Lars Regestein

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.

Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors

BackgroundConventional experiments in small scale are often performed in a ‘Black Box’ fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device.ResultsThe same ‘mastermix’ (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects).ConclusionsThe parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.

Potentials and limitations of miniaturized calorimeters for bioprocess monitoring.

In theory, heat production rates are very well suited for analysing and controlling bioprocesses on different scales from a few nanolitres up to many cubic metres. Any bioconversion is accompanied by a production (exothermic) or consumption (endothermic) of heat. The heat is tightly connected with the stoichiometry of the bioprocess via the law of Hess, and its rate is connected to the kinetics of the process. Heat signals provide real-time information of bioprocesses. The combination of heat measurements with respirometry is theoretically suited for the quantification of the coupling between catabolic and anabolic reactions. Heat measurements have also practical advantages. Unlike most other biochemical sensors, thermal transducers can be mounted in a protected way that prevents fouling, thereby minimizing response drifts. Finally, calorimetry works in optically opaque solutions and does not require labelling or reactants. It is surprising to see that despite all these advantages, calorimetry has rarely been applied to monitor and control bioprocesses with intact cells in the laboratory, industrial bioreactors or ecosystems. This review article analyses the reasons for this omission, discusses the additional information calorimetry can provide in comparison with respirometry and presents miniaturization as a potential way to overcome some inherent weaknesses of conventional calorimetry. It will be discussed for which sample types and scientific question miniaturized calorimeter can be advantageously applied. A few examples from different fields of microbiological and biotechnological research will illustrate the potentials and limitations of chip calorimetry. Finally, the future of chip calorimetry is addressed in an outlook.

Comparison of oxygen enriched air vs. pressure cultivations to increase oxygen transfer and to scale‐up plasmid DNA production fermentations

Escherichia coli producing a plasmid DNA (pDNA) vaccine was cultivated in fed‐batch mode at small scale (1 L) using oxygen‐enriched air, and at pilot scale (50 L) using a pressurized bioreactor, to maintain aerobic conditions. In the small scale, the attained oxygen transfer rate (OTRMAX) using an oxygen concentration in the inlet gas of 68.2%, reached 0.42 mol L−1 h−1. The OTRMAX in the pressurized reactor with an overpressure of 8 bar was 0.5 mol L−1 h−1. In the small‐ and pilot‐scale cultivations, the final biomass concentrations (74 and 79 g/L, respectively), pDNA concentrations (236 and 215 mg/L), overall productivity and pDNA topology were very similar. Therefore, the pressurized cultivation is a viable option to scale up pDNA production processes.

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.

Comparison of two methods for designing calorimeters using stirred tank reactors

Calorimetry is a robust method for online monitoring and controlling bioprocesses in stirred tank reactors. Up to now, reactor calorimeters have not been optimally constructed for pilot scale applications. Thus, the objective of this paper is to compare two different ways for designing reactor calorimeters and validate them. The “heat capacity” method based on the mass flow of the cooling liquid in the jacket was compared with the “heat transfer” method based on the heat transfer coefficient continuously measured in the cultivation of Escherichia coli VH33 in a 50 L stirred tank reactor. It was found that the values of the “heat transfer” method agreed very well with the calculated values from the oxygen consumption. By contrast, the curve of the “heat capacity” method deviated from that of the oxygen consumption calculated with the oxycaloric equivalent. In conclusion, the “heat transfer” method has been proven to have a higher degree of validity than the “heat capacity” method. Thus, it is a better and more robust means to measure heat generation of fermentations in stirred tank bioreactors on a pilot scale. Biotechnol. Bioeng. 2013; 110: 180–190.

Online monitoring of fermentation processes via non‐invasive low‐field NMR

For the development of biotechnological processes in academia as well as in industry new techniques are required which enable online monitoring for process characterization and control. Nuclear magnetic resonance (NMR) spectroscopy is a promising analytical tool, which has already found broad applications in offline process analysis. The use of online monitoring, however, is oftentimes constrained by high complexity of custom‐made NMR bioreactors and considerable costs for high‐field NMR instruments (>US

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

200,000). Therefore, low‐field 1H NMR was investigated in this study in a bypass system for real‐time observation of fermentation processes. The new technique was validated with two microbial systems. For the yeast Hansenula polymorpha glycerol consumption could accurately be assessed in spite of the presence of high amounts of complex constituents in the medium. During cultivation of the fungal strain Ustilago maydis, which is accompanied by the formation of several by‐products, the concentrations of glucose, itaconic acid, and the relative amount of glycolipids could be quantified. While low‐field spectra are characterized by reduced spectral resolution compared to high‐field NMR, the compact design combined with the high temporal resolution (15 s–8 min) of spectra acquisition allowed online monitoring of the respective processes. Both applications clearly demonstrate that the investigated technique is well suited for reaction monitoring in opaque media while at the same time it is highly robust and chemically specific. It can thus be concluded that low‐field NMR spectroscopy has a great potential for non‐invasive online monitoring of biotechnological processes at the research and practical industrial scales. Biotechnol. Bioeng. 2015;112: 1810–1821.

Impact of butyric acid on butanol formation by Clostridium pasteurianum.

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. Biotechnol. Bioeng. 2013; 110: 1386–1395.

In Situ Product Recovery of Single-Chain Antibodies in a Membrane Bioreactor

The butanol yield of the classic fermentative acetone-butanol-ethanol (ABE) process has been enhanced in the past decades through the development of better strains and advanced process design. Nevertheless, by-product formation and the incomplete conversion of intermediates still decrease the butanol yield. This study demonstrates the potential of increasing the butanol yield from glycerol though the addition of small amounts of butyric acid. The impact of butyric acid was investigated in a 7L stirred tank reactor. The results of this study show the positive impact of butyric acid on butanol yield under pH controlled conditions and the metabolic stages were monitored via online measurement of carbon dioxide formation, pH value and redox potential. Butyric acid could significantly increase the butanol yield at low pH values if sufficient quantities of primary carbon source (glycerol) were present.