Biomedical subjects

Jochen Büchs

Publications and source records attributed to Jochen Büchs.

At least 19 recordsLinked to original sources

PubMed2007

Modelling and advanced understanding of unsteady-state gas transfer in shaking bioreactors.

In shaken bioreactors equipped with a sterile closure (e.g. a cotton plug), a realistic understanding and estimation of gas transfer is advantageous to avoid oxygen limitation or carbon dioxide inhibition of a microbial culture. Therefore, in the present study, an unsteady-state gas-transfer model for shake flasks was developed and experimentally investigated for a wide range of gas-transfer resistances (k(plug)). The introduced approach is based on the model of Henzler and co-workers [Henzler and Schedel (1991) Bioprocess Eng. 7, 123-131; Mrotzek, Anderlei, Henzler and Büchs (2001) Biochem. Eng. J. 7, 107-112], which describe the spatially resolved gas partial pressures inside the sterile closure, affecting the local gas diffusion coefficients and convective Stefan flow. For further easy processing the resulting total mass-transfer resistance (k(plug)) is described as a function of the mass flow through the sterile plug (OTR(plug)) by an empirical equation. This equation is introduced into a simulation model which calculates the gas partial pressures in the head space of the flask. Additionally, the gas-transfer rates through the sterile closure and gas/liquid interface inside the flask is provided. Simulations indicate that neglecting the oxygen in the head space volume of the flask under initial conditions (simple steady-state assumption) may lead to an underestimation of the oxygen transfer into the liquid phase. The extension of error depends on the conditions. A good agreement between the introduced unsteady-state model and experimental results for the sulfite oxidation as a chemical model system confirmed the validity and usefulness of the proposed unsteady-state approach.

Bioreactors↗

PubMed2006

An experimental comparison of respiration measuring techniques in fermenters and shake flasks: exhaust gas analyzer vs. RAMOS device vs. respirometer.

Respiration measurement is applied as a universal tool to determine the activity of biological systems. The measurement techniques are difficult to compare, due to the vast variety of devices and analytical procedures commonly in use. They are used in fields as different as microbiology, gene engineering, toxicology, and industrial process monitoring to observe the physiological activity of living systems in environments as diverse as fermenters, shake flasks, lakes and sewage plants. A method is introduced to determine accuracy, quantitation limit, range and precision of different respiration measurement devices. Corynebacterium glutamicum cultures were used to compare an exhaust gas analyzer (EGA), a RAMOS device (respiration measurement in shake flasks) and a respirometer. With all measuring devices it was possible to determine the general culture characteristics. The EGA and the RAMOS device produced almost identical results. The scatter of the respirometer was noticeably higher. The EGA is the technique of choice, if the reaction volume is high or a short reaction time is required. The possibility to monitor cultures simultaneously makes the RAMOS device an indispensable tool for media and strain development. If online monitoring is not compulsive, the respiration of the investigated microbial system extremely low, or the sample size small, a respirometer is recommended.

Bacteriological Techniques↗

PubMed2006

Assaying CO2 release for determination of formate dehydrogenase activity in entrapment matrices and aqueous-organic two-phase systems.

Formate dehydrogenase is an important enzyme for NADH-regeneration in enzyme-catalysed reductions. Methods to determine the activity of this biocatalyst during reaction in aqueous-organic two-phase systems or after immobilisation were therefore investigated. Determination of gaseous CO2 in the headspace of reaction vessels either by gas chromatography or by pressure sensors was found to be a suitable way for deduction of FDH-activity in either of these reaction systems. In the presence of organic solvents, gas chromatography yielded more precise data than pressure sensors, while pressure measurements offer the opportunity to assay continuously the activity of entrapped FDH throughout the whole course of reaction.

Algorithms↗

PubMed2006

Metabolic activity of Corynebacterium glutamicum grown on L: -lactic acid under stress.

Respiration measurement in shake flasks is introduced as a new method to characterize the metabolic activity of microorganisms during and after stress exposure. The major advantage of the new method is the possibility to determine the metabolic activity independent of manual sampling without the necessity to change the culture vessel or the cultivation medium. This excludes stress factors, which may be induced by transferring the microorganisms to plates or respirometers. The negative influence, which interruptions of the shaker during sampling times may have on the growth of microorganisms was demonstrated. The applicability of the method was verified by characterizing the behavior of Corynebacterium glutamicum grown on the carbon source L: -lactic acid under stress factors such as carbon starvation, anaerobic conditions, lactic acid, osmolarity, and pH. The following conditions had no effect on the metabolic activity of C. glutamicum: a carbon starvation of up to 19 h, anaerobic conditions, lactic acid concentrations up to 10 g/l, 3-(N-morpholino) propanesulfonic acid buffer concentrations up to 42 g/l, or pH from 6.4 to 7.4. Lactic-acid concentrations from 10 to 30 g/l lead to a decrease of the growth rate and the biomass substrate yield without effecting the oxygen substrate conversion. Without adaptation, the organism did not grow at pH< or =5 or > or =9.

Anaerobiosis↗

PubMed2006

Hydromechanical stress in shake flasks: correlation for the maximum local energy dissipation rate.

Shake flasks are widely used in biotechnological process research. Bioprocesses for which hydromechanical stress may become the rate controlling parameter include those where oils are applied as carbon sources, biotransformation of compounds with low solubility in the aqueous phase, or processes employing animal, plant, or filamentous microorganisms. In this study, the maximum local energy dissipation rate as the measure for hydromechanical stress is characterized in shake flasks by measuring the maximum stable drop size. The theoretical basis for the method is that the maximum stable drop diameter in a coalescence inhibited liquid/liquid dispersion is only a function of the maximum local energy dissipation rate and not of the dispersing apparatus. The maximum local energy dissipation rate is obtained by comparing the drop diameters in shake flasks to those in a stirred tank reactor. At the same volumetric power consumption, the maximum energy dissipation rate in shake flasks is about 10 times lower than in stirred tank reactors explaining the common observation of considerable differences in the morphology of hydromechanically sensitive cells between these two reactor types. At the same volumetric power consumption, the maximum local energy dissipation rate in baffled and in unbaffled shake flasks is very similar. A correlation is presented to quantify the maximum local energy dissipation rate in shake flasks as a function of the operating conditions. Non-negligible drop viscosity may be considered by known literature correlations. Further, from dispersion experiments a critical Reynolds number of about 60,000 is proposed for turbulent flow in unbaffled shake flasks.

Algorithms↗

PubMed2006

Oxygen limitation is a pitfall during screening for industrial strains.

Oxygen supply is a key parameter in aerobic fermentation processes like the industrial production of amino acids. Although the oxygen transfer rate (OTR; or the volumetric oxygen transfer coefficient k(L)a) is routinely analyzed by engineers during stirred tank fermentations, it is often not taken into account by biologists conducting screening experiments in shake flasks. To show the importance of knowing how to avoid oxygen transfer limitations during primary screenings, Corynebacterium glutamicum ATCC 13032 (wild-type strain) and DSM 12866 (lysine-producing strain) were cultivated in shake flasks with different culture liquid volumes and under different shaking conditions. With the Respiration Activity Monitoring System, the OTR was determined quasi-continuously. Optical density as well as concentrations of lysine and byproducts (lactate, acetate, succinate) were determined off-line and correlated with the OTR signal. From the results, design criteria for improved screening in shaken bioreactors that help to avoid selection of suboptimal strains during early process development steps can be derived. Finally, the suitability of DSM 12866 as a strain for industrial processes with a high space-time yield is discussed.

Acetic Acid↗

PubMed2005

Oxygen transfer phenomena in 48-well microtiter plates: determination by optical monitoring of sulfite oxidation and verification by real-time measurement during microbial growth.

Oxygen limitation is one of the most frequent problems associated with the application of shaking bioreactors. The gas-liquid oxygen transfer properties of shaken 48-well microtiter plates (MTPs) were analyzed at different filling volumes, shaking diameters, and shaking frequencies. On the one hand, an optical method based on sulfite oxidation was used as a chemical model system to determine the maximum oxygen transfer capacity (OTR(max)). On the other hand, the Respiration Activity Monitoring System (RAMOS) was applied for online measurement of the oxygen transfer rate (OTR) during growth of the methylotropic yeast Hansenula polymorpha. A proportionality constant between the OTR(max) of the biological system and the OTR(max) of the chemical system were indicated from these data, offering the possibility to transform the whole set of chemical data to biologically relevant conditions. The results exposed "out of phase" shaking conditions at a shaking diameter of 1 mm, which were confirmed by theoretical consideration with the phase number (Ph). At larger shaking diameters (2-50 mm) the oxygen transfer rate in MTPs shaken at high frequencies reached values of up to 0.28 mol/L/h, corresponding to a volumetric mass transfer coefficient (k(L)a) of 1,600 1/h. The specific mass transfer area (a) increases exponentially with the shaking frequency up to values of 2,400 1/m. On the contrary, the mass transfer coefficient (k(L)) is constant at a level of about 0.15 m/h over a wide range of shaking frequencies and shaking diameters. However, at high shaking frequencies, when the complete liquid volume forms a thin film on the cylindric wall of the well, the mass transfer coefficient (k(L)) increases linearly to values of up to 0.76 m/h. Essentially, the present investigation demonstrates that the 48-well plate outperforms the 96-well MTP and shake flasks at widely used operating conditions with respect to oxygen supply. The 48-well plates emerge, therefore, as an excellent alternative for microbial cultivation and expression studies combining the advantages of both the high-throughput 96-well MTP and the classical shaken Erlenmeyer flask.

Bioreactors↗

PubMed2005

The oxygen mass transfer, carbon dioxide inhibition, heat removal, and the energy and cost efficiencies of high pressure fermentation.

This study focuses on the enhancement of oxygen transfer in aerobic fermentation processes through the use of the reactor headspace pressure as a process variable. Our investigation was performed using an example 5 m3 fermentor, and by applying the mass transfer (sorption) characteristics obtained from measurements with Pseudomonas putida CA-3 in a mineral medium. By applying reactor pressures up to 11 bar (10 bar overpressure) the oxygen transfer capacities of stirred tank reactors can theoretically be increased to values above 1 kmol m(-3) h(-1), although the problem of heat removal limits that value to levels of 0.7 kmol m(-3) h(-1). However, this is still sufficient for the oxygenation of most aerobic fermentation processes, even at very high cell densities. Possible inhibition by the carbon dioxide partial pressure at elevated total pressures is discussed. It is shown that increasing reactor pressure enhances the energy efficiency, defined as the ratio of the oxygen transfer capacity to the total power consumption of the reactor system. The cost efficiency of the oxygen mass transfer, defined as the ratio of the oxygen transfer capacity to the overall cost of the system (overall purchase cost divided by a project lifetime of ten years, plus total energy cost) also increases with reactor pressure up to 5.6 bar.

Bioreactors↗

PubMed2005

Transponder-based sensor for monitoring electrical properties of biological cell solutions.

An inductive passive remote sensor circuit for monitoring fermentation processes is presented. The sensor circuit consists of an interdigital capacitor and a planar coil structured on a glass laminated FR4-printed circuit board. This circuit resonates at frequencies between 2 and 4 MHz. After the resonant sensor circuit is immersed in a fermentation vessel with a cell solution, the resonant frequencies are detected by measuring the impedance of an external loop antenna. A new theory is presented to describe the behavior of the sensor circuit. In combination with a proposed equivalent circuit, the theory enables the calculation of the permittivity and conductivity of the cell solution under test by determining the resonant frequencies of the sensor without the need for any additional fitting functions. The influence of the relaxation behavior of living cells on the sensor signal with respect to the conductivity of the solution is discussed in detail. To prove the new theory, the determined permittivity is compared with the optical density of a cell solution, an indicator of cell concentration. The performed measurements show the expected correlation between the determined permittivity and optical density. The solution under test is a yeast culture in YPG medium.

Biosensing Techniques↗

PubMed2005

Optimization of adsorptive immobilization of alcohol dehydrogenases.

In this work, a systematic examination of various parameters of adsorptive immobilization of alcohol dehydrogenases (ADHs) on solid support is performed and the impact of these parameters on immobilization efficiency is studied. Depending on the source of the enzymes, these parameters differently influence the immobilization efficiency, expressed in terms of residual activity and protein loading. Residual activity of 79% was achieved with ADH from bakers' yeast (YADH) after optimizing the immobilization parameters. A step-wise drying process has been found to be more effective than one-step drying. A hypothesis of deactivation through bubble nucleation during drying of the enzyme/glass bead suspension at low drying pressure (<45 kPa) is experimentally verified. In the case of ADH from Lactobacillus brevis (LBADH), >300% residual activity was found after drying. Hyperactivation of the enzyme is probably caused by structural changes in the enzyme molecule during the drying process. ADH from Thermoanaerobacter species (ADH T) is found to be stable under drying conditions (>15 kPa) in contrast to LBADH and YADH.

Adsorption↗

PubMed2005

Method for quantitative determination of spatial polymer distribution in alginate beads using Raman spectroscopy.

A new method based on Raman spectroscopy is presented for non-invasive, quantitative determination of the spatial polymer distribution in alginate beads of approximately 4 mm diameter. With the experimental setup, a two-dimensional image is created along a thin measuring line through the bead comprising one spatial and one spectral dimension. For quantitative analysis of the Raman spectra, the method of indirect hard modeling was applied to make use of the information contained in the entire recorded spectra. For quantification of the alginate signals from within the beads, a calibration curve acquired from sodium alginate solutions was used after it was shown that only negligible differences occur between signals from alginate solutions and alginate gels. The distribution of alginate over the bead gel matrix was acquired with high spatial (51 microm) and time (12 s) resolution. The inhomogeneous distribution obtained using the new measuring technique is qualitatively in excellent agreement with data from the literature. In contrast to known measuring techniques, correct quantitative information about the spatial polymer distribution within the matrix was derived. It gave an alginate mass fraction of approximately 0.045 g/g at the edges and 0.02 g/g in the center of the beads. Next to the determination of mere polymer concentrations, the excellent time resolution of the presented method will enable investigation of the dynamic process of gel formation and it will also serve as a basis for investigation of mass transfer of small diffusing molecules in alginate matrices.

Alginates↗

PubMed2004

New insights in the spatially resolved dynamic pH measurement in macroscopic large absorbent particles by confocal laser scanning microscopy.

Both, experimental investigation of protein adsorption processes and mathematical models describing such processes indicate, that the pH in the absorbent particle might be the key factor for an improved understanding of these chromatographic processes. Thus, a technique aiming at the spatially resolved pH measurement in macroscopic large absorbent particles is presented. The first application of this method, being based on confocal laser scanning microscopy (CLSM), revealed an apparent dependence of the pH calibration curve on the scanning depth. By a model-based approach, factors distorting the measurement signal are identified: The wavelength-dependent light scattering and the re-absorption of emitted light. The resulting consequences for further development and application of CLSM based techniques to measure pH in macroscopic large absorbent particles are illustrated and discussed.

Adsorption↗

PubMed2003

The oxygen transfer rate as key parameter for the characterization of Hansenula polymorpha screening cultures.

Screening cultures are usually non-monitored and non-controlled due to a lack of appropriate measuring techniques. A new device for online measurement of oxygen transfer rate (OTR) in shaking-flask cultures was used for monitoring the screening of Hansenula polymorpha. A shaking frequency of 300 rpm and a filling volume of 20 ml in 250-ml flasks ensured a sufficient oxygen transfer capacity of 0.032 mol (l h)(-1) and thus a respiration not limited by oxygen. Medium buffered with 0.01 mol phosphate l(-1) (pH 6.0) resulted in pH-inhibited respiration, whereas buffering with 0.12 mol phosphate l(-1) (pH 4.1) resulted in respiration that was not inhibited by pH. The ammonium demand was balanced by establishing fixed relations between oxygen, ammonium, and glycerol consumption with 0.245+/-0.015 mol ammonium per mol glycerol. Plate precultures with complex glucose medium reduced the specific growth rate coefficient to 0.18 h(-1) in subsequent cultures with minimal glycerol medium. The specific growth rate coefficient increased to 0.26 h(-1) when exponentially growing precultures with minimal glycerol medium were used for inoculation. Changes in biomass, glycerol, ammonium, and pH over time were simulated on the basis of oxygen consumption.

Antineoplastic Agents↗

PubMed2003

Characterization of gas-liquid mass transfer phenomena in microtiter plates.

Gas-liquid mass transfer properties of shaken 96-well microtiter plates were characterized using a recently described method. The maximum oxygen transfer capacity (OTR(max)), the specific mass transfer area (a), and the mass transfer coefficient (k(L)) in a single well were determined at different shaking intensities (different shaking frequencies and shaking diameters at constant filling volume) and different filling volumes by means of sulfite oxidation as a chemical model system. The shape (round and square cross-sections) and the size (up to 2 mL maximum filling volume) of a microtiter plate well were also considered as influencing parameters. To get an indication of the hydrodynamic behavior of the liquid phase in a well, images were taken during shaking and the liquid height derived as a characteristic parameter. The investigations revealed that the OTR(max) is predominantly dependent on the specific mass transfer area (a) for the considered conditions in round-shaped wells. The mass transfer coefficient (k(L)) in round-shaped wells remains at a nearly constant value of about 0.2 m/h for all shaking intensities, thus within the range reported in the literature for surface-aerated bioreactors. The OTR(max) in round-shaped wells is strongly influenced by the interfacial tension, determined by the surface tension of the medium used and the surface properties of the well material. Up to a specific shaking intensity the liquid surface in the wells remains horizontal and no liquid movement can be observed. This critical shaking intensity must be exceeded to overcome the surface tension and, thus, to increase the liquid height and enlarge the specific mass transfer area. This behavior is solely specific to microtiter plates and has not yet been observed for larger shaking bioreactors such as shaking flasks. In square-shaped microtiter plate wells the corners act as baffles and cause a significant increase of OTR(max), a, and k(L). An OTR(max) of up to 0.15 mol/L/h can be reached in square-shaped wells.

Bacteriological Techniques↗

PubMed2003

Necessity of a two-stage process for the production of azadirachtin-related limonoids in suspension cultures of Azadirachta indica.

The effect of major nutrients on growth and azadirachtin-related limonoids (AZRL) production in plant cell culture of Azadirachta indica (neem) was studied with the objective to increase the yield of AZRL, one of the major group of pesticidal compounds found in intact neem trees. We report the novel online monitoring of plant cell respiration activities in a new parallel shake flask measuring device. Results obtained using three standard plant cell culture media showed non-growth-associated production characteristics for AZRL. These findings were supported by the oxygen uptake rate data. Further investigations on AZRL production in a modified MS medium with different concentrations of nitrogen and phosphorus sources resulted in 0.25 mg.g(-1) dry weight of AZRL, compared to no detectable AZRL production in standard MS media. These characteristics suggest the necessity of a two-stage process for the production of AZRL in plant cell culture. Compared to the single-stage process, an almost twofold increase in the volumetric productivity of AZRL was achieved using the two-stage process.

Journal Article↗

PubMed2003

Effect of oxygen supply on passaging, stabilising and screening of recombinant Hansenula polymorpha production strains in test tube cultures.

Twenty-four Hansenula polymorpha transformants were passaged and stabilised in glucose medium and screened in glycerol medium for recombinant phytase in shaken test tubes. The cultivations were performed under either limited or non-limited oxygen supply. Maximum oxygen transfer capacities of test tubes were assessed by sulfite oxidation. Oxygen-limited glucose cultures resulted in a partially anaerobic metabolism and formation of 4.1 g ethanol l(-1), which was subsequently aerobically metabolised. Non-limited oxygen supply led to overflow metabolism and to accumulation of 2.1 g acetic acid l(-1), reducing the biomass yield. The use of glycerol in the screening main cultures prevented by-product formation irrespective of oxygen supply. Preculturing in glucose medium under non-limited oxygen supply resulted in a 20-h lag phase of the screening main culture. This lag phase was not observed when preculturing was performed under oxygen limitation. Phytase activity was on average 25% higher in cultures passaged, stabilised and screened under limited oxygen supply than in cultures under non-limited oxygen supply.

6-Phytase↗

PubMedDate not supplied

Effect of oxygen limitation and medium composition on Escherichia coli fermentation in shake-flask cultures.

Shake-flask cultures are widely used for screening of high producing strains. To select suitable strains for production scale, cultivation parameters should be applied that provide optimal growth conditions. A novel method of measuring respiratory activity in shake-flask cultures was employed to analyze Escherichia coli fermentation under laboratory conditions. Our results suggest that the length of fermentation, choice of medium, and aeration do not normally satisfy the requirements for unlimited growth in shake flasks. Using glycerol rather than glucose as a carbon source greatly reduced the accumulation of overflow and fermentative metabolites when oxygen supply was unlimited. A rich buffered medium, Terrific Broth (TB), yielded 5 times more biomass compared to LB medium but also caused oxygen limitation in standard shake-flask cultures at shaking frequencies below 400 rpm. These results were used to optimize the production of benzoylformate decarboxylase from Pseudomonas putida in E. coli SG13009, resulting in a 10-fold increase in volumetric enzyme production. This example demonstrates how variation of medium composition and oxygen supply can be evaluated by the measurement of the respiratory activity. This can help to efficiently optimize screening conditions for E. coli.

Carboxy-Lyases↗

PubMedDate not supplied

A novel parallel shaken bioreactor system for continuous operation.

A novel continuous bioreactor system was developed as a shaken culture vessel for the investigation of the growth kinetics and product formation of microorganisms in milliscale. The novel bioreactor system mainly consists of a specially designed 250-mL shake flask with two inlets, one for gas supply and one for medium supply, and one combined outlet on the side of flask for exhaust gas and culture liquid. As a result of the circulating motion of the fermentation broth in the shake flask, the maximum liquid height reaches the edge of the outlet and the fermentation broth is accelerated into the outlet by centrifugal force. Additionally, the excess fermentation broth leaving the culture vessel is continuously driven by the exhaust gas. Because of the small scale and the simple handling it is possible to operate many of these shaken bioreactor vessels simultaneously. By using parallel vessels operated at different dilution rates on the same shaker, the data for a complete biomass over dilution rate (X-D) diagram of a biological culture can be evaluated in an efficient manner, thus saving money, materials, and time. Continuous fermentations of the yeast Saccharomyces cerevisiae H1022 (ATCC 32167) in the shaken bioreactor system and in a conventional stirred tank fermentor showed very similar results.

Bioreactors↗