PubMed Health⌕ Search

Biomedical subjects

T Scheper

Publications and source records attributed to T Scheper.

At least 19 recordsLinked to original sources
PubMed2005

Two-dimensional fluorescence spectroscopy: a novel approach for controlling fed-batch cultivations.

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

Aerobiosis↗
PubMed2003

Visualizing transport processes at liquid-liquid interfaces--the application of laser-induced fluorescence.

Modeling of liquid-liquid extraction processes involves the concentration of the extracted component directly at the interface. Currently, only very few and specialized methods are available for the direct measurement of these concentrations. Therefore a new, fluorescence-based measurement system with a high spatial resolution and a broad application spectrum was developed and tested. The detection principle is based on the use of fluorescent dyes excited by an argon ion laser. The intensity of the emitted light is dependent on the concentration of the extracted component in the very near surroundings of the dye. This intensity distribution is reproduced by an optical, microscope-based system onto a highly sensitive camera with a spatial resolution of 1 mum. This distribution is converted into a concentration profile at the interface using a calibration function and digital image processing routines. Measurements were performed in a commonly used stirred two-phase reactor modified to meet the requirements of an optical measurement system. It was shown that the concentration profiles at moving and nonmoving interfaces could be visualized with a resolution of 1 mum. The profiles formed at the interface differ significantly according to the kinetic of the used extraction system and the flow profiles in the reactor and can be used for further modeling of the extraction processes.

Journal Article↗
PubMed2003

Multi parameter in vitro testing of ratjadone using flow cytometry.

Ratjadone, isolated from the myxobacterium Sorangium cellulosum, belongs to the family of so-called orphan ligands, which includes leptomycin, callystatin and other compounds. In previous screening tests, ratjadone revealed a growth inhibitory effect against bacteria, yeast and human cancer cells. Following these first results, ratjadone was tested on several human tumour cell lines (Jurkat, HepG2, U87-MG) and, as a control, on a non-tumour cell line (RLC18) for its mode of action. The cell analysis was carried out by flow cytometry. This comprised cell density measurements, live-dead analysis, cell-cycle analysis and detection of apoptosis. First experiments confirmed the growth inhibitory effect on any chosen tumour cell line. Following these results a dose effect relationship was monitored, confirming the high effectiveness of ratjadone against cell growth at nanomolar concentration. Cell cycle analysis has shown that ratjadone intervenes in the cell cycle by arresting the cells in G1-phase. Biological testing of additional ratjadone derivatives with changed configuration and stereochemistry, identified the pharmacophoric site of the molecule.

Antineoplastic Agents↗
PubMed2003

Chemometric modelling based on 2D-fluorescence spectra without a calibration measurement.

MOTIVATION: 2D fluorescence spectra provide information from intracellular compounds. Fluorophores like trytophan, tyrosine and phenylalanin as well as NADH and flavins make the corresponding measurement systems very important for bioprocess supervision and control. The evaluation is usually based on chemometric modelling using for their calibration procedure off-line measurements of the desired process variables. Due to the data driven approach lots of off-line measurements are required. Here a methodology is presented, which enables to perform a calibration procedure of chemometric models without any further measurement. RESULTS: The necessary information for the calibration procedure is provided by means of the a priori knowledge about the process, i.e. a mathematical model, whose model parameters are estimated during the calibration procedure, as well as the fact that the substrate should be consumed at the end of the process run. The new methodology for chemometric calibration is applied for a batch cultivation of aerobically grown S. cerevisiae on the glucose Schatzmann medium. As will be presented the chemometric models, which are determined by this method, can be used for prediction during new process runs. AVAILABILITY: The MATHLAB routine is free available on request from the authors.

Algorithms↗
PubMed2002

In-situ-fluorescence-probes: a useful tool for non-invasive bioprocess monitoring.

Optical sensors appear to be very promising for different applications in modern biotechnology. They offer the possibility to interface all the well known optical analysis techniques to bioprocesses via fiber optical cables. Thus, high sophisticated and sensitive optical analysis techniques can be coupled to a bioprocess via these light signal transporting fibers. A wide variety of sensor types for application in biotechnology has been described. Normally these sensors are non-invasive and the response times are nearly instantaneous. In particular, the use of glass fiber technology makes these sensors small, robust and reduces their costs.

Biomass↗
PubMed2001

The enantioselective hydrolysis of 3-hydroxy-5-phenyl-4-pentenoicacidethylester in supercritical carbon dioxide using lipases.

A new experimental high-pressure-unit was constructed for the enantioselective enzymatic hydrolysis of 3-hydroxy-5-phenyl-4-pentenoicacidethylester (a precursor for biological interesting substances) in a biphasic buffer/SCCO(2)-system. One objective is to take advantage of the solubility differences of the substrate and the produced acid. Thus the different solubilities of the substrates and the products in the different phases were studied regarding to an overall process integration. One ester enantiomer is preferably hydrolyzed, the other remains in the supercritical phase. And the produced acid enantiomer is concentrated in the buffer phase. The decrease in pressure is followed by an extraction process of the remaining substrate-enantiomer, in consequence it will be possible to combine an enzymatic reaction with a separation step. The catalysis was optimized in regard to enantioselectivity, enantiomeric excess, conversion and reaction time. A high enantioselectivity is achieved for the aromatic substrate using the lipase of Pseudomonas cepacia. The results show that this unconventional reaction system offers tremendous advantages for enzyme process development.

Journal Article↗
PubMed2001

Oxygen monitoring in supercritical carbon dioxide using a fibre optic sensor.

Investigations of enzymatic reactions in supercritical CO(2) are often hindered by the high pressure involved in these processes, making reaction monitoring extremely difficult. This paper describes the implementation of a fiber optic based oxygen sensor into a high pressure reactor for supercritical carbon dioxide. The sensor is pressure resistant, working in supercritical carbon dioxide and reusable after depressurisation. The sensor signal is found to be affected by pressure changes, but stable at constant pressure. Oxygen concentration in supercritical CO(2) is monitored using the disproportionation of hydrogen peroxide as a simple oxygen producing reaction.

Journal Article↗
PubMed2001

Basic examinations on chemical pre-oxidation by ozone for enhancing bioremediation of phenanthrene contaminated soils.

Biological treatment of polycyclic aromatic hydrocarbons (PAH) has been demonstrated to be a feasible and common remediation technology which has been successfully applied to the clean-up of contaminated soils. Because bioavailability of the contaminants is of great importance for a successful bioremediation, a chemical pre-oxidation step by ozone was tested to enhance the subsequent biodegradation steps. Oxidation of PAH by ozone should result in reaction products that have a better solubility in water and thus a better bioavailability. A major part of this work was done by examinations of the model substance phenanthrene as a typical compound of PAH. After initial ozonation of phenanthrene, analysis by GC-MS showed at least seven identified conversion-products of phenanthrene. In comparison with phenanthrene these conversion products were more efficiently biodegraded by Sphingomonas yanoikuyae or mixed cultures when the ozonation process resulted in monoaromatic compounds. Primary ozonation products with biphenylic structures were found not to be biodegradable. Investigations into the toxicity of contaminated and ozonated soils were carried out by well-established toxicity assays using Bacillus subtilis and garden cress. The ozonated soils surprisingly showed higher toxic or inhibitory effects towards different organisms than the phenanthrene or PAH itself. The microbial degradation of phenanthrene in slurry reactors by S. yanoikuyae was not enhanced significantly by preozonation of the contaminated soil.

Bacillus subtilis↗
PubMed2001

Application of oxygen vectors to Claviceps purpurea cultivation.

The application of a two-phase fermentation system for the production of ergot peptide alkaloids by Claviceps purpurea is described. Perfluorocarbons (PFC) are used as oxygen vectors in Claviceps fermentation for the first time. In shake-flask cultivations, the inclusion of PFC in the medium brings about a five-fold increase in the total alkaloid production and a six-fold increase in the pharmaceutically important component, ergotamine. This rise cannot be correlated with the concentration of the added PFC and it is thought that the enhancement is due to a combination of factors, including the influence of PFC. Other oxygen vectors, such as several hydrocarbons, prove to be poor oxygen carriers in our study. Cultivations with PFC in a bioreactor are reproducible, the maximum total alkaloid and ergotamine production being attained on the 11th and 9th days, respectively. The relatively lower increase in the total alkaloid production in the bioreactor as compared to the shake-flasks is attributed to the unequal oxygen availability in the reactor. Processes with PFC offer the operational advantage of a five-fold reduction in aeration rate.

Alkaloids↗
PubMed2001

Flow cytometry in biotechnology.

Flow cytometry is a general method for rapidly analyzing large numbers of cells individually using light-scattering, fluorescence, and absorbence measurements. The power of this method lies both in the wide range of cellular parameters that can be determined and in the ability to obtain information on how these parameters are distributed in the cell population. Flow cytometric assays have been developed to determine both cellular characteristics such as size, membrane potential, and intracellular pH, and the levels of cellular components such as DNA, protein, surface receptors, and calcium. Measurements that reveal the distribution of these parameters in cell populations are important for biotechnology, because they better describe the population than the average values obtained from traditional techniques. This Mini-Review provides an overview of the principles of flow cytometry, with descriptions of methods used to measure various cellular parameters and examples of the application of flow cytometry in biotechnology. Finally, a discussion of the challenges and limitations of the method is presented along with a future outlook.

Animals↗
PubMed2000

Microbial degradation of phenanthrene by addition of a sophorolipid mixture.

The influence of sophorolipids on microbial degradation of poorly soluble phenanthrene in liquid and soil suspension culture was evaluated in the work presented. Experiments were carried out in two parts. In the first part, important basic physico-chemical characteristics of the biosurfactant and the pollutant used were determined. The critical micelle concentration (CMC) and the solubilization ratio of the biosurfactant were found to be in a good range compared with synthetic surfactants. Also, a reduction to 71% of the detectable amount of phenanthrene was measured within 4 d in soil suspension without any biotic influence. In the second part, culture experiments were done with Sphingomonas yanoikuyae, the bacterium used throughout the work presented here with the aim to assess the toxicity of the sophorolipids on these bacteria and the effect of the surfactant on biodegradation. In exponential growth tests, no toxicity up to 1 g l(-1) sophorolipids could be detected, whereas in an agar plate test, slight growth hindrance was measured at a lower concentration of 250 mg l(-1). The above mentioned data were important for planning further experiments. In the following cultivations with liquid and soil suspension media, enhancements of the biodegradation with surfactant addition were measurable. Fluorescence measurements showed that this effect was not due to an increasing biomass, but to an augmentation of bioavailability of the phenanthrene through increasing the apparent dissolved pollutant. Surfactant addition had the consequence of decreasing the residual detectable pollutant concentration (after 36 h 0.5 compared with 2.3 mg l(-1) soil suspension) and increasing the maximal degradation rate (127 instead of 80 mg l(-1) soil suspension x 10 h). Therefore, the two main problems of biological soil remediation techniques, longer process time and residual pollutants, may be solved by the use of surfactants.

Biodegradation, Environmental↗
PubMed2000

Monitoring and control of industrial downstream processing of sugar beet molasses.

In present work the determination of several amino acids during the industrial chromatographic desugarisation of molasses is presented. The use of innovative biosensor systems for highly specific detection of serine is described. Using two-dimensional fluorescence spectrometry, a non-invasive method for the determination of several product fractions could be established in an industrial chromatographic procedure.

Biosensing Techniques↗
PubMed2000

Studies on nutritional and oxygen requirements for production of L-asparaginase by Enterobacter aerogenes.

The carbon and nitrogen sources most suitable for L-asparaginase production by Enterobacter aerogenes were selected and their concentrations optimized in shake-flask cultures. Sodium citrate (1.0%) and diammonium hydrogen phosphate (0.16%) proved to be the best sources of carbon and nitrogen, respectively. Nitrogen catabolite repression of enzyme formation was absent in this bacterium. Cultivation in a reactor showed that the dissolved oxygen level is the limiting factor for L-asparaginase production by E. aerogenes. Glucose was found to be a repressor of enzyme synthesis. Asparagine was absent intracellularly when the L-asparaginase level was high. An increase in the extracellular alanine level when the dissolved oxygen remained low indicated a shift from aerobic to fermentative metabolism.

Aerobiosis↗
PubMed1999

A mathematical model for the intracellular circadian rhythm generator.

A mathematical model for the intracellular circadian rhythm generator has been studied, based on a negative feedback of protein products on the transcription rate of their genes. The study is an attempt at examining minimal but biologically realistic requirements for a negative molecular feedback loop involving considerably faster reactions, to produce (slow) circadian oscillations. The model included mRNA and protein production and degradation, along with a negative feedback of the proteins upon mRNA production. The protein production process was described solely by its total duration and a nonlinear term, whereas also the feedback included nonlinear interactions among protein molecules. This system was found to produce robust oscillations in protein and mRNA levels over a wide range of parameter values. Oscillations were slow, with periods much longer than the time constants of any of the individual system parameters. Circadian oscillations were obtained for realistic values of the parameters. The system was readily entrainable to external periodic perturbations. Two distinct classes of phase response curves were found, viz. with or without a time domain within the circadian cycle in which external perturbations fail to induce a phase shift ("dead zone"). The delay and nonlinearity in the protein production and the cooperativity in the negative feedback (Hill coefficient) were for this model found to be necessary and sufficient to generate robust circadian oscillations. The similarities between model outcomes and empirical findings establish that circadian rhythmicity at the cellular level can plausibly emerge from interactions among molecular systems which are not in themselves rhythmic.

Biological Transport↗
PubMed1999

Thermal biosensors in biotechnology.

The application of enzyme thermistor devices for the continuous monitoring of enzymatic processes is described. Different hardware concepts are presented and discussed, practical results are also given. These devices were used to analyze the enantiomeric excess in biotransformation processes and for thermal immunoanalysis. In addition, the biosensors were applied for the monitoring and control of an L-ornithine producing process and for the application in hemodialysis monitoring. A review section discusses the use of thermal biosensors for monitoring biotechnological processes in general.

Biosensing Techniques↗
PubMed1999

A simple method for the isolation and purification of L-asparaginase from Enterobacter aerogenes.

L-Asparaginase from Enterobacter aerogenes was purified by a simple method involving sonication of the crude cell mass, gel filtration with Sephacryl S-100 as the separating material, followed by ultrafiltration. Recent methods involve complex purification procedures of 5-6 steps. The isolation process resulted in 10-fold purification of the enzyme with a specific activity of 55 IU/mg protein and recovery of 54%. The purity was tested by capillary electrophoresis, used for the first time for documenting the purification of L-asparaginase. The choice of the column material was critical in the purification process.

Asparaginase↗
PubMed1999

Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining.

The fluorescence properties of one chemically and seven biologically produced polyhydroxyalkanoic acid were investigated as film castings and in living cells respectively after staining with Nile red. All these polyesters show a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm. This could be shown by the use of two-dimensional fluorescence spectroscopy and flow cytometry. The examination of native poly(3-hydroxybutyric acid), poly(3HB), granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 micrograms Nile red is necessary for total staining of 1.0 mg granules. The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the poly(3HB) concentration of grana suspensions at different grana concentrations. These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a high potential for the quantitative determination of hydrophobic bacterial polyhydroxyalkanoic acids.

Bacteriological Techniques↗