the membrane showed good stability over 6 months or for 3000 measurements. Selectivity with respect to other amino-acids was evaluated.
l-Lysine Electrode Immobilized enzyme

"A Flow Injection Analysis System For Fermentation Monitoring And Control"
Biotechnol. Bioeng. 1989 Volume 34, Issue 4 Pages 423-428
Morten Garn *, Marcus Gisin, Christian Thommen, Pierre Cevey

Abstract: An automated analysis system for on-line fermentation monitoring is presented. The modular system consists of an in-line sterilizeable crossflow microfilter, a selection valve that allows injection of sample or standards, a degassing unit, a dilution module, and a FIA manifold with a spectrophotometric UV/VIS detector. In the dilution module samples are conditioned and diluted depending upon concentration of analyte and the working range of the analyzer. Methods for the monitoring of glucose, ethanol, ammonia and phosphate are described. Results from the monitoring of glucose and their use in fermentation control are presented. The maximal analysis frequency is 30 samples per hour including the dilution of 1 : 200. Detection limits are 5 mg/L for ethanol and glucose, 1 mg/L for phosphate and 50 mg/L for ammonia.
Process monitoring Process control

"Online Fermentation Monitoring Using Flow Injection Analysis"
Biotechnol. Bioeng. 1990 Volume 36, Issue 6 Pages 647-651
F. Valero, J. Lafuente *, M. Poch, C. Solà, A. N. Araujo, J. L. F. C. Lima

Abstract: An automated system is described to determine 0.5 to 40 g L-1 of glucose in Candida rugosa batch fermentation. A sample stream from the reactor is passed through a micro-filtration unit (0.45 µm), and 75 µL amounts of the filtrate are injected into a carrier stream via a four-way rotary valve. After passage through a mixing coil, a stream-splitter device is used to adjust the rectilinear working range according to the expected glucose concentration. The glucose in the sample stream is determined by successive reaction with glucose oxidase to give gluconic acid, followed by reaction with H2O2, 4-aminoantipyrine and phenol in the presence of peroxidase. The colored product is monitored spectrophotometrically at 490 nm. The system, which is capable of analyzing 30 samples per h, is computer-controlled and makes use of 0.5 mL of reactor solution and 2.9 mL of enzyme solution for each analysis. A coefficient of variation of 3.22% and a detection limit of 0.2 g L-1 of glucose are reported. Agreement of the results with those by an HPLC method confirms the reliability of the method.
Glucose HPLC Spectrophotometry Automation Filtration Valve Enzyme Computer Method comparison

"Anomeric Specificity Of Glucose Uptake Systems In Lactococcus Cremoris, Escherichia Coli, And Saccharomyces Cerevisiae: Mechanism, Kinetics, And Implications"
Biotechnol. Bioeng. 1992 Volume 40, Issue 1 Pages 137-146
Stig Benthin*, Jens Nielsen, John Villadsen

Abstract: The mechanism and kinetics of the glucose uptake systems of three representative microorganisms are studied during cultivation in a chemostat. The three microorganisms are Lactococcus cremoris, Escherichia coli, and Saccharomyces cerevisiae. Two models describing respectively competitive and independent uptake of the two glucose anomers are tested on experimental data where α- and β-glucose are determined by flow injection analysis after pulse addition of the pure anomers to a chemostat. The very accurate experimental results are used to give a convincingly clear model discrimination for all three microorganisms. The uptake of glucose by S. cerevisiae occurs by a competitive mechanism with preference for α-glucose (Kα = 32 mg/L and K(beta) = 48 mg/L). Surprisingly, the glucose uptake by the two bacteria is shown to be mediated by anomer specific transport systems with no competitive inhibition from the other glucose anomer. This novel finding has not been described in the literature on the phosphotransferase system. In L. cremoris the relative uptake rates of the glucose anomers match the equilibrium composition exactly (36% α- glucose). In E. coli the relative uptake rate of α- glucose at glucose unlimited growth is 26%, which means preference for β-glucose. However, the saturation constants of the two sites in E. coli are Kα = 2 mg/L and K(beta) = 15 mg/L, and a preference for α-glucose is exhibited at very low glucose concentrations. The results are of considerable importance in relation to enzyme based on- line measurements during fermentations as well as to the modeling of glucose limited growth and product formation.
β-d-Glucose α-d-Glucose

"Monitoring Glutamine In Animal Cell Cultures Using A Chemiluminescence Fibre-optic Biosensor"
Biotechnol. Bioeng. 1993 Volume 41, Issue 6 Pages 659-665
M. V. Cattaneo, J. H. T. Luong

Abstract: Cell culture supernatants or standard solution containing glutamine (I) were measured using a flow injection fiber-optic system with flow rates maintained at 1 mL min-1. Samples (13 µL) were injected into 1 mM acetate buffer of pH 5.3 and passed through a column of AG1-X8 anion-exchange resin in order to remove endogeneous glutamate, urate and aspartate. Samples were then mixed with a stream of 20 mM acetate buffer of pH 5.3 containing 100 mM NaCl and passed through an enzyme reactor containing glutaminase and glutamate oxidase immobilized on aminopropyl glass beads. The eluant, containing hydrogen peroxide product, was mixed with 0.125 mM luminol and 30 mM ferricyanide in 200 mM glycine buffer of pH 11.2. The resulting chemiluminescence was monitored in a 100 µL detection chamber with a 3 ft glass fiber-optic bundle coupled to a photomultiplier tube (applied voltage, 950 V) and readout device. The calibration graph was rectilinear from 1 to 100 µM of I, whilst the reproducibility ranged from ±1.1% to ±4.4%.
Glutamine Chemiluminescence Sensor Optical fiber Buffer Resin Interferences Glass beads Immobilized enzyme

"Simultaneous Enzymic - Electrochemical Determination Of Glucose And L-glutamine In Hybridoma Media By Flow Injection Analysis"
Biotechnol. Bioeng. 1993 Volume 41, Issue 10 Pages 964-969
Mark E. Meyerhoff *, Marek Trojanowicz, Bernhard O. Palsson

Abstract: A flow injection system is described (with diagram) that incorporates an immobilized glucose oxidase flow-through amperometric glucose sensor and a flow-through potentiometric ammonium sensor. Serum-free cell culture medium (20 µL) was injected into water and the stream was split. One stream was merged with 0.1 M phosphate buffer solution (pH 6.5) and passed through the glucose sensor. The other stream was mixed with 10 mM Li acetate buffer solution (pH 4.9), passed through a cation-exchange unit operated at 40°C with 0.5 M Li acetate buffer solution (pH 4.9) and passed through a glutaminase enzyme reactor and then to the ammonium sensor. Calibration graphs were rectilinear from 0.03 to 30 mM glucose and from 0.1 to 10 mM L-glutamine (log - linear graph).
Glucose l-Glutamine Potentiometry Electrode Buffer

"Model System For A Fluorimetric Biosensor Using Permeabilized Zymomonas Mobilis Or Enzymes With Protein-confined Dinucleotides"
Biotechnol. Bioeng. 1993 Volume 42, Issue 3 Pages 387-393
O. Thordsen, S. J. Lee, A. Degelau, T. Scheper *, H. Loos, B. Rehr, H. Sahm

Abstract: A biosensor for the determination of glucose, fructose, gluconolactone and sorbitol is described. The sensor used either permeabilized Zymomonas mobilis or glucose - fructose oxidoreductase isolated from Z. mobilis. The sensing element was confined to a small measuring chamber fitted with a membrane. This was incorporated into a FIA system. The principle of the system was that the NADP(H) cofactor molecule, which is confined in a protein complex, would be oxidized or reduced during enzymatic reactions, and could be made to undergo fluorescence by excitation at 360 nm (measurement at 450 nm). Using this sensor, changes in fluoresence intensity were related to substrate concentration and the range of sensitivity found to be from 0.001 to 100 g L-1 of analyte. The system was shown to be capable of monitoring bioprocesses by using it to determine the relevant analytes in samples from a Pseudomonas pseudoflava cultivation.
Glucose Fructose Gluconolactone Sorbitol Fluorescence Sensor Enzyme

"Online Monitoring Of Monoclonal Antibodies In Animal Cell Culture Using A Grating Coupler"
Biotechnol. Bioeng. 1993 Volume 42, Issue 11 Pages 1287-1292
R. Polzius *, F. F. Bier, U. Bilitewski, V. Jäger, R. D. Schmid

Abstract: A grating coupler was used for the online determination of monoclonal antibodies produced in a perfused animal cell bioreactor. The device was connected with the culture vessel via a flow injection analysis (FIA) system, which was controlled automatically. Specific antimouse IgG antibodies were immobilized on the surface of the sensor-chip. After injection of the sample, the binding of mouse IgG was observed in real time. The regeneration of the binding sites of the immobilized antibodies using an acidic solution allowed the online detection of produced monoclonal antibodies in the range of 10 to 150 µg/mL. In contrast to other techniques coupled to bioprocesses, the developed method represents a regenerable direct immunosensor. Results were compared with standard ELISA techniques (off-line) and a competitive immunochemical assay using the grating coupler (off-line).
Immunoassay Immobilized enzyme Method comparison

"Semi-online Analysis For Fast And Precise Monitoring Of Bioreaction Processes"
Biotechnol. Bioeng. 1996 Volume 52, Issue 2 Pages 237-247
L. H. Christensen *, J. Marcher, U. Schulze, M. Carlsen, R. W. Min, J. Nielsen, J. Villadsen

Abstract: Monitoring of substrates and products during fermentation processes can be achieved either by online, in situ sensors or by semi-online analysis consisting of an automatic sampling step followed by an ex situ analysis of the retrieved sample. The potential risk of introducing time delays and signal bias during sampling makes it necessary to distinguish between real-time, online, in situ methods and semi-online analysis. In addition, semi-online analyzers are often mechanically complex-a circumstance which has to be given special attention during their industrial use on a routine basis. This review on semi-online analysis will focus both on the dynamics and precision of aseptic sampling devices and on the performance of flow injection analysis (FIA) and sequential injection analysis (SIA), especially with regard to their robustness when used in industry.
Review Process monitoring Sequential injection

"Online Monitoring Of Glucose In Mammalian Cell Culture Using A Flow Injection Analysis (FIA) Mediated Biosensor"
Biotechnol. Bioeng. 1997 Volume 55, Issue 3 Pages 497-504
Keith B. Male, P. Octavian Gartu, A. Amine Kamen, John H. T. Luong

Abstract: A flow injection analysis (FIA) biosensor system has been developed for on-line determination of glucose during mammalian cell cultivation. The culture sample was peristaltically withdrawn from the bioreactor and after cell separation by a steam sterilizable ceramic microfilter, the filtrate was continuously fed to the FIA mediated-biosensor system at 4 mLh-1, whereas the cell-containing retentate was recirculated to the bioreactor. In the amperometric biosensor system, glucose oxidase was covalently immobilized onto a preactivated nylon membrane and attached to the sensing area of a platinum working electrode. The enzyme reaction was coupled with the mediator 1,1-dimethylferricinium (DMFe+)-cyclodextrin inclusion complex to recycle the reduced glucose oxidase to its original active state. 1,1-Dimethylferrocene (DMFe) was then reoxidized to DMFe+ at the surface of the platinum electrode poised at + 0.15 V vs silver/silver chloride. The FIA mediated-biosensor was linear up to 6 mM glucose, with a detection limit of 0.1 mM, and possessed excellent reproducibility (± 0.4 %, 95 % confidence interval) over 123 repeated analyzes during a 62 h continuous operation. The immobilized glucose oxidase was stable for up to 7 days when applied to glucose measurement during 5-10 day fed-batch cultivation of 293S mammalian cells. The results obtained from the mediated-biosensor system compared well with the hexokinase and HPLC data.
Glucose Sensor Process monitoring

"A Novel Feeding Strategy For Enhanced Plasmid Stability And Protein-production In Recombinant Yeast Fedbatch Fermentation"
Biotechnol. Bioeng. 1997 Volume 56, Issue 1 Pages 23-31
Chinyuan Cheng, Yu Liang Huang, Shang-Tian Yang

Abstract: A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of β-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.
Cells Glucose Computer Process monitoring

"Optimal Design Of An Enzymatic Reactor For Flow Injection Analysis"
Biotechnol. Prog. 1993 Volume 9, Issue 5 Pages 473-480
M. Poch, J. L. Montesinos, M. del Valle, J. Alonso, A. Araujo, and J. L. F. C. Lima

Abstract: A simulation procedure for the optimization of enzymatic reactors used in sandwich flow injection systems is evaluated. The system is modeled as a plug-flow reactor with axial dispersion. To calibrate it, dispersion coefficients can be evaluated using residence time distribution techniques; meanwhile, enzymatic kinetics must be determined for the system considered, according to the values of the substrate conversion attained. The model has been linked to an optimization routine based on the Powell algorithm. The proposed approach has been evaluated in a system performing simultaneous determinations of glucose and glycerol, considered the common carbon sources in a fermentation process.
Glucose Glycerol Modeling Enzyme Optimization Plug flow Powell

"Control Of Long-term Perfusion Chinese Hamster Ovary Cell Culture By Glucose Auxostat"
Biotechnol. Prog. 1996 Volume 12, Issue 1 Pages 100-109
Konstantin B. Konstantinov, Yeong-shou Tsai, Donald Moles, and Ricaredo Matanguihan

Abstract: The strategies for control of the feed rate in high-density perfusion cultures of animal cells are limited to several simple schemes. While in an industrial environment simplicity is seen as a major advantage, the need for more elaborate closed-loop control methods that can improve process stability in long-term continuous cultures is also well understood. What has prevented the application of the advanced control strategies known from theory is the lack of reliable real-time information that can be used to close the feedback loop. Among the variables that are appropriate for direct feedback control of the perfusion rate, high priority should be given to the glucose concentration. Unlike some other environmental variables, such as dissolved oxygen and pH, it provides unambiguous information which facilitates the selection of the right feed rate. The present paper describes the application of a closed loop control scheme, known as a 'glucose-stat', to the long-term cultivation of Chinese hamster ovary cells in a high-density (35-40 million cells/mL) perfusion process. The monitoring and control system worked successfully for more than 2.5 months without any signs of performance degradation. In targeting industrial application, issues such as reliability, sterility, and accuracy, are given high priority. The implementation of the glucose monitoring system, which is the main part of the control complex, is addressed in details. The performance of the perfusion culture was evaluated at four different glucose set points, providing essential information about process optimization. It became evident that the perfusion culture was operated in the so-called 'high-gain' zone (where the system is highly sensitive to the dilution rate), which justifies the application of a feedback control. The online glucose concentration was also used by an embedded expert system which drove the process through the batch and the perfusion phase, achieving total computer control of the feed rate. In summary, the proposed glucose monitoring and control technique proved to be a reliable biotechnology tool which can be applied with confidence at an industrial scale to either microbial or mammalian cell cultures.
Glucose Spectrophotometry Biotechnology Process monitoring Closed loop Optimization Perfusion

"Assay Of β-galactosidase Activity By Flow Injection Analysis"
Biotechnol. Techniq. 1991 Volume 5, Issue 5 Pages 389-392
J. Cairó, M. Vidal, A. Villaverde, F. Valero, F. J. Lafuente and C. Solà

Abstract: A novel method to analyze β-galactosidase by Flow Injection Analysis is presented with a linear working range extended to at least 2150 U/mL, being the detection limit 25 U/mL with 55 samples per hour frequency and a RSD of 0.54% versus 2.4% obtained by manual assay. The method was tested with optimal results with samples from Escherichia coli cultures producing β-galactosidase.
Enzyme, galactosidase Spectrophotometry Buffer Heated reaction pH

"Simultaneous Online Monitoring Of Intracellular β-galactosidase Activity And Biomass Using Flow Injection Analysis In Escherichia Coli Batch Fermentations"
Biotechnol. Techniq. 1992 Volume 6, Issue 3 Pages 213-218
Valero F., Lafuente F. J., Solà C., Benito A., Vidal M., Cairó J. and Villaverde A.

Abstract: Fermentation sample was sonicated at 4°C for 45 s and subjected to flow injection analysis after mixing with phosphate buffer solution (pH 7.0) containing 2-β-mercaptoethanol. The solution was mixed with 2-nitrophenyl-β-D-galactopyranoside at 47°C before addition of 1 M Na2CO3 and 0.5% Na dodecyl sulfate and detection at 420 nm. Biomass determination was achieved offline at 550 nm. The limit of detection was 25 iu mL-1 of β-galactosidase and the calibration graph was rectilinear for 5100 iu mL-1. The coefficient of variation was 1.5%. A novel method to monitor online intracellular β-galactosidase activity and biomass simultaneously, using flow injection analysis (FIA), was developed. The automatic ultrasonic cell disruption and FIA anal. allow the processing of 10 samples/h with a wide and variable linear working range of β-galactosidase activity and biomass and a max. relative standard deviation of 1.5%. The system was optimized by monitoring biomass and intracellular β-galactosidase activity in E. coli batch fermentation
Enzyme, galactosidase Spectrophotometry Process monitoring

"An Improved FIA System For Measuring α-amylase In Cultivation Media"
Biotechnol. Techniq. 1994 Volume 8, Issue 7 Pages 479-482
Morten Carlsen, Jacob Marcher, and Jens Nielsen

Abstract: Samples (60 µL) were injected into a carrier stream (1.3 ml/min) of water containing 0.1 g/l of BSA and 0.1 g/l of CaCl2.H2O, which merged with a stream (1.3 ml/min) of starch reagent of pH 5 (20 g of soluble starch in 1 l of water containing 2 g of potassium sorbate, diluted 20-fold with water containing 12.1 g/l of Tris and 9.8 g/l of maleic acid anhydride). After passing through a reaction coil (375 µL; 0.8 mm i.d.; 40°C) the stream merged with a stream (1.3 ml/ml) of I2 solution prepared by diluting 9 mL of stock solution (H2O containing 4 g/l of I2 and 12.6 g/l of KI) to 1 l with water. After passing through a mixing coil (500 µL; 0.8 mm i.d.; 40°C) the absorbance was measured at 660 nm. The calibration graph was linear from 0.01-0.1 units/ml of α-amylase (1 unit breaks down 5.26 g of starch/h; cf. Hansen, Anal. Chim. Acta, 1984 158, 375) and the RSD was 1%. The method was applied to Aspergillus oryzae cultivation media diluted 2-fold; it gave a detection limit of ~0.02 units/ml.
α-Amylase Spectrophotometry

"Potentiometric Sensor For On Line Glucose Determination"
Biotechnol. Techniq. 1996 Volume 10, Issue 11 Pages 867-870
R. O. M. Folly, B. Valdman, F. Valero and C. Solà

Abstract: An enzymatic biosensor, with a flow-through sensor for continuous monitoring of glucose coupled to a flow injection analysis (FIA) sampling line has been used to determine glucose over the range of 5 to 120 g/l. without sample dilution. The system can analyze 10 samples per hour. Calibration and reproducibility analysis have shown a good linearity and excellent results as compared to a commercial glucose analyzer..
Glucose Potentiometry Sensor Method comparison

"Online Monitoring Of Mammalian-cell And Yeast Fermentations With A Commercial Biochemical Analyzer"
Biotechnol. Techniq. 1997 Volume 11, Issue 6 Pages 427-430
N. Vriezen and J.P. van Dijken

Abstract: A commercial analyzer was tested for online monitoring of fermentations. A new sample block was constructed to effectively degas the fermentation broth. The robust analyzer accurately measured glucose up to 110 mmol/l and lactate up to 21 mmol/l at a frequency of 1 measurement per 2 minutes directly in suspensions of mammalian and yeast cells. 8 References
Glucose Lactate Process monitoring Suspension

"Process Monitoring Of Acetic Acid In Escherichia Coli Cultivation Using Electrochemical Detection In A Flow Injection System"
Biotechnol. Techniq. 1997 Volume 11, Issue 9 Pages 683-687
X.-J. Tang, A. Tocaj, O. Holst and G. Johansson

Abstract: A flow injection system for determination of acetic acid in Escherichia coli cultivations with electrochemical detection based on immobilized acetate kinase (AK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) was developed to cope with the problems related to measurements under process conditions such as interferences from pyruvate, drift of electrode baseline and making the cultivation medium and reagents compatible. The results obtained by flow injection analysis were compared with those obtained with an enzymatic test kit.
Acetic acid Electrode Process monitoring Interferences

"Measurement Of Intracellular Adenosine Triphosphate Using An Online FIA System For Determination Of Micro-organism Cells Number"
Bunseki Kagaku 1989 Volume 38, Issue 11 Pages T183-T186
Haketa, Y.;Motohashi, R.;Kajiwara, K.;Matsunaga, N.;Gonda, K.

Abstract: A flow injection analysis system was developed for the determination of ATP (free and intracellular) in micro-organisms, e.g., yeast and E. coli cultures, by a bioluminescent reaction using luciferin and Photinus-luciferin 4-monooxygenase (ATP-hydrolysing). Luminescence response was rectilinear from 1 nM to 10 µM-ATP. There was good correlation between intracellular ATP concentration. and viable cell numbers (r = 0.992). Results agreed well with those by conventional methods.
Bioluminescence Method comparison

"Multivariate Approach For The Simultaneous Determination Of Total Biomass And Glucose From A Yeast Fermentation By Sequential Injection Analysis"
Chem. Anal. 1995 Volume 40, Issue 4 Pages 455-471
Baxter, P.J.;Christian, G.D.;Ruzicka, J.

Abstract: The sequential injection system (diagram given) was incorporated into the circulation line of the fermentation unit. Samples (50 µL) of fermentation liquor were carried to a flow cell (Analyst, 1994, 119, 1807) in Trinder reagent (2 ml/min). The flow and stirring were stopped for 3.5 min, resumed for 20 s to dilute the sample, then stopped again while spectra were recorded from 402-800 nm with 2 nm resolution. The flow cell was then rinsed with 6 mL carrier. The assay time was 8 min. A calibration set of samples in which glucose was determined using a manual Trinder assay, and biomass was determined by the dry weight method, was used to calculate linear partial least squares calibration models. The models had excellent predictive ability for 1 g/kg biomass (average errors 3.68-6.15%). The predictive ability for 10 mg/dl glucose was less good, but identification of pulse spikes in the fermentation broth was possible.
Biomass Glucose Spectrophotometry Calibration Chemometrics Sequential injection Simultaneous analysis Partial least squares

"FIA System For Fast Glucose Measurement In Bioprocesses"
Chem. Ing. Tech. 1998 Volume 70, Issue 3 Pages 297-299
Karsten Schöngarth, Priv.-Doz. Dr. Bernd Hitzmann, Karl Friehs

Abstract: A flow injection analysis system is presented for the determination of glucose concentration. online in bioprocesses without a sampler. It is based on the injection of a glucose oxidase solution into the fluid and the determination of the O consumption in the glucose turnover. In a culture of Escherichia coli the measuring error was ≤5% compared to the off-line values. The measuring time was 47 s.
Glucose Process monitoring

"An Expert System As A Tool For The Specification And Intelligent Control Of A Flow Injection Analysis System"
Chemom. Intell. Lab. Syst. 1993 Volume 21, Issue 2-3 Pages 243-247
M. Perisa,*, A. Maquieiraa, R. Puchadesa, V. Chirivellab, R. Orsb, J. Serranob and A. Bonastreb

Abstract: A conceptual three-level system is described. The highest is represented by the expert system, which passes decisions to and receives information from the middle level. The middle level consists of a set of programs that serve the expert system, and passes orders to and receives results from the FIA system at the lowest level. The user interacts routinely with middle level by passing parameters to it and receiving reports from it. Both a more detailed block structure of the system and a pictorial scheme showing the connections to the different devices are presented, together with the operating flow diagram of the expert system. The performance of the whole system is illustrated by the monitoring of total acidity, pH, reducing sugars and ethanol in a fermentation broth.
Acidity pH Sugars, reducing Ethanol

"Study On Multiple-enzyme Electrode For Sucrose Determination"
Shengwu Gongcheng Xuebao 1991 Volume 7, Issue 4 Pages 339-344
Hu W, Zhang X, Zhang X, Hu S.

Abstract: Invertase (INV), mutarotase (MUT), glucose oxidase (GOD) and BSA were coimmobilized via glutaraldehyde-bridged covalent bonding, and directly absorbed on the teflon membrane. This membrane was covered with a nylon mesh and placed over an oxygen electrode. An enzyme electrode for flow injection analysis system (EFIA) was adopted. The optimum enzyme composition (IU) for immobilization on the teflon membrane of INV-MUT-GOD was found to be in the ratio 72:48:2.4, with a recovery activity INV-MUT of more than 42.9%. pH 5.8-6.5 was the most suitable range of acidity for the sensor activity. The optimum temperature was 35-45°C. The system exhibited good linearity in the range of 5 x 10^-4 approximately 10^-1 M sucrose (kinetic method) and 10^-5 approximately 2 x 10^-3 M sucrose (steady state method), in short response time (20 seconds for kinetic method, 2 minutes for steady state method), CV = 1.7% (kinetic method). The sensor had been used for determining sucrose concentration in fermentation broth, with an average recovery rate of 98%. The interference caused by the presence of glucose derived from decomposition of sucrose was eliminated by calibration with a GOD sensor. No significant loss of the enzyme electrode activity was observed after 120 hours of the continuous-flow of fresh 1 mM sucrose. The multiple-enzyme membrane showed a relatively long lifetime (compared with 14 hours as reported previously) and good storage stability (30 days, stored in distilled water at 4°C).
Sucrose Electrode Heated reaction Immobilized enzyme Interferences Kinetic pH Teflon membrane Steady state

"A Flow Dialysis Cell For Online Measurement Of Glucose In Fermentation Broth"
Shengwu Gongcheng Xuebao 1993 Volume 9, Issue 3 Pages 210-215
Zhang Xianen Hu Weiping Zhao Guoqiang Zhang Zhiping Zhang Xiaomei Gui Yiqun Zhang Xin Wei Hongping

Abstract: A plate flow dialysis cell, employing an acetate cellulose ultrafiltration membrane, was designed and tested for applicability to online sampling in fermentation. The glucose contained in effluents of both sample stream channel (Channel A) and carrier stream channel (Channel B) was determined with an enzyme electrode flow injection analysis system. Glucose penetration rate was defined as Rp, Rp = Gb/(Ga + Gb), here Ga and Gb are glucose concentrations of effluent of channel A and B respectively. A higher penetration rate was obtained when using phosphate buffer (0.01 M) as carrier solution instead of using distilled water. Operating pressure differences, temperature and residential time affected glucose penetration. Under the condition of 0.02 MPa pressure differences and 0.23 min of residential time (12.8 mL/L), Rp was about 12% in the range of 10^-70 mM with CV <4%. When sample stream was yeast broth, the glucose penetration rate Rp was stable for at least 48 hr. Good relationship was observed between online and off-line sampling for glucose determination in yeast fermentation, the correlation coefficient r was 0.985.
Glucose Dialysis

"The Glutamate Biosensor And Its Application To Flow Injection Analysis System"
Shengwu Gongcheng Xuebao 1994 Volume 10, Issue 2 Pages 83-89
Ye Bangce Li Qingshan Li Yourong Yu Juntang

Abstract: A micro-enzyme electrode was fabricated by cross-linking L-glutamate oxidase with glutaraldehyde on aminopropyl-platinized platinum wire. A flow injection analysis system with glutamate sensor was used for L- glutamate determination. The peak current is linearly related to the L- glutamate concentration in the range of 0.02-2.0 mM, with good performance, accuracy (CV = 0.4%), fast response (< 60s), and stability (> 20 days). The system was applied to determine the concentration of L- glutamate in a fermentation broth. The recovery rate was in the range of 98.7-107.5%.
l-Glutamate Sensor Electrode Apparatus

"Determination Of L-glutamate Using Flow Injection Analysis With Immobilized L-glutamate Oxidase Reactor"
Shengwu Gongcheng Xuebao 1994 Volume 10, Issue 4 Pages 351-355
Li Qingshan Ye Bangce Zhang Siliang Yu Juntang

Abstract: L-Glutamate oxidase (GOD) and horseradish peroxidase (HRP) were covalently coupled on alkylamine pretreated controlled pore glass (CPG) by means of glutaraldehyde. The immobilized enzymes were packed into a teflon tube and used in flow injection analysis (FIA) system for L- glutamate determination. A good linearity range was obtained at 0.1-2.0 mM, and the coefficient of variation was 0.7% (n = 8). More than 80 samples were measured within an hour. The stability of the immobilized GOD reactor was good, retaining 50% of its initial activity after 4 months storage in buffer at 4°C. When the concentration of L- glutamate remained lower than 2.5 mM, the determination of L-glutamate in this system was not affected by pH and temperature within the range of 6.0-8.0 and 20-35°C, respectively. The system was applied to determine L-glutamate in broth samples during L-glutamate fermentation and good correlations were achieved between results obtained with the FIA system, L-glutamate oxidase kit and Warburg's method.
l-Glutamate Immobilized enzyme Method comparison Controlled pore glass

"Rapid HPLC Determination Of Narasin In Fermentation Broth"
Chromatographia 1991 Volume 31, Issue 5-6 Pages 277-280
F. L. Neely

Abstract: A rapid HPLC assay with post-column derivatization with vanillin was developed for the determination of narasin (I) in fermentation broth. Separation was carried out on an ODS (3 µm) column (5 cm x 4.6 mm o.d.) with a mobile phase (2 mL min-1) of 93% methanol - 7% 0.1 M K2HPO4 (pH 4). The post-column reagents (viz, methanolic H2SO4 and vanillin) were mixed (1 mL min-1) at a tee fitting before mixing with the column eluate. Detection was at 520 nm. Response was rectilinear from 1 to 300 ppm of I. The coefficient of variation were 2% and recoveries were 101 to 120%. No matrix interference was observed.
Narasin HPLC Interferences Post-column derivatization

"Online Monitoring Of Fermentation Processes By Ultrafiltration And Column Liquid Chromatography"
Chromatographia 1992 Volume 33, Issue 11-12 Pages 525-532
N. C. van de Merbel, I. M. Kool, H. Lingeman, U. A. Th. Brinkman, A. Kolhorn and L. C. de Rijke

Abstract: The cited system (diagram given) was used to monitor a E. coli batch culture, with brain - heart infusion, as a complex fermentation mixture at 37°C, for lactose, fructose and glucose. The fermentation broth was filtered by continuously pumping through a hollow-fiber ultrafiltration module at 75 mL min-1. The retentate was pumped back to the fermenter and the filtrate transferred online to the HPLC system. Once the 35 µL injection loop was filled, a six-part valve was switched and the filtrate stream was returned to the fermenter. HPLC was on a column (30 cm x 0.375 in.) packed with carbohydrate cation-exchange resin operated at 90°C with water as mobile phase (0.5 mL min-1). For sugar concentration. 0.1 g L-1 post-column derivatization was used and p-aminobenzoic acid hydrazide was added (0.4 mL min-1) for derivatization in a stainless-steel reaction coil (3 m x 0.54 mm) followed by UV detection at 190 or 410 nm. Above 0.1 g L-1 of analyte was detected by differential refractometry. For the determination of non-reducing sugars a solid-phase catalytic reactor was used. Effects of several parameters on the ultrafiltration module were studied. Results gave good correlation with an off-line method.
Lactose Fructose Glucose HPLC Spectrophotometry Refractometry Method comparison Heated reaction Post-column derivatization

"Automated Flow Injection Procedures For The Determination Of Hydrolytic Enzymes In Bioreactor Preparations"
Chromatographia 1992 Volume 33, Issue 1-2 Pages 49-52
H. F. Pfeiffer, H. Waldhoff, P. J. Worsfold and I. R. C. Whiteside

Abstract: The cited procedures were used for the determination of amylase, xylanase, polygalacturonase and protease, acting on macromolecular substrates. Peptides produced by the protease were derivatized with trinitrobenzene sulfonic acid; for the other enzymes, the reducing sugars produced were derivatized with 4-aminobenzoylhydrazide. Two flow injection manifold designs were proposed; one optimizes sample throughput at a high sensitivity level by incorporating several parallel incubation coils, the other minimizes sample volume at a low sensitivity level and facilitates automation; detection ranged from 0.1 to several 100 iu mL-1. Automated spectrophometric flow injection (FI) procedures for the quant. determination of the hydrolytic enzymes amylase, xylanase, polygalacturonase, and protease, acting on macromol. substrates, are described. The peptide produced by the protease are derivatized with trinitrobenzenesulfonic acid. For the other enzymes, the reducing sugars produced are derivatized with p-aminobenzoylhydrazide. The FI manifold design allows the choice of any required detection range between 0.1 and several hundred U/mL. Two FI manifold designs are proposed; 1 optimizes sample throughput at a high sensitivity level by incorporating several parallel incubation coils, the other minimizes sample volume at a low sensitivity level and facilitates automation. The instrumentation is largely based on commercial HPLC equipment.
α-Amylase Enzyme, xylanase Enzyme, polygalaturonase Enzyme, protease Spectrophotometry Optimization

"Fermentation Monitoring"
Curr. Opin. Biotechnol. 1992 Volume 3, Issue 1 Pages 40-44
Gokaraju K. Raju and Charles L. Cooney

Abstract: Fermentation monitoring continues to be the focus of much research. Over the last year, important strides were made in improving bioprocess monitoring using NADH fluorescence, viscosity, affinity techniques, enzyme and microbial sensors, calorimetry, flow injection analysis and bioluminescence. Better fermentation monitoring is important for improving understanding, operation, development and control of the process. We expect progress in these areas of research to continue. In addition, we highlight some non-conventional approaches.
Biotechnology Bioluminescence Review Process monitoring Viscosity

"Biosensors For Fermentation Control"
Curr. Opin. Biotechnol. 1993 Volume 4, Issue 2 Pages 183-187
Marie-No&euml;lle Pons

Abstract: Over the past year, biosensor development has been an active area of research. The actual application of biosensors in process monitoring and control is limited to a few cases, mainly as a result of difficulties relating to their long-range stability and their sensitivity to interfering compounds. Steam sterilization is no longer a problem though, as the great majority of sensors are part of flow injection analysis systems.
Sensor Interferences Process monitoring

"Online Monitoring Of Monoclonal Antibody Formation In High Density Perfusion Culture Using FIA"
Cytotechnology 1991 Volume 6, Issue 1 Pages 55-63
Christel Fenge, Elisabeth Fraune, Ruth Freitag, Thomas Scheper and Karl Sch&uuml;gerl

Abstract: An automated flow injection system for online analysis of proteins in real fermentation fluids was developed by combining the principles of stopped-flow, merging zones flow injection analysis (FIA) with antigen-antibody reactions. IgG in the sample reacted with its corresponding antibody (a-IgG) in the reagent solution. Formation of insoluble immunocomplexes resulted in an increase of the turbidity which was determined photometrically. This system was used to monitor monoclonal antibody production in high cell density perfusion culture of hybridoma cells. Perfusion was performed with a newly developed static filtration unit equipped with hydrophilic microporous tubular membranes. Different sampling devices were tested to obtain a cell-free sample stream for online product analysis of high molecular weight (e.g., monoclonal antibodies) and low molecular weight (e.g., glucose, lactate) medium components. In fermentation fluids a good correlation (coefficient: 0.996) between the FIA method and an ELISA test was demonstrated. In a high density perfusion cultivation process mAb formation was successfully monitored online over a period of 400 h using a reliable sampling system. Glucose and lactate were measured over the same period of time using a commercially available automatic analyzer. based on immobilized enzyme technology.
Proteins Spectrophotometry Turbidimetry Hydrophilic membrane Merging zones Microporous membrane Stopped-flow Tubular membrane Immobilized enzyme Perfusion

"Determination Of Ammonium And L-glutamine In Hybridoma Cell Cultures By Sequential Flow Injection Analysis"
Cytotechnology 1994 Volume 14, Issue 3 Pages 177-182
Carles Campmaj&oacute;, Jordi Joan Cair&oacute;, Anna Sanfeliu, Esteve Mart&iacute;nez, Salvador Alegret and Francesc G&oagrave;dia

Abstract: A flow injection analytical system based on a gas diffusion membrane module for ammonia and an ammonium flow-through potentiometric detector has been set up for measurement of L-glutamine and ammonium ions in hybridoma cell cultures. The main feature of the system is that the same basic analytical concept and equipment is used in both measurements, the only difference being for the determination of L- glutamine, in which the sample flows through an immobilized glutaminase cartridge. The conditions to enable the performance of both analysis consecutively, avoiding potential interferences by unwanted deamination of other compounds in the samples, have been determined. Finally, the proposed system has been compared with reference analytical methods for batch hybridoma cell culture experiments.
Ammonium l-Glutamine Potentiometry Gas diffusion Method comparison Immobilized enzyme Interferences

"Automated Imunoanalysis Systems For Monitoring Mammalian Cell Cultivation Processes"
Cytotechnology 1994 Volume 15, Issue 1-3 Pages 259-269
Birgitt Schulze, Cornelia Middendorf, Martin Reinecke, Thomas Scheper, Wolfgang No&eacute; and Michael Howaldt

Abstract: Two different automated immunoanalysis systems are presented. Both are based on the principles of flow injection analysis and were developed to provide reliable, rapid monitoring of relevant proteins in animal cell cultivation processes. One system uses a turbidimetric analysis, and the other employs a heterogeneous chemistry with immobilized immunocomponents. For both systems, the analysis time is in the range of a few minutes, and a complete analysis cycle, including triplicate analyzes and various washing steps, is in the range of 20-30 minutes. Samples from cultivation processes can be analyzed directly without dilution. Quantitation of proteins such as rt-PA or monoclonal antibodies can be performed over an analyte concentration range of 1- 1000 mg/L. Both systems were compared to conventional ELISA assays on microtiter plates. The turbidimetric analysis system also included a biosensor for simultaneous glucose determination.
Glucose Immunoglobulin G Turbidimetry Immunoassay Sensor Simultaneous analysis

"Development Of A Turbidimetric Immunoassay For Online Monitoring Of Proteins In Cultivation Processes"
Enzyme Microb. Technol. 1991 Volume 13, Issue 12 Pages 969-975
Ruth Freitag, Thomas Scheper and Karl Sch&uuml;gerl

Abstract: An online assay for a thermostable pullulanase and antithrombin III (AT III) is described. The assay is based on the formation of aggregates between the protein to be measured and antibodies raised against this protein. Assay automation was achieved by utilizing the flow injection analysis (FIA) principles. The apparatus, a stopped-flow, merging-zone manifold, is described in detail. Since the reaction used in an FIA system does not have to reach equilibrium, it was possible to reduce the time for an assay cycle to 2.5 min. A method for simulating cultivation conditions was developed for assay optimization. Using this method, a detection limit of 1 mg L-1 together with a standard deviation of 1.5 was found. A sandwich ELISA was used as reference assay in the case of AT III and an enzymatic activity assay in the case of pullulanase. Correlation coefficients of 0.988 (AT III) and 0.976 (pullulanase) were determined. The turbidimetric assay was successfully used for pullulanase monitoring during a 240-h cultivation of Clostridium thermosulfurogenes.
Proteins Immunoassay Turbidimetry Merging zones Stopped-flow Optimization Process monitoring

"Uses Of β-galactosidase Tag In Online Monitoring Production Of Fusion Proteins And Gene Expression In Escherichia Coli"
Enzyme Microb. Technol. 1993 Volume 15, Issue 1 Pages 66-71
A. Benito, F. Valero, J. Lafuente, M. Vidal, J. Cairo, C. Sol&agrave; and A. Villaverde

Abstract: A simple method for monitoring and quantifying automatically the production by fermentation of β-galactosidase fusion proteins, making use of the remaining activity of the β-galactosidase part, is considered. A hybrid protein carrying the major antigenic domain of foot-and-mouth disease virus C1 joined at the N-terminus of β- galactosidase has been expressed in Escherichia coli. The yield of the chimeric protein has been monitored by flow injection analysis (FIA) during batch fermentations at laboratory scale, and a high correlation between values of product concentration from FIA and from immunological quantizations has been obtained. Because of the possibility of employing FIA in large-scale experiments, and the high sampling frequency, versatility, and reproducibility offered by this method, we propose FIA as a general, simple, quick, flexible, and reliable instrument for both monitoring the yield of recombinant proteins produced industrially, and performing basic research at laboratory scale.
Proteins Process monitoring

"Novel Flow Injection Analysis Amperometric Biosensor System For The Determination Of Glutamine In Cell-culture Systems"
Enzyme Microb. Technol. 1993 Volume 15, Issue 1 Pages 26-32
K. B. Male, J. H. T. Luong*, R. Tom and S. Mercille

Abstract: A flow injection analysis (FIA) biosensor system has been developed for determining glutamine in insect cell and in murine hybridoma cell cultures. Glutamate oxidase and glutaminase, respectively, were covalently immobilized onto porous aminopropyl glass beads to form an immobilized enzyme column. The hydrogen peroxide produced by the enzyme reactions was detected by an amperometric electrode (platinum vs. silver/silver chloride) posied at +0.7 V. Among several anion exchange resins tested, endogenous glutamate was completely adsorbed onto acetate anion exchange resins. Such an ion exchanger also effectively adsorbed aspartic acid, uric acid, ascorbic acid, and acetaminophen. The FIA biosensor was linear up to 1 mM glutamine, with a lower detection limit of 10 µM, and possessed good reproducibility (relative error of ± 1.2%). Each analysis could be performed in 3.5 min including sampling and washing with a corresponding throughput of 17 h-1. The anion exchange column could be operated continuously for 12 h for 200 analyzes with diluted cell culture. The immobilized enzyme column was stable for at least 500 repeated analyzes without significant loss of activity. When the biosensor system was applied to glutamine measurement in insect cell and mammalian cell cultures, the results obtained compared well with those of HPLC.
Glutamine Amperometry Sensor

"Monitoring Of The Glucose Concentration During Microbial Fermentation Using A Novel Mass-producible Biosensor Suitable For Online Use"
Enzyme Microb. Technol. 1997 Volume 20, Issue 8 Pages 590-596
Ibtisam E. Tothill, Jeffrey D. Newman, Stephen F. White and Anthony P. F. Turner

Abstract: A flow injection analysis system was combined with a mass producible, disposable biosensor and was used to monitor glucose concentrations during several microbial fermentations. The biosensor was manufactured using thick film, screen printing technology. Unlike previous devices of this type, the biosensor was designed to make multiple measurements over extended periods rather than to operate as a ''one-shot'' sensor. One yeast, one lactic acid, and three E. coli bioreactor cultures were tested using either defined or complex media. Results from the sensor were compared with a standard spectrophotometric test kit. In samples containing glucose concentrations within the range of the biosensor and the test kit, good correlations were obtained between the two methods. In addition to glucose, microbial growth and pH were recorded. (C) 1997 by Elsevier Science Inc. 17 References
Glucose Sensor Amperometry Electrode Process monitoring Method comparison Interferences

"Micro-enzyme Electrode And Flow Injection Analysis System For Fermentation Monitoring"
Fenxi Huaxue 1993 Volume 21, Issue 12 Pages 1449-1451
Ye, B.C.;Xie, X.Z.;Li, Y.R.

Abstract: A platinized electrode, treated with glucose oxidase and 2% glutaraldehyde, showed a linear response to glucose up to 10 mM; the 100% response time was 20 s, and other sugars produced little interference. This electrode was incorporated into the FIA system (diagram given) for the online monitoring of glucose in fermentation processes. The analysis rate was 45 runs/h.
Glucose Electrode Enzyme Interferences

"A New Approach To Enhance The Useful Lifetime Of A Glucose Oxidase-mediated Electrode"
Fenxi Huaxue 1995 Volume 23, Issue 4 Pages 426-429
Li, Q.S.;Jiang, B.T.;Zhang, S.L.;Yu, J.T.

Abstract: Using a flow injection analysis system, the leaching of mediators from the glucose biosensor was investigated by selecting less paraffin-soluble mediators, such as tetrathiafulvalene, tetracyanoquinodimethane and 1,1'-dimethylferrocene, or by coating DEAE-dextran on the electrode surface. Results indicated that electrodes coated with DEAE-dextran had a longer lifespan, lower background current and better reproducibility. However, the response time was longer and the response value was smaller. The DEAE-dextran - modified glucose oxidase-mediated electrode was used for determination of glucose in lincomycin fermentation liquor.
Glucose Electrode Sensor Optimization

"Development Of A Robust Online Monitoring And Control System For Nutrients And Metabolites In Fermentations. 1. System Design And Configuration"
Genet. Eng. Biotechnol. 1995 Volume 15, Issue 4 Pages 217-223
Keay, P.J.;Wang, Y.;Seddon, C.;Seddon, T.

Abstract: A computer-based fully automated system is described for online monitoring of fermentation variables (measured every few min), with real-time process control from a simulated instrument panel on the monitor. The system contains 7 operational channels (4 channels for flow injection analysis; one for biomass estimation via turbidity; one for pH and oxygen measurement and control; one with an online sampling device connected to a spectrophotometer) and further control for up to 29 devices. Sample withdrawal and operation of the analytical system is controlled by an Apple Macintosh computer (Quadra 950), which also performs data acquisition, calibration and calculation. An easy-to-use computer interface program is written in the visual programming language LabVIEW. The software is powerful, flexible and suitable for use in rapid development of new fermentation control techniques.
Spectrophotometry Turbidimetry Electrode Electrode Computer Apparatus Process control Process monitoring

"Development Of A Robust Online Monitoring And Control System For Nutrients And Metabolites In Fermentations. 2. System Calibration"
Genet. Eng. Biotechnol. 1995 Volume 15, Issue 4 Pages 225-233
Keay, P.J.;Wang, Y.;Seddon, C.;Seddon, T.

Abstract: Calibration of the gas-driven flow injection system described in Part 1 is described. The dispersion coefficient for the system was measured and the performance (reproducibility) of the system was tested using phosphate determination. The possibility of online flow dilution confers wide flexibility and can make physical dilution unnecessary. The spectrophotometric determination of glucose and lactate by enzymatic methods and of ammonia via indophenol formation are described. The stability of these assays was measured; graphs of absorbance vs. concentration are included for each analyte.
Glucose Lactate Ammonia Spectrophotometry Dilution Apparatus Process control Process monitoring

"Rise To Today's Challenge With The Aid Of Modular Systems"
GIT Fachz. Lab. 1990 Volume 34, Issue 5 Pages 570-572
Flachsbarth, W.

Abstract: The modular concept of the EVA (Eppendorf Variable Analysis system) FIA system is described; it allows flexibility and the use of tailor-made configurations for particular applications. Modules described are a master module (controller, detector and data processor), peristaltic pump, injector, selector, reaction manifold and sampler. A number of different types of detector can be fitted into the modules or connected externally. Examples are presented of application of the apparatus involving the determination of polymeric β-glucan and the online determination of glucose in fermentation liquors.
Glucose β-Glucan Detector Injector

"From FIA To Bio-probes - Biochemical Analytics In Examples Of Application"
GIT Fachz. Lab. 1990 Volume 34, Issue 9 Pages 1045-1046
Bilitewski, U.;Schmid, R.D.

Abstract: A review is presented on the application of online analytical systems (FIA and in situ electrodes) for the enzymatic or immunochemical analysis of e.g., fermentation liquors, drinking water and foodstuffs. (22 references).
Electrode Sensor Review Enzyme

"Complete System For Fermentation Monitoring"
Int. Biotechnol. Lab. 1986 Volume 4, Issue 2 Pages 33-40
PARKER C. P. ; GARDELL M. G. ; DI BIASIO D.

Abstract: The Control Equipment Corporation MCA-103 MultiFlow Carbohydrate Analyzer is described. It is a fully automated monitoring device based on flow injection analysis with a gas-displacement system to deliver the solvents, and has two detectors, the first of which monitors the concentration. of a specific carbohydrate or alcohol with an integral detector by using immobilized oxidase enzymes and the second of which performs a broad range of chemical analyzes by using spectrophotometric detection. Its application is illustrated by the monitoring of glucose and phosphate levels in fermentation broths. Glucose was determined in the range 0.5 to 2.0% with glucose oxidase trapped between polycarbonate and cellulose acetate membranes stretched over a platinum anode. Phosphate (10 to 100 mg l-1) was determined by mixing the eluate from the glucose detector with molybdovanadate reagent and measuring the absorbance at 400 nm.
Carbohydrates Phosphate Glucose Electrode Spectrophotometry Cellulose acetate Immobilized enzyme

"Flow Injection Amperometric Determination For Sucrose And Glucose In Sugarcane Juice And Molasses"
Int. Sugar J. 1998 Volume 100, Issue 1195 Pages 320-324
Luiz De Mattos, I.;Zagatto, E.A.G.;De Oliveira Neto, G.

Abstract: A flow injection system for amperometric determination of sucrose (I) and glucose (II) employing mutarotase and glucose oxidase immobilized on controlled-pore glass was developed. A Pt microelectrode polarized at 650 mV vs Ag/AgCl was used as the working electrode. The proposed system allows sequential determination of I (0-8.0 mM, r = 0.99973, RSD = 1.5%) and II (0 - 0.60 mM, R = 0.99852, RSD = 1.2%) and was applied to the anal. of sugarcane juice and molasses. Results were compared with those provided by HPLC, and the agreement between them corroborated the applicability of the proposed procedure for large scale anal. and(or) for quality control of ethanolic fermentation Sampling frequency was 50/h.
Sucrose Glucose Amperometry Immobilized enzyme Controlled pore glass Method comparison

"Application And Automation Of Flow Injection Analysis (FIA) Using Fast Responding Enzyme Glass Electrodes To Detect Penicillin In Fermentation Broth And Urea In Human Serum"
J. Autom. Methods Manag. Chem. 1992 Volume 14, Issue 4 Pages 137-143
HELMUT MEIER, FRAN&Ccedil;OIS LANTREIBECQ, and CANH TRAN-MINH

Abstract: Penicillinase and urease were cross-linked as very fine films directly onto the sensitive ends of pH glass electrodes and incorporated into a flow injection analysis system (diagram given). Samples were injected into a carrier stream (4 mL min-1) of buffer solution and passed to the detection cell containing the electrodes. Calibration graphs were rectilinear for 100 mM penicillin and for 10 mM urea; corresponding coefficient of variation were 0.94 and 1.94%. An enzyme immobilization technique was developed to determine the concentration. of biological compounds This technique was applied to penicillinase and urease, which are crosslinked as very fine films directly onto the sensitive ends of pH glass electrodes, thereby dispensing with the need of an online enzyme reactor. The biosensor is incorporated in a FIA system within a magnetically stirred detection cell. Penicillin-V in fermentation broth and urea in human serum samples was detected and the results were compared with HPLC and spectrophotometric methods. Online measurement is achieved through the automation of this FIA system.
Penicillin Urea Electrode Electrode Sensor Method comparison

"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 Electrode Electrode Electrode Gas diffusion Selectivity Silicone membrane Immobilized enzyme Dual detection Linear dynamic range Glass Calibration Optimization

"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 Biochemical analysis Immunoassay Enzyme Review

"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.
Sensor Process control Immobilized enzyme Computer Calibration Review

"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 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 Electrode Biotechnology Enzyme Method comparison Process monitoring

"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 Reactor Process monitoring Computer

"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 Amperometry Electrode Sensor Immobilized enzyme Process monitoring

"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 Electrochemical analysis Process monitoring

"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 Immunoassay Turbidimetry Process monitoring

"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 Sample preparation Immobilized enzyme Heated reaction Pervaporation Process monitoring Extraction

"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 Sensor Electrode Electrode Method comparison Process monitoring Controlled pore glass

"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 Chemiluminescence Optrode Sensor Computer Process monitoring Optimization

"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 Capacitance Immunoassay Fluorescence Perfusion

"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 Biotechnology Immobilized enzyme Method comparison Process monitoring Interferences Dialysis Multichannel

"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 Biotechnology Immobilized cell Kalman filter Perfusion Interferences Reactor Multichannel

"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 Turbidimetry Fluorescence Well stirred mixing chamber Multichannel Process monitoring Closed loop

"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 Biotechnology Process monitoring Kinetic

"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 Spectrophotometry Method comparison Photodiode Light emitting diode

"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 Sensor Immobilized enzyme Kinetic Reactor

"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.
Kalman filter Signal processing Quality

"Flow Injection Analysis With Immobilized Enzymes For Process Control Of Pullulan Production By Fermentation"
J. Chem. Technol. Biotechnol. 1992 Volume 53, Issue 4 Pages 397-400
Ursula Englbrecht, Hanns-Ludwig Schmidt

Abstract: Pullulan (I) and glucose (II) are determined in filtered Aureobasidium pullulans fermentation broth after hydrolysis with α-dextrin endo-1,6-α-glucosidase and glucan 1,4-α-glucosidase immobilized on controlled-pore glass. Total II (i.e., that formed by hydrolysis and that present as such in the initial sample) is determined in a FIAstar 5020 analyzer. by oxidation with glucose 1-dehydrogenase in the presence of NAD+ and spectrophotometric monitoring of the NADH formed. Free II was determined similarly without enzymatic hydrolysis, and the I content was calculated by difference. The calibration graphs for both I and II were rectilinear over the range 2 to 20 mg l-1. One analysis takes only 10 min and the system response remains constant for 30 samples. A flow injection system is described for the parallel determination of pullulan and glucose during a fermentation of the fungus Aureobasidium pullulans. The polysaccharide was hydrolyzed by pullulanase and amyloglucosidase immobilized to controlled-pore glass. The glucose produced was oxidized by glucose dehydrogenase, and the NADH formed was determined photometrically. The pullulan concentration. was calculated from the difference to the response obtained for free glucose. The calibration curves for monomer and polymer were both linear between 2 and 20 mg/L. The determination of glucose and pullulan took ~10 min per sample.
Pullulan Glucose Sample preparation Spectrophotometry Process control Controlled pore glass Immobilized enzyme Tecator

"An Automatic Dehydrogenase-based Flow Injection System: Application For The Continuous Determination Of Glucose And Lactate In Mammalian Cell- Cultures"
J. Chem. Technol. Biotechnol. 1993 Volume 58, Issue 2 Pages 183-190
T. Becker, W. Schuhmann, R. Betken, H.-L. Schmidt*, M. Leible, A. Albrecht

Abstract: A concept for the development of an automatic flow injection analyzer with integrated dehydrogenase columns and its application in the control of industrial processes is presented. The system is based upon a kernel consisting of a nested-loop injection unit, pumps for the filling of the injection loops and the transport of buffer and values for switching on the one hand between sample and standard solutions and on the other hand between different enzyme columns. A Microsoft Windows 3.x application 'WIN-FIA' controls interactively the whole system and can be easily adapted to a specific solution of an analytical problem. As an example, the flow injection system was used for the continuous determination of glucose and lactate, using glucose dehydrogenase (GDH) and lactate dehydrogenase (LDH) as indicator enzymes, in a mammalian cell-culture fermentation process. The resulting concentration values are in good agreement with those obtained by discontinuously taken standard spectrophotometric enzyme assays. A general concept is presented for the development of dehydrogenase-based flow injection systems for the control of industrial fermentation processes. The proposed system is based on a kernel consisting of a nested-loop injection unit, pumps for the filling of the injection loops and the transport of buffer, and valves for switching between process streams. The flow injection system was demonstrated for the continuous determination of glucose and lactate in a mammalian cell-culture system, using glucose dehydrogenase and lactate dehydrogenase as indicator enzymes. Values obtained for the concentration were found to be in good agreement with those obtained by discontinuously taken standard spectrophotometric enzyme assays.
Glucose Lactate Method comparison Immobilized enzyme Computer

"Simultaneous Online Monitoring Of Glucose And Total Malto Sugar In Fermentation Processes Using An FIA System"
J. Chem. Technol. Biotechnol. 1996 Volume 67, Issue 3 Pages 276-280
Enobong F. Umoh, Anton B. v. Putten, Karl Sch&uuml;gerl

Abstract: flow injection analysis (FIA) system is presented which is able to monitor simultaneously and on-line the concentrations of glucose and total malto sugars during fermentation or hydrolysis processes. The system consists of glucoamylase (GA), glucose oxidase (GOD) EC 1.1.3.4 and mutarotase (MUT) EC 5.1.3.3 immobilized on synthetic polymer beads contained in cartridges serving as reactors. A high degree of selectivity towards non-malto sugars and carbohydrates, amino acids, and other possible components in the fermentation medium was exhibited by the system. The measurements delivered by the system during the monitoring of Bacillus licheniformis fermentation for protease production in complex media described the actual progress of the process. A correlation of the on-line measurements with the off-line enzymatic and off-line colorimetric measurements rendered correlation coefficients (r2) of 0.997 and 0.995, respectively. The enzyme reactors could be used on a long-term basis at 30°C and a pH of 5.5.
Glucose Sugars, reducing, total Simultaneous analysis Process monitoring

"Stability And Stabilization Of Enzymes From Mesophilic And Thermophilic Organisms In Respect To Their Use In FIA Systems For The Determination Of L-lactate And Acetate"
J. Chem. Technol. Biotechnol. 1997 Volume 69, Issue 2 Pages 161-166
Christine Lehn*, Hanns-Ludwig Schmidt

Abstract: The stabilization effect of 'bilayer encagement' on enzymes from mesophilic organisms and their 'thermophilic' counterparts was compared. Lactate dehydrogenases from pig heart and from a thermophilic bacterium (Clostridium thermohydrosulfuricum), respectively, showed stabilization factors of 4.5 (at 47°C) and 12.8 (at 70°C), respectively. For 'thermophilic' acetate kinase no stabilization effect of encagement was observed. Lactate dehydrogenase and acetate kinase from Clostridium thermohydrosulfuricum were immobilized to controlled porous glass and tested for their long-term stability. The 'thermophilic' enzymes showed by far a longer half-life as compared with the corresponding enzymes from pig heart and Escherichia coli, respectively, the half-life time of the flow injection system response with thermophilic lactate dehydrogenase at 35°C attaining 250 h (mesophilic enzyme 89 h), and with thermophilic acetate kinase 79 h (mesophilic enzyme 24 h). 12 References
Enzyme, activity Immobilized enzyme Heated reaction

"Online Monitoring Of Product Concentration By Flow-ELISA In An Integrated Fermentation And Purification Process"
J. Ferment. Bioeng. 1994 Volume 78, Issue 5 Pages 356-360
Mats Nilsson, Arumugaswamy Rajendran Vijayakumar, Olle Holst, Christina Schornack, H&aring;kan H&aring;kanson and Bo Mattiasson*

Abstract: Process integration involving fermentation with cell recycling, affinity adsorption to remove the product, and online process monitoring of the product were investigated. α-Amylase was produced in continuous and batch fermentations of Bacillus amyloliquefaciens with complete cell recycling, which was achieved using a membrane unit coupled to the fermenter. An affinity column of cross-linked starch was employed in the recycling loop to adsorb the α-amylase before the cell-free broth was recycled to the fermenter. The concentration of α-amylase in the cell-free broth prior to and after the affinity separation step was continuously registered using a flow injection, immunochemical monitoring system. The different unit operations were integrated pair-wise, and eventually as a complete system. The complete feasibility of monitoring a macromolecular product online during a cultivation/adsorption process was also demonstrated.
α-Amylase Process monitoring

"Colominic Acid Production From Escherichia-coli In A Fed-batch Culture Under The Control Of Ammonium-ions Using An FIA System"
J. Ferment. Bioeng. 1997 Volume 83, Issue 1 Pages 59-63
Hiroyuki Hondaa, Takuo Nakazekoa, Koji Ogisoa, Yuji Kawaseb, Nobuhiro Aokib, Mitsuo Kawaseb and Takeshi Kobayashia,*

Abstract: A fed-batch culture was applied for the production of colominic acid from Escherichia coli. It was found that the copper ion, which is a trace element in medium and an essential element in N-acetylneuraminic acid synthase, affected the colominic acid production and the highest production was obtained using 1.6 mg/l CuCl2. Ammonia formed from nitrogen source utilization greatly affected colominic acid production and only slightly affected the cell growth. A flow injection analysis (FIA) system was fabricated and applied to monitor and control the ammonia concentration in the culture broth. It was found that a concentration of 0.3 g/l of ammonia was optimal for colominic acid production where 1.9 g/l was obtained. 7 References
Ammonia Enzyme Process control Process monitoring

"α-Amylase Production In Recombinant Aspergillus Oryzae During Fed-batch And Continuous Cultivations"
J. Ferment. Bioeng. 1998 Volume 86, Issue 1 Pages 49-56
Anders Spohr*, Morten Carlsen, Jens Nielsen and John Villadsen

Abstract: Three a-amylase producing strains of Aspergillus oryzae used for recombinant protein production have been studied with respect to growth and protein production in fed-batch and continuous cultures. The three strains are (i) a wild-type, (ii) a transformant strain of the wild-type strain containing additional copies of the a-amylase gene and (iii) a morphological mutant of the transformant strain. It is found that production of a-amylase is a growth related process. Comparison of the three strains with respect to morphology and protein production reveals that changes in the morphology of the fungus influence the a-amylase production during submerged growth.
α-Amylase Process monitoring

"Determination Of Monensin In Fermentation Broth By HPLC With Post-column Derivatization"
J. Liq. Chromatogr. Relat. Technol. 1992 Volume 15, Issue 9 Pages 1513-1522
Frank L. Neely

Abstract: Samples of fermentation broth were diluted tenfold with methanol and the suspension obtained was homogenized with a grinder and allowed to settle for 1 h at 5°C, followed by filtration of the supernatant, if necessary. For HPLC a Regis Little Champ column of ODS silica was used with a mobile phase (1.5 mL min-1) of aqueous NH4H2PO4 (adjusted to pH 3.0 with H3PO4) - methanol (3:17), post-column reaction at 120°C with 3% vanillin in methanolic 3% H2SO4 in a coil (10 ft x 0.01 in.) and detection at 520 nm. A rectilinear calibration graph was obtained for 0.25 to 2.0 mg mL-1 of monensin with a response ratio of 0.86 for A to B form. Recovery of added monensin was >97%, and the mean coefficient of variation of determination was 1.2%.
Monensin HPLC Spectrophotometry Post-column derivatization Heated reaction

"Continuous-flow NMR Bioreactor For Invivo Studies Of Microbial Cell-suspensions With Low Biomass Concentrations"
J. Magn. Reson. 1992 Volume 98, Issue 3 Pages 654-659
A. A. de Graaf, R. M. Wittig, U. Probsta, J. Strohhaecker, S. M. Schoberth and H. Sahm

Abstract: The authors have initiated in vivo NMR studies using microbial cultures with a low biomass concentration (up to 10 mg dry weight (dw)/ml), because these suspensions can be handled quite well in a reproducible manner. To keep the measuring time within acceptable limits, the authors had to develop a new experimental setup that has an NMR sensitivity (defined as signal-to-noise ratio per unit time) as high as possible.
Nuclear magnetic resonance In vivo monitoring

"Biosensors In Flow Injection Systems For Biomedical Analysis, Process And Environmental Monitoring"
J. Mol. Recognit. 1996 Volume 9, Issue 5-6 Pages 658-663
Canh Tran-Minh

Abstract: This paper presents the construction of various biosensors using thin- film layers incorporated in flow injection devices, providing automated systems for biomedical analysis, process and environmental monitoring. A urease sensor has been developed in conjunction with a flow injection system for the automatic determination of urea. Use of the spraying immobilization technique gives rise to a response time of a few seconds, which allows sample throughputs up to 200 h-1. With a penicillin biosensor adapted in an appropriate cell detection, online measurements of penicillin V in the fermentation broth are achieved during the whole fermentation process; the results are compared with the HPLC method. Linearity, sensitivity and reproducibility of the biosensor are studied with regards to sample dilution in a stirred flow detection cell to provide optimal operating conditions. Measurements without any change in parameters are obtained during the whole fermentation process. Acetylcholinesterase sensors have been used in batch systems for the determination of pesticides, but they require large amounts of substrate. When those enzyme sensors are combined with flow injection systems, only small volumes (100 µL) of substrate are injected into the carrier stream and an automated system can be obtained for continuous control of water quality.
Urea Penicillin V Sensor Process monitoring

"Physiological Effects Of Nitrogen Starvation In An Anaerobic Batch Culture Of Saccharomyces Cerevisiae"
Microbiology 1996 Volume 142, Issue 8 Pages 2299-2310
U Schulze, G Liden, J Nielsen and J Villadsen

Abstract: The effects of nitrogen starvation on the anaerobic physiology of Saccharomyces cerevisiae were studied in cells cultivated in a bioreactor. The composition of the mineral medium was designed such that the nitrogen source became depleted while there was still ample glucose left in the medium. The culture was characterized by acoustic gas analysis, flow injection analysis and HPLC analysis of extracellular substrates and metabolites. During the cultivation, the macromolecular composition of the cells was analyzed with respect to the cellular content of RNA, protein, trehalose and glycogen. During exponential growth under anaerobic conditions, the maximum specific growth rate conditions. Depletion of ammonium in the medium led to an abrupt decrease (mumax) of S. cerevisiae CBS 8066 (0.46 h-1) was identical to the mumax determined under aerobic in the flux through glycolysis. Subsequently, a continuous decrease in the carbon dioxide evolution rate, caused by catabolite inactivation of the hexose- transport system, was observed. The apparent half-life of the transport system under nitrogen starvation was 13 h. During the exponential growth phase, the cellular content of RNA and protein was 15% (w/w) and 60% (w/w), respectively. At the end of the cultivation where the cells had been starved of nitrogen for 18 h, the cellular content of RNA and protein had decreased to 4% (w/w) and 22% (w/w), respectively. The intracellular carbohydrate content increased dramatically as trehalose and glycogen accumulated to final concentrations of 7% (w/w) and 25% (w/w), respectively. Glycerol formation during nitrogen starvation was higher than that accounted for by the formation of organic acids, suggesting a protein turnover of approximately 6% h-1. The growth energetics of S. cerevisiae CBS 8066 also changed as a result of nitrogen starvation, and YxATP was observed to increase from 80 mmol g- 1 during the exponential growth phase to more than 130 mmol g-1 towards the end of the cultivation. The presented results illustrate the effect of nitrogen starvation on glycerol formation, protein turnover, catabolite inactivation of the sugar-transport system, the cellular composition, the cell cycle and growth energetics.
Biotechnology

"Flow Injection Analysis Of Coumarins In Cell Suspension Cultures Of Angelica Archangelica"
Pharmazie 1998 Volume 53, Issue 4 Pages 273-274
Siatka, T.; Solich, P.; Kotyk, R.

Abstract: Coumarins in culture medium and methanolic extracts of A. archangelica cells were determined in buffer ph 6.0 as a carrier in the flow injection system, the fluorescence intensity was measured at the excitation max. of 345 nm. The content of coumarins was expressed as µmol scopoletin.
Coumarins Fluorescence Process monitoring Buffer

"Theoretical And Experimental Studies On An Immobilized L-glutamate Decarboxylase Electrode"
Proc. Natl. Sci. Counc. 1989 Volume 13, Issue 3 Pages 120-129
Lu, W.M.;Chen, W.C.

Abstract: An L-glutamate decarboxylase electrode has been developed for a continuous-flow measuring system. A microcomputer was applied for automatic control and calibration of the system. The electrode response was rectilinear from 0.1 to 1.0 g L-1 of L-glutamic acid (I) and reached its max. response potential within 5 min at 7.0 cm min-1 fluid velocity. The electrode was stable for 700 continuous assays of pure I solution and 250 assays of dilute fermentation broth. Reproducibility and stability after 45 days storage was good. A double-layer model was proposed for study of the effect of diffusion resistances of CO2 within the enzyme and gas permeable membranes. The diffusion resistance of CO2 in the gas permeable membrane is much larger than that in the enzyme layer and cannot be neglected.
l-Glutamic acid Sensor Electrode Computer Automation Enzyme Membrane

"Application Of Biosensors With An Electrolyte Isolator Semiconductor Capacitor (EIS-CAP) Transducer For Process Monitoring"
Process Biochem. 1998 Volume 33, Issue 2 Pages 175-180
C. Menzel, T. Lerch, K. Schneider, R. Weidemann, C. Tollnick, G. Kretzmer, T. Scheper and K. Sch&uuml;ger*

Abstract: A fluoride sensitive (pF), buffer capacity insensitive electrolyte isolator semiconductor capacitor (pF-EIS-CAP) chip was used as a transducer for biosensors with co-immobilized enzymes. Hydrogen peroxide forms fluoride ions in stoichiometric amounts in the presence of p-fluoraniline and peroxidase (POD). This was measured by the pF-EIS-CAP-sensor. The combination of this sensor with various oxidases allows the measurement of the concentration. of several analytes. Sugar syrup (glucose, maltose, maltotriose and other oligosaccharide) concentrations. were analyzed with co-immobilized β-amylase, amyloglucosidase, mutarotase, glucose oxidase and peroxidase. The glucose concentration. was analyzed with co-immobilized glucose oxidase and peroxidase, the ethanol concentration. with co-immobilized alcohol oxidase and peroxidase and the phosphate concentration. by co-immobilized nucleoside phosphorylase, xanthine oxidase and peroxidase. These (Bio-pF-EIS-CAP) sensors were integrated in flow injection analysis systems. The medium components were monitored online during Saccharomyces cerevisiae, Acremonium chrysogenum and recombinant baby hamster kidney (rBHK) cell cultivations. The agreement between the off- and online concentrations. of the medium components was within 10%.
Hydrogen peroxide Glucose Maltose Maltotriose Oligosaccharides Sensor Sensor Method comparison

"Bioreactor Monitoring Using Flow Injection - Membrane-introduction Mass Spectrometry With An Ion-trap Detector"
Process Control Qual. 1991 Volume 1, Issue 2 Pages 105-116
M.J. Hayward, D.E. Riederer, T. Kotiaho, R.G. Cooks, G.D. Austin, M.-J. Syu and G.T. Tsao

Abstract: Plugs (250 µL) of the fermentation broth were injected into a stream (1 mL min-1) of water, followed after 2 min by a plug of standard solution The flow injection system delivered the sample and standard to a special membrane probe (described with diagram) to introduce analytes into an ion-trap mass spectrometer. The major liquid phase products (e.g., butane-2,3-diol, acetoin, acetic acid and ethanol) were monitored by scanning from m/e 45 to 95 by water CI at 20 µTorr. The monitoring sequence was consecutive plugs of fermentation broth, standard, broth acidified with 0.1 M HCl (1:1) and acidified standard and monitoring at m/e 47, 61, 73 and 89. Acidification was required for acetic acid to permeate the membrane.
2,3-dihydroxybutane Acetoin Acetic acid Ethanol Mass spectrometry Mass spectrometry Membrane

"Online Monitoring Of Microbial Processes By Flow Injection Analysis"
Process Control Qual. 1992 Volume 2, Issue 4 Pages 371-384
J. Nielsen

Abstract: The application of flow injection analysis in biotechnological analysis is described and different sampling methods are discussed.
Process monitoring

"Sequential Injection Methodology For Fermentation Monitoring"
Process Control Qual. 1992 Volume 3, Issue 1-4 Pages 115-125
S.C. Chung, G.D. Christian and J. Ruzicka

Abstract: Sequential injection methodology was developed for the online monitoring of glucose during a yeast fermentation. The glucose assay was based on the Trinder reaction; standard glucose solution were used for calibration. The analyzer. consisted of a sinusoidal piston pump, a single multi-port selection valve and a detector. By the appropriate switching of the valve, a single stroke of the piston drew 2 mL of deionized water as carrier, 400 µL of Trinder reagent and 50 µL of sample solution into the holding conduit. The piston then moved in the reverse direction to transfer the solution to the detector cell of the spectrophotometer. The flow of liquid was stopped for 30 s while the absorbance at 504 nm was monitored. From the results, the rate of reaction and hence the concentration. of glucose was calculated. The calibration graph for glucose was rectilinear from 0.04 to 0.4 g l-1.
Glucose Spectrophotometry Sequential injection Optimization

"Direct Analysis Of Organic-compounds In Water By Membrane Introduction Mass-spectrometry Advances In Selectivity And Interfaces"
Process Control Qual. 1995 Volume 7, Issue 3-4 Pages 117-131
J.S. Patrick, P. Wong, C. Xu, M. Soni, N. Kasthurikrishnan, N. Srinivasan and R.G. Cooks

Abstract: An overview of recent advances in membrane introduction mass spectrometry (MIMS) is presented. Particular attention is given to developments in interfaces, improvements in sensitivity and enhancements in selectivity. A new interface is employed which uses pneumatically assisted transport of the analyte to a jet separator interfaced to a benchtop ion trap mass spectrometer. Affinity MIMS, a new experiment which allows selective trapping of analytes (here benzaldehydes) at a chemically modified membrane, is described. Extended loading times followed by release using a chemical agent yields a limit of detection of 10 ppb for benzaldehyde. Notched broad-band excitation using stored waveform inverse Fourier transform (SWIFT) experiments on a benchtop ion trap is also used to enhance selectivity by permitting injection or retention of selected ions within the ion trap. The combination of SWIFT, signal averaging and extended ionization times has allowed the detection of trans-dichloroethene and toluene at 500 parts-per-quadrillion levels in water with no sample pre-concentration. Tandem mass spectrometry of analytes, introduced by MIMS, is also performed using SWIFT on the benchtop ion trap instrument. Both unimolecular dissociations and ion/molecule reactions can be used to characterize analytes. Trace level(10 ppb) analysis of volatile organics in a seawater matrix is described using flow injection sample handling; the matrix causes no changes in permeation rate or detector response relative to an aqueous solution. Methods are discussed for the detection of glycerol and 5-hydroxymethylfurfuraldehyde, a dehydration product of glucose, using MIMS. Finally, information acquired by MIMS is used to provide feedback control of a yeast fermentation. Each of these advances in MIMS finds application in online monitoring of chemical process streams. (52 references)
Organic compounds Mass spectrometry Interface Membrane Process control Fourier transform Review PPQ Preconcentration

"Membrane Introduction Mass Spectrometry In A Pilot Plant: Online Monitoring Of Fermentation Broths"
Rapid Commun. Mass Spectrom. 1997 Volume 11, Issue 4 Pages 363-367
Rudolph C. Johnson, N. Srinivasan, R. Graham Cooks*, D. Schell

Abstract: The performance of a membrane introduction mass spectrometry (MIMS) system has been tested under pilot plant conditions. The work employed a Finnigan ITS-40 ion trap instrument, adapted to MIMS experiments by the addition of an external membrane/jet separator interface and packaged to operate in an industrial production environment. A representative sample from a 9000 L fermentation reactor was taken continuously and monitored on-line, using a stainless steel tangential filter as the only sample pretreatment. This filter was capable of withstanding the temperature and pressure of 30 psig steam sterilization as well as the 10^-30 psig operating pressure from a sample stream containing in excess of 10% solids. The filtrate was sampled using a flow injection analysis system which allowed quantification using external standards. Calibration experiments established that the system displayed a linear response to ethanol at concentrations between 1 and 10% by volume. During subsequent on-line experiments, ethanol standard and sample streams were examined alternately. The response of the standard solutions was used to quantitate the response of the sample stream and reduce errors associated with long term instrumental drift. Ethanol concentrations were found to be 2.97±0.07% and were quantitatively in agreement with off-line high-performance liquid chromatographic data. Minor components identified during on-line reactor monitoring included acetic and lactic acid. After one week of off-line operation in the pilot plant, on-line monitoring was continued for a period of four days.
Acetic acid Lactic acid Mass spectrometry Process monitoring Silicone membrane Method comparison

"Study Of The Enzyme Electrode Based On Glutamate Oxidase"
Shengwu Gongcheng Xuebao 1993 Volume 9, Issue 3 Pages 277-281
Yang Qingling, Bi Kewan

Abstract: A diffusion-controlled enzyme electrode was constructed for L-glutamate determination. The glutamate oxidase was immobilized between the cellulose acetate membrane and polycarbonate membrane using serum albumine and glutaraldehyde. The enzyme membrane was attached on the hydrogen peroxide probe surface moistened with electrolyte. The linear range of the enzyme electrode extends up to 1000 mg/L. A FIA system using the enzyme electrode was developed. The system exhibited good linearity (5-8000 mg/L with r = 0.9998), rapid response time (less than 20 s, and good selectivity. The electrode can be used continually for more than 2 weeks. The coefficient of variation (CV) of 41 times measurements at the glutamate concentration of 4000 mg/L is 2.8%. So its application to fermentation process control and food analysis is very promising.
Glutamate Electrode Process control

"A Stop-flow Sequential Injection Analysis Using Immobilized D-lactate Dehydrogenase For Online D-lactic Acid Monitoring During A Fermentation Process"
Biosensors for Food Analysis 1998 Volume 167, Issue 1 Pages 144-153
H.-C. Shu

Abstract: An automatic title system was set up to monitor the prodn. of D-lactic acid during batch fermentation of Lactobacillus delbrueckii. Samples were taken from the flux of a membrane filtration unit. Sequential injection analysis technol. proved to be a promising system to monitor D-lactic acid prodn. online during the fermentation process.
d-Lactic acid Sequential injection Process monitoring Immobilized enzyme Stopped-flow

"Biological Reaction Calorimetry: Development Of High Sensitivity Bio-calorimeters"
Thermochim. Acta 1998 Volume 309, Issue 1-2 Pages 157-173
I. Marison, J. -S. Liua, S. Ampuero, U. Von Stockar and B. Schenker

Abstract: A review of different types of biological reaction calorimetry systems currently used together with the operating principles is presented. The average resolution of these systems is approximately 20 to 1000 mW L-1, sufficient for studies of a wide range of cell culture processes. Poorly exothermic and endothermic processes require the development of even higher resolution systems. To this end, the Mettler-Toledo RCl calorimeter has been extensively studied to determine the factors which limit the resolution. By changing both the hardware and software, the resolution has been increased to 2-5 mW L-1 for non-aerated processes and to 10^-15 mW L-1 for aerated systems. The changes include a switchable electrical heater for the oil circulation thermostat, a new higher resolution AID board, PI controller and a thermostat reactor housing. The online measurement of the power input through agitation is proposed to be essential for low heat output biological processes, even under conditions where the rheological properties of the culture are not believed to be changing. The results show that it is possible to develop high-resolution systems capable of operating under standard laboratory bioreactor conditions; however, it is felt that the limits to the instrument resolution have been attained and that the calorimetric signal resolution is limited by the requirement of high agitation, nutrient feeds, gassing, pH control and other external effects which can only be overcome by heat-balancing methods.
Cells Calorimetry Review Process control Process monitoring

"Online Bioprocess Monitoring - An Academic Discipline Or An Industrial Tool?"
Trends Anal. Chem. 1998 Volume 17, Issue 2 Pages 88-95
Lisbeth Olsson*, Ulrik Schulze and Jens Nielsen

Abstract: Bioprocess monitoring capabilities are gaining increasing importance both in physiological studies and in bioprocess development, The present article focuses on online analytical systems since these represent the backbone of most bioprocess monitoring systems, both in academia and in industry. We discuss advantages and drawbacks of various of the most frequently used components (sampling units, flow systems and detection unit) and analytical techniques, The differences between academia and industry in the use of bioprocess monitoring are discussed, based on the key drivers determining implementation of analytical systems in each of these fields.
Sensor Process monitoring Sequential injection Review

"What’s Cooking? Optimizing Bioprocess Monitoring"
Trends Biotechnol. 1992 Volume 10, Issue 7 Pages 230-232
Michael J. Geisow

Abstract: Biotechnology products derived from microbial, eukaryotic cell, and even whole plant and animal systems are increasing sharply in number. The scale (e.g. fermenter size) and complexity (e.g. glycoprotcins from hollow-fiber cell culture) of products arc also increasing. Such cultures demand close, interactive monitoring. This is needed not only to preclude serious economic losses from large-scale cultures, but also to ensure that product quality is consistent (e.g. in glycoprotcins produced from mammalian cell culture).
HPLC Optimization Process monitoring

"Online And In-situ Monitoring Of Biomass In Submerged Cultivations"
Trends Biotechnol. 1997 Volume 15, Issue 12 Pages 517-522
Lisbeth Olsson and Jens Nielsen

Abstract: Biomass is one of the most important variables in submerged-cultivation processes and, in recent years, many different sensors have been introduced for online or in situ monitoring of this variable. This article provides an overview of the principles behind these sensors and discusses their application in both laboratory and industrial processes. 34 References
Biomass Fluorescence Sensor Review

"Short-term Toxicity Test Using Escherichia-coli - Monitoring Carbon Dioxide Production By Flow Injection Analysis"
Water Res. 1990 Volume 24, Issue 3 Pages 351-354
W. F. Jardim, C. Pasquini, J. R. Guimar&atilde;es and L. C. de Faria

Abstract: Short-term toxicity tests using Escherichia coli were carried out for different stressing agents such as metal ions [Cd(II), Cu(II) and Hg(II)], sediments and one type of antibiotic (Bactrin). Inhibition of the microbial respiration was monitored using flow injection analysis (FIA) with a conductometric detector. Inhibition in the respiration of E. coli suspensions were detected within 20 min for Hg(II) ions and the antibiotic, whereas sediment samples from eutrophic water bodies stimulated CO2 production.
Carbon dioxide Bacteria, echerichia coli

"Photocatalytic Degradation Of Aromatic Chlorinated Compounds Using TiO2 Toxicity Of Intermediates"
Water Res. 1997 Volume 31, Issue 7 Pages 1728-1732
W. F. Jardim, S. G. Moraes and M. M. K. Takiyama

Abstract: The photocatalytic degradation of four chlorinated aromatic compounds, namely pentachlorophenol (PCP), 2,4,-dichlorophenol (2,4,-DCP), 3,5,-dichlorophenol (3,5,-DCP), and 2,3,5,-trichlorophenol (2,3,5,-TCP), was investigated in the presence of an aqueous suspension of TiO2 irradiated with near-UV light. The toxicity of the transient species was compared to that observed for the parent compound. The comparison was carried out by monitoring the inhibition in bacterial respiration (Escherichia coil) by measuring the CO2 in the aqueous culture medium using flow injection analysis (FIA). Only in the case of PCP and 2,3,5,-TCP were the intermediates. identified by HPLC and GC/MS, found to be more toxic than the parent compound. The principal intermediates observed during the photocatalytic oxidation process of PCP were 2,3,5,6,-tetrachloro-1,4,-hydroquinone, 2,3,5,6,-tetrachloro-1,4,-benzoquinone and 2, 3,5,6,-tetrachlorophenol. In the case of 2,3,5,-TCP and 3,5,-DCP, the identified intermediates were 2,3,5,-trichloro-1,4,-hydroquinone and 2,5,dichloro-1,4,-hydroquinone, respectively. The intermediates of 2,4,-DCP were not identified. (C) 1997 Elsevier Science Ltd. 18 References
Carbon dioxide

"Determination Of Cephalosporin C Potency By Autoanalyser"
Zhongguo Yiyao Gongye Zazhi 1987 Volume 18, Issue 4 Pages 163-166
CHEN Li-juan XIE Xue-min

Abstract: Cephalosporin C(I) forms a reaction product with nicotinamide, which further forms a carbonyl addition product with dihydroxyacetone in alkaline medium. The UV absorbance of the final product is proportional to the concentration. of I. The potency of I in fermentation broths is determined by continuous-flow automatic analysis based on the above reactions; 30 samples can be analyzed in 1 h. The range of determination is up to 25,000 int. units mL-1, and results agree with those by HPLC. A flow diagram for the method is given. The method is simple, rapid, specific and sensitive.
Cephalosporin C Spectrophotometry Method comparison

"Extraction And Determination Of Biogenic Amines In Fermented Sausages And Other Meat Products Using Reversed-phase-HPLC"
Z. Lebensm. Unters. Forsch. 1993 Volume 197, Issue 3 Pages 230-232
Bernhard Straub, Margit Schollenberger, Martin Kicherer, Bernd Luckas and Walter P. Hammes

Abstract: A convenient method is described for the analysis of biogenic amines (BA) by means of reversed-phase-HPLC. The method is characterized by multi-channel UV detection (diodearray), subsequent post-column derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid, and fluorescence detection. For the analysis of meat products and especially fermented sausages an optimized perchloric acid extraction process was introduced to determine putrescine, cadaverine, histamine, tyramine and 2-phenylethylamine. BA recoveries from meat ranged between 96 and 113% with a detection limit for amines of 0.5 mg/kg. Sausage (10 g) was homogenized with 50 mL of 0.6 M HClO4 for 1 min and a portion of the aqueous layer was centrifuged at 9000 g for 10 min at 0°C. The supernatant solution was filtered, the filtrate was diluted with water (1:10) and a 20 µL portion of the resulting solution was analyzed by HPLC on a column (25 cm x 4.6 mm) of Nucleosil 100 7C18, equipped with a similar guard column (3 cm). Gradient elution (1 ml/min) was effected with 0.05 M hexanesulfonic acid, 0.1 M potassium dihydrogen phosphate and acetonitrile (details given) with post-column derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid (details given) and fluorimetric detection at 455 nm (excitation at 340 nm). Recoveries ranged from 96-113% and the detection limit was 0.5 mg/kg of biogenic amines in the sample. The method may be applied to the analysis of other meat products.
Amines, biogenic HPLC Fluorescence Sample preparation Reverse Gradient technique

"Monitoring Alcoholic Fermentation Of Red Wine By Electrochemical Biosensors"
Am. J. Enol. Vitic. 2003 Volume 54, Issue 1 Pages 39-45
Marco Esti, Giulia Volpe, Dario Compagnone, Giacomo Mariotti, Danila Moscone, and Giuseppe Palleschi

Abstract: Amperometric biosensors for the determination of glucose, fructose, ethanol, and glycerol were used to monitor alcoholic fermentation during red wine production in industrial-scale plants. Platinum-based probes, covered with appropriate enzyme membranes, were assembled in electrochemical flow-injection analysis systems. Before use, the biosensors were validated in must and wine samples by spectrophotometric procedures. The biosensors were applied during alcoholic fermentation in three wineries that used different red winemaking techniques. Results are reported and compared. Glucose, fructose, ethanol, and glycerol content varied with different kinetics corresponding to three characteristic phases: an early phase with no detectable variation of the analytes, an intermediate phase characterized by rapid sugar use, and a late phase with little variation of the concentration levels. In particular, glucose was consumed more rapidly than fructose, confirming that the former is the preferential sugar source for Saccharomyces cerevisiae. Experimental data demonstrated that the biosensors are useful tools for the close monitoring of alcoholic fermentation during industrial mass production of wine.
Glucose Ethanol Glycerol Fructose Electrochemical analysis Sensor Biochemical analysis Sensor Internal standard Process monitoring Process control

"Development Of A Sequential Injection Analysis System For The Simultaneous Biosensing Of Glucose And Ethanol In Bioreactor Fermentation"
Food Chem. 2003 Volume 81, Issue 1 Pages 141-146
Rui A. S. Lapa, Jos&eacute; L. F. C. Lima and Ivone V. O. S. Pinto

Abstract: An on-line sequential injection analysis system using amperometric detection for the simultaneous monitoring of glucose and ethanol in fermentations media is presented. The automatic analytical procedure developed is based in a sequential injection analysis (SIA) strategy and uses catalytic reactors of oxidase enzymes immobilized on controlled-pore glass. The proposed method allows the simultaneous determination of glucose (linear range between 5 and 750 mg L-1 with a RSD better than 1.2%) and ethanol (linear range between 0.15 and 30 mg L-1 with a RSD better than 2.2%). It was applied to monitor both species in beer fermentation. In a way to adjust the levels of ethanol present in the broth to the characteristics of the proposed system a dialysis unit was used between fermenter and SIA manifold. The sampling rate optimized was 50 samples h-1.
Glucose Ethanol Amperometry Controlled pore glass Immobilized enzyme Sequential injection

"An Effective Automated Glucose Sensor For Fermentation Monitoring And Control"
World J. Microbiol. Biotechnol. 1992 Volume 8, Issue 1 Pages 7-13
I. Queinnec, C. Destruhaut, J. B. Pourciel, and G. Goma

Abstract: An industrial glucose analyzer. was partnered to an automated injection system to evaluate glucose in the culture medium of a bioreactor. This sensor has been validated on continuous cultures ofSchizosaccharomyces pombe and continuous and fed-batch cultures of Saccharomyces cerevisiae. In addition to the advantage of a more accurate process monitoring, the main interest of this sensor deals with the control of the substrate concentration to a prespecified reference signal. Several experiments have been carried out first to validate the sensor, then to control the process evolution.
Glucose Sensor

"Hydrogen Peroxide As An Oxygen Source For Immobilized Gluconobacter Oxydans Converting Glycerol To Dihydroxyacetone"
Appl. Microbiol. Biotechnol. 1985 Volume 22, Issue 6 Pages 383-388
Olle Holst , Hans Lundb&auml;ck and Bo Mattiasson

Abstract: A flow injection analysis (FIA) system with amperometric detection was developed for measuring hydrogen peroxide which was used as an oxygen source for immobilized cells. A constant concentration of peroxide in the reactor was maintained by processing the analytical signal in a computer programmed as a PI-regulator. The concentration of dissolved oxygen was followed using a commercial Clark-electrode. The simultaneous measurements of hydrogen peroxide and dissolved oxygen are discussed with respect to process control. Conversion of glycerol to dihydroxyacetone by Gluconobacter oxydans immobilized in calcium alginate was used as a model system. Initial specific productivity increased with increasing hydrogen peroxide concentration. However, decreases in viable counts, enzymatic activities and overall productivities were noted. Various techniques for improving operational stability are discussed.
Hydrogen peroxide Amperometry Optimization Process control