On-line simultaneous monitoring of glucose and acetate with FIA during high cell density fermentation of recombinant E. coli (original) (raw)
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Analytica Chimica Acta, 1996
Screen printed ampemmetric glucose biosen~ors were constructed and optimised for use in a flow injection analysis (FIA) system. The sensors were fabricated using a catalytic metallised carbon based ink, allowing a working potential of + 350 mV (Ag/AgCI). Overall the sensors displayed a high degree of reliability and robustness. A linear response was obtained over the range 0.1 to 25 mM glucose with a slope of 1.35 + 0.20/tA/mM. The within electrode coefficient of variation at each concentration tested (n = 10) was less than 2% (or 0.1 mM for concentrations below 5 raM). In addition, it was demonstrated that a single sensor can be used in the HA system over a seven day period indicating a high level of operational staldlity, l'hese sensors fulfil the criteria necessary for a mass produced device, which would be suitable for the on-line monitoring of glucose consumption during microbial fermentation. ateywoms: Flow htj~tio~, Biose~ots, FcL6~at~tion * Corresponding author.
Development of an On-line Glucose Sensor for Fermentation Monitoring
Biosensors, 1987
A dimethylferrocene-mediated enzyme electrode for glucose (Cass et al., 1984) was evaluated for application to microbial fermentation monitoring and control. The deficiencies revealed-insufficient stability, progressive increase in response time and progressive decrease in linear range-were investigated using electrochemical and radiochemical techniques. The enzyme immobilization was improved with a novel procedure using periodate-oxidized glucose oxidase and an alkylamine electrode coating. An in-situ probe and a computer-controlled analytical system were developed for fermentation monitoring. The glucose concentration of an Escherichia coli batch culture was successfully monitored on line using this analytical system.
A system for the on-line monitoring of the major state variables during the production of recombinant proteins with E. coli was developed. Substrate (glucose) and the major by-product (acetate) were measured with a Flow Injection Analysis system, while the carbon dioxide and oxygen transfer rates were calculated from data obtained with exhaust gas analysis. The fermentation culture weight was also continuously assessed with a balance placed below the fermenter, allowing the use of more precise mass-based concentrations. The graphical programming environment LabVIEW was used to acquire and integrate these variables in a supervisor computer, allowing to perform integrated monitoring and control of the process. This framework is illustrated with different experiments for model calibration, state estimation by software sensors and process performance optimisation by exponential feed control.
Journal of Fermentation and Bioengineering, 1994
Process integration involving fermentation with cell recycling, affinity adsorption to remove the product, and on-line process monitoring of the product were investigated, a-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 fermentor. An affinity column of crosslinked starch was employed in the recycling loop to adsorb the a-amylase before the cell-free broth was recycled to the fermentor. The concentration of a-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 on-line during a cultivation/adsorption process was also demonstrated.
Enzyme and Microbial Technology, 1997
A jlow-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. Resultsffom 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.
Analytica Chimica Acta, 1995
The development of a highly flexible sequential-injection system for the on-line control of fermentation processes is described. The developed system shows a significantly improved flexibility and adaptability to the needs of a particular fermentation process to be monitored as compared with conventional flow-injection systems. The number of necessary hardware components has also been drastically decreased, and the minimal injection volume decreased to 3-5 ~1 allowing improved dilution of an injected sample plug and improved mixing of coinjected reagents. A dilution factor can be adjusted to between 1 and 100 due to controlled dispersion of the injected sample plug within the tube system by choosing an appropriate injection volume. Due to the symmetrical design of the injection manifold and the versatile control software, complex injection profiles can be obtained that are necessary to coinject a sample together with a reagent or a sample together with one or two standard solutions for immediate recalibration. The system is fully automated, and the specially developed software package comprises automatic calibration and recalibration with up to 5 standard solutions, real-time calculation of the actual concentration values based on the most recent calibration data, documentation of the fermentation course, and injection of samples from up to 4 sampling sites with variable time basis. 0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0003-2670(95)00300-Z 114 W. Schuhmann et al./Analytica Chimica Acta 315 (1995) 113-122
Flow Injection Analysis for Amperometric Detection of Glucose with Immobilized Enzyme Reactor
Biotechnology & Biotechnological Equipment, 2010
GOD was covalently immobilized on polymer/silica gel hybrid support prepared by coating high surface area of silica gel with modified acrylonitrile co-polymer. The relationship between immobilization factors and enzyme activity were examined by the series of contour plots. The selections of the immobilization variable range were extremely precise in the 3-level-3factor fractional design. The results indicated that the optimal conditions for GOD immobilization were: 0.1% enzyme solution, immobilization temperature-4 o C and immobilization time-24 h. Immobilized GOD was applied to amperometric determination of glucose using flow-injection analysis. The optimal flow rate was determined as 4.0 ml/min when injecting 100 µl sample volumes. The linear response range for the on-line detection of glucose using immobilized GOD in column minibioreactor was estimated to be from 0.01 mM to 20 mM (a correlation coefficient of 0.985). Moreover, its experimental detection limit is 10 µM (S/N=3) and the apparent Michaelis-Menten constant was calculated to be 20.15 mM. The proposed method for glucose measurement was validated in real samples of fruit juices.
Monitoring of fed-batch E. coli fermentations with software sensors
Bioprocess and Biosystems Engineering, 2009
Accurate monitoring and control of industrial bioprocess requires the knowledge of a great number of variables, being some of them not measurable with standard devices. To overcome this difficulty, software sensors can be used for on-line estimation of those variables and, therefore, its development is of paramount importance. An Asymptotic Observer was used for monitoring Escherichia coli fed-batch fermentations. Its performance was evaluated using simulated and experimental data. The results obtained showed that the observer was able to predict the biomass concentration profiles showing, however, less satisfactory results regarding the estimation of glucose and acetate concentrations. In comparison with the results obtained with an Extended Kalman Observer, the performance of the Asymptotic Observer in the fermentation monitoring was slightly better.