Non-invasive online detection of microbial lysine formation in stirred tank bioreactors by using calorespirometry.

Non-invasive methods for online monitoring of biotechnological processes without compromising the integrity of the reactor system are very important to generate continuous data. Even though calorimetry has been used in conventional biochemical analysis for decades, it has not yet been specifically applied for online detection of product formation at technical scale. Thus, this article demonstrates a calorespirometric method for online detection of microbial lysine formation in stirred tank bioreactors. The respective heat generation of two bacterial strains, Corynebacterium glutamicum ATCC 13032 (wild-type) and C. glutamicum DM1730 (lysine producer), was compared with the O2 -consumption in order to determine whether lysine was formed. As validation of the proposed calorespirometric method, the online results agreed well with the offline measured data. This study has proven that calorespirometry is a viable non-invasive technique to detect product formation at any time point.

Linear correlation between online capacitance and offline biomass measurement up to high cell densities in Escherichia coli fermentations in a pilot-scale pressurized bioreactor.

To yield high concentrations of protein expressed by genetically modified Escherichia coli, it is important that the bacterial strains are cultivated to high cell density in industrial bioprocesses. Since the expressed target protein is mostly accumulated inside the E. coli cells, the cellular product formation can be directly correlated to the bacterial biomass concentration. The typical way to determine this concentration is to sample offline. Such manual sampling, however, wastes time and is not efficient for acquiring direct feedback to control a fedbatch fermentation. An E. coli K12-derived strain was cultivated to high cell density in a pressurized stirred bioreactor on a pilot scale, by detecting biomass concentration online using a capacitance probe. This E. coli strain was grown in pure minimal medium using two carbon sources (glucose and glycerol). By applying exponential feeding profiles corresponding to a constant specific growth rate, the E. coli culture grew under carbon-limited conditions to minimize overflow metabolites. A high linearity was found between capacitance and biomass concentration, whereby up to 85 g/L dry cell weight was measured. To validate the viability of the culture, the oxygen transfer rate (OTR) was determined online, yielding maximum values of 0.69 mol/l/h and 0.98 mol/l/h by using glucose and glycerol as carbon sources, respectively. Consequently, online monitoring of biomass using a capacitance probe provides direct and fast information about the viable E. coli biomass generated under aerobic fermentation conditions at elevated headspace pressures.

Online determination of viable biomass up to very high cell densities in Arxula adeninivorans fermentations using an impedance signal.

Up to now biomass has been measured online by impedance analysis only at low cell densities in yeast fermentations. As industrial fermentation processes focus, for example, on producing high target concentrations of biocatalysts or pharmaceutical proteins, it is important to investigate cell growth under high cell-density conditions. Therefore, for the first time, biomass has been measured online using impedance analysis in a 50L high-pressure stirred tank reactor. As model organism the yeast Arxula adeninivorans was cultivated in two chemically defined mineral media at a constant growth rate in fed-batch mode. To ensure aerobic culture conditions over the entire fermentation time, the fermentations were conducted at an elevated headspace overpressure of up to 9.5bar. The highest oxygen transfer rate value of 0.56molL(-1)h(-1) ever reported for yeast fermentations was measured in these investigations. Unlike previous findings, in this study a linear correlation was found between capacitance and biomass up to concentrations of 174gL(-1) dry cell weight.

High cell-density processes in batch mode of a genetically engineered Escherichia coli strain with minimized overflow metabolism using a pressurized bioreactor.

A common method to minimize overflow metabolism and to enable high cell-density is to operate microbial processes in fed-batch mode under carbon-limiting conditions. This requires sophisticated process control schemes with expensive hardware equipment and software and well-characterized processes parameters. To generate high-cell density, a more simplified strategy would be beneficial. Therefore, a genetically engineered Escherichia coli strain with a modified glucose uptake system was cultivated in batch mode. In the applied strain, the usual phosphotransferase system of a K12-derived strain was inactivated, while the galactose permease system was amplified. Upon cultivating this E. coli strain in pure minimal media, the acetate concentration did not exceed values of 0.35 g L(-1), even when the batch fermentation was started with a glucose concentration of 130 g L(-1). Finally, maximum biomass concentrations of 48 g L(-1) dry cell weight and maximum space-time yields of 2.10 g L(-1) h(-1) were reached. To provide an unlimited growth under fully aerobic conditions (DOT>30%) at comparatively low values for specific power input (3-4 kW m(-3)), a pressurized bioreactor was used. Consequentially, to our knowledge, this study using a bioreactor with elevated headspace pressure generate the highest oxygen transfer rate (451 mmol L(-1) h(-1)) ever reached in batch cultivations.

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.