Carsten Lindemann

Two-Dimensional Fluorescence Spectroscopy: A New Tool for On-Line Bioprocess Monitoring

Two‐dimensional fluorescence spectroscopy is presented as a new method for bioprocess monitoring. It covers a wide range of excitation and emission wavelengths and is a further development of the fluorescence measurements performed so far, which concentrated mainly on NAD(P) H culture fluorescence. Biogenic fluorophores such as proteins, coenzymes, and vitamins can simultaneously be detected qualitatively and quantitatively inside and outside the cells. This optical method is noninvasive, suitable for in vivo measurements. One whole spectrum (excitation, 250–550 nm; emission, 260–600 nm) with the described parameters is performed within 1 min, which allows an almost continuous monitoring of the bioprocess. The technique is ideal for on‐line, in situ measurements via fiber optical systems. Results are presented for cultivations of Claviceps purpurea, Escherichia coli, Saccharomyces cerevisiae, and Sphingomonas yanoikuyae. Cell growth and the metabolism of the cells (changes from aerobic to anaerobic conditions and uncoupling of the oxidative phosphorylation) could be detected.

Optical sensor systems for bioprocess monitoring

Bioreactors are closed systems in which microorganisms can be cultivated under defined, controllable conditions that can be optimized with regard to viability, reproducibility, and product-oriented productivity. To drive the biochemical reaction network of the biological system through the desired reaction optimally, the complex interactions of the overall system must be understood and controlled. Optical sensors which encompass all analytical methods based on interactions of light with matter are efficient tools to obtain this information. Optical sensors generally offer the advantages of noninvasive, nondestructive, continuous, and simultaneous multianalyte monitoring. However, at this time, no general optical detection system has been developed. Since modern bioprocesses are extremely complex and differ from process to process (e.g., fungal antibiotic production versus mammalian cell cultivation), appropriate analytical systems must be set up from different basic modules, designed to meet the special demands of each particular process. In this minireview, some new applications in bioprocess monitoring of the following optical sensing principles will be discussed: UV spectroscopy, IR spectroscopy, Raman spectroscopy, fluorescence spectroscopy, pulsed terahertz spectroscopy (PTS), optical biosensors, in situ microscope, surface plasmon resonance (SPR), and reflectometric interference spectroscopy (RIF).

On-line bioprocess monitoring with a multi-wavelength fluorescence sensor using multivariate calibration.

Cultivations of Pseudomonas fluorescens were monitored with a multi-wavelength on-line fluorescence sensor. The multi-wavelength fluorometer used excitation light from 270 to 550 nm with 20 nm steps and measured fluorescence emission from 310 to 590 nm. The fluorescence, on-line exhaust gas measurements and off-line analysis of nitrate, succinate, optical density and protein were compared chemometrically by multivariate calibration, i.e. computing partial least square (PLS) regression models. Based on the multivariate regression models, it was possible to determine CO2 and O2 composition in the exhaust gas (the correlation coefficients, R2 between the predicted values by the PLS model and the measured values was 0.97 for CO(2) and 0.97 for O2, respectively). Also to make quantitative determinations of succinate (R(2) = 0.97), protein (R(2) = 0.94), optical density (R(2) = 1.0) and nitrate (R(2) = 0.98) in the medium based on the fluorescence spectra. Only a limited data set was available but the results indicated that the sensor could indirectly determine non-fluorescent compounds, i.e. nitrate and succinate, which probably is due to the stoichiometric relationship between fluorescent cellular components and non-fluorescent compounds. Consequently multi-wavelength fluorescence is an interesting technique for a wide range of applications.

Application of fluorescence spectroscopy for on-line bioprocess monitoring and control

12 Modern bioprocess control requires fast data acquisition and in-time evaluation of bioprocess variables. On-line fluorescence spectroscopy for data acquisition and the use of chemometric methods accomplish these requirements. The presented investigations were performed with fluorescence spectrometers with wide ranges of excitation and emission wavelength. By detection of several biogenic fluorophors (amino acids, coenzymes and vitamins) a large amount of information about the state of the bioprocess are obtained. For the evaluation of the process variables partial least squares regression is used. This technique was applied to several bioprocesses: the production of ergotamine by Claviceps purpurea, the production of t-PA (tissue plasminogen activator) by animal cells and brewing processes. The main point of monitoring the brewing processes was to determine the process variables cell count and extract concentration.

Chemometric Models for the On-Line Estimation of Bioprocess Variables from 2-D-Fluorescence Spectra

Abstract Measurements of a multi wavelength excitation/emission spectrofluorometer employed for monitoring of various bioprocesses, such as the cultivation of Sphingomonas yanoikuyae, Claviceps purpurea, Escherichia coli and Saccharomyces cerevisiae are presented. Applying principal component analysis as a chemometric modeling tool, the spectra have been correlated to various bioprocess variables such as substrate, biomass and product concentration. It is demonstrated that the data reduction ratio is less than 1 %. Employing cross validation as well as external validation the chemometric models were able to predict important bioprocess variables with a mean error less than 5 %. The advantages as well as the disadvantages of chemometric modeling will be discussed.

Two-dimensional fluorescence spectroscopy for online monitoring of chromatographic separation of molasses

Monitoring and control of industrial or biotechnological processes invariably require reliable and fast analytical systems. Two-dimensional spectral fluorescence allows real time automatic measurements directly inside the process and provides a continuous stream of information compared to discrete information available from repeated sampling and offline analysis. Normally, chromatographic molasses desugarization processes are typically monitored with a combination of an online-refractometer, polarimeter and the measurement of density and conductivity. Additional information of the separation profile could be obtained by multiple sampling during the separation cycle followed by offline laboratory analysis. An optical sensor (BioViewR) allows online fluorescence measurements for a continuous monitoring directly at the outlet of the separation columns. It was the aim to predict the amino acid serine during the chromatographic cycle. Based on fluorescence, it was possible to monitor a fluorophor, which eluted a few minutes before the serine fraction during molasses desugarization. The application of fluorescence measurements for monitoring and control of chromatographic separation process may improve yield and purity of the separating fractions and can lower the costs for the next downstream processing of by-products.

New Sensor Application in European Industries for Detailed Bioprocess Monitoring

Abstract The European commission has set up tremendous efforts in order to emphasize a cooperation between universities and European industries in order to establish an efficient transfer of basic research into industrial application. Different areas of science are involved in this program. On major focus in future biotechnology is the establishment of new powerful biosensor systems for an optimized bioprocess monitoring in regard of bioprocess optimization, bioprocess control and quality documentation. In an Europe-wide cooperation between four industrial partners and one university partner a straightforward research project was set up to transfer basic research technology in biosensing and modern data processing into the practical use in production plants of biotechnological industries. Within this 3 year research project a new optical sensor system was designed, set up, tested and applied in pilot and production plants. The sensor system can be used for non-invasive monitoring of bioprocesses in real time application. It offers the possibility to monitor intra- and extracellular components in a broad variety of bioprocesses with high specificity, resulting in detailed information about the processes, and a broad area of application. The potential of the system for bioprocess monitoring will be given in this paper.