A fully online sensor-equipped,disposable multiphase microbioreactor as a screening platform for biotechnological applications

A new disposable, multiphase, microbioreactor (MBR; with a working volume of 550 μl) equipped with online sensors is presented for biotechnological screening research purposes owing to its high-throughput potential. Its design and fabrication, online sensor integration, and operation are described. During aerobic cultivation, sufficient oxygen supply is the most important factor that influences growth and product formation. The MBR is a microbubble column bioreactor (μBC), and the oxygen supply was realized by active pneumatic bubble aeration, ensuring sufficient volumetric liquid-phase mass transfer (k _L a) and proper homogenization of the cultivation broth. The μBC was equipped with miniaturized sensors for the pH, dissolved oxygen, optical density and glucose concentration that allowed real-time online monitoring of these process variables during cultivation. The challenge addressed here was the integration of sensors in the limited available space. The MBR was shown to be a suitable screening platform for the cultivation of biological systems. Batch cultivations of Saccharomyces cerevisiae were performed to observe the variation in the process variables over time and to show the robustness and operability of all the online sensors in the MBR.     

Characterisation of operation conditions and online monitoring of physiological culture parameters in shaken 24-well microtiter plates

A new online monitoring technique to measure the physiological parameters, dissolved oxygen (DO) and pH of microbial cultures in continuously shaken 24-well microtiter plates (MTP) is introduced. The new technology is based on immobilised fluorophores at the bottom of standard 24-well MTPs. The sensor MTP is installed in a sensor dish reader, which can be fixed on an orbital shaker. This approach allows real online measurements of physiological parameters during continuous shaking of cultures without interrupting mixing and mass transfer like currently available technologies do. The oxygen transfer conditions at one constant shaking frequency (250 1/min) and diameter (25 mm) was examined with the chemical sulphite oxidation method. Varied filling volumes (600–1,200 μL) of Escherichia coli cultures demonstrated the importance of sufficient oxygen transfer to the culture. Cultures with higher filling volumes were subjected to an oxygen limitation, which influenced the cell metabolism and prolongated the cultivation time. The effects could be clearly monitored by online DO and pH measurements. A further study of different media in an E. coli fermentation elucidated the different growth behaviour in response to the medium composition. The MTP fermentations correlated very well with parallel fermentations in shake flasks. The new technique gives valuable new insights into biological processes at a very small scale, thus enabling parallel experimentation and shorter development times in bioprocessing.     

Development of a novel membrane aerated hollow-fiber microbioreactor

A new challenge in biotechnological processes is the development of flexible bioprocessing platforms, allowing strain selection, facilitating scale-up and integrating separation steps. Miniaturization of such a cultivation system allows parallel use and the saving of resources but makes the supply of oxygen to the cells difficult. In this work we present a membrane aerated hollow-fiber microbioreactor (HFMBR) which consists of an acrylic glass module equipped with two different types of membrane fibers. Fibers of polyethersulfone and polyvinyldifluoride were used for substrate and oxygen supply, respectively. Cultivation of E. coli as model organism and production of His-tagged GFP were carried out in the extracapillary space of the membrane aerated HFMBR and compared with cultivations in shaking flask which are commonly used for screening experiments. The measurement of the oxygen transfer capacity and the online monitoring of the dissolved oxygen during the cultivation were performed using a fiber optic oxygen sensor. Online measurement of the optical density was also integrated to the bioreactor. Due to efficient oxygen transfer, a better cell growth than in the shaking flask experiments was achieved, while no negative influence on the GFP productivity was observed in the membrane aerated bioreactor. Thus the feasibility of a future integrated downstreaming could also be demonstrated.     

Glucose-containing polymer rings enable fed-batch operation in microtiter plates with parallel online measurement of scattered light,fluorescence, dissolved oxygen tension,and pH

Bioprocesses operated in batch mode can induce adverse effects like overflow metabolism, substrate inhibition, osmotic inhibition, oxygen limitation, and catabolite repression. To avoid these adverse effects, fed-batch is the predominant operation mode in industrial production. Nevertheless, screening for optimal production strains is usually performed in microtiter plates and shake flasks operated in batch mode without any online monitoring. Recently, a polymer-based controlled-release fed-batch microtiter plate with stable glucose release characteristics was described. In this study, a glucose-containing polymer matrix was used to manufacture polymer rings that were placed at the bottom of a 48-well microtiter plate. Thereby, the liquid content of the well became accessible for optical measurement by the BioLector device. Reflections caused by the polymer ring were minimized by adjusting the scattered-light measurement position. Influences on the measurement of the dissolved oxygen tension and pH could be avoided by choosing appropriate polymer-ring geometries. These adjustments enabled parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH of Escherichia coli BL21 (DE3) fed-batch cultivations. The online monitoring and fed-batch operation capabilities of the fed-batch microtiter plate presented in this study finds optimal application in screenings and initial process development.     

Multiplex bacterial growth monitoring in 24-well microplates using a dual optical sensor for dissolved oxygen and pH

Non-invasive, simultaneous optical monitoring of oxygen and pH during bacterial cultivation in 24-well microplates is presented using an integrated dual sensor for dissolved oxygen and pH values. The dual sensor is based on oxygen-sensitive organosilica microparticles and pH-sensitive microbeads from a polymethacrylate derivative embedded into a polyurethane hydrogel. The readout is based on a phase-domain fluorescence lifetime-based method referred to as modified frequency domain dual lifetime referencing using a commercially available detector system for 24-well microplates. The sensor was used for monitoring the growth of Pseudomonas putida bacterial cultures. The method is suitable for parallelized, miniaturized bioprocessing, and cell-based high-throughput screening applications.     

Design, operation, and modeling of a membrane photobioreactor to study the growth of the Cyanobacterium Arthrospira platensis in space conditions

A membrane photobioreactor was designed, implemented and used to grow the cyanobacterium Arthrospira platensis PCC 8005 in batch mode. Growth was followed directly by monitoring optical density and indirectly by measuring pressure increase due to the oxygen produced and separated from the liquid phase by diffusion through a hydrophobic membrane, and pH increase due to carbon consumption. When the pressure attained an upper limit, valves opened automatically, and the oxygen in the gas chamber was flushed out with nitrogen. As expected, two growth phases were observed, a short exponential phase followed by a linear phase, indicating limitation by light transfer. Growth rate during the second phase was measured easily and accurately, and consistency of optical density, pressure and pH data values was checked using a model of the system. Pressure measurement was found best suited to monitoring and measuring growth rate in space in terms of accuracy, precision and reliability.     

Comparisons of optical pH and dissolved oxygen sensors with traditional electrochemical probes during mammalian cell culture

Small-scale upstream bioprocess development often occurs in flasks and multi-well plates. These culturing platforms are often not equipped to accurately monitor and control critical process parameters; thus they may not yield conditions representative of manufacturing. In response, we and others have developed optical sensors that enable small-scale process monitoring. Here we have compared two parameters critical to control in industrial cell culture, pH and dissolved oxygen (DO), measured with our optical sensors versus industrially accepted electrochemical probes. For both optical sensors, agreement with the corresponding electrochemical probe was excellent. The Pearson Correlations between the optical sensors and electrochemical probes were 98.7% and 99.7%, for DO and pH, respectively. Also, we have compared optical pH sensor performance in regular (320 mOsm/kg) and high-osmolality (450 mOsm/kg) cell culture media to simulate the increase in osmolality in pH-controlled cultures. Over a pH range of 6.38-7.98 the average difference in pH readings in the two media was 0.04 pH units. In summary, we have demonstrated that these optical sensors agree well with standard electrochemical probes. The accuracy of the optical probes demonstrates their ability to detect potential parameter drift that could have significant impact on growth, production kinetics, and protein product quality. We have also shown that an increase in osmolality that could result from controlling pH or operating the reactor in fed-batch mode has an insignificant impact on the functionality of the pH patches.     

Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes

Background

Bioprocesses depend on a number of different operating parameters and temperature is one of the most important ones. Unfortunately, systems for rapid determination of temperature dependent reaction kinetics are rare. Obviously, there is a need for a high-throughput screening procedure of temperature dependent process behavior. Even though, well equipped micro-bioreactors are a promising approach sufficient temperature control is quite challenging and rather complex.

Results

In this work a unique system is presented combining an optical on-line monitoring device with a customized temperature control unit for 96 well microtiter plates. By exposing microtiter plates to specific temperature profiles, high-throughput temperature optimization for microbial and enzymatic systems in a micro-scale of 200 μL is realized. For single well resolved temperature measurement fluorescence thermometry was used, combining the fluorescent dyes Rhodamin B and Rhodamin 110. The real time monitoring of the microbial and enzymatic reactions provides extensive data output. To evaluate this novel system the temperature optima for Escherichia coli and Kluyveromyces lactis regarding growth and recombinant protein production were determined. Furthermore, the commercial cellulase mixture Celluclast as a representative for enzymes was investigated applying a fluorescent activity assay.

Conclusion

Microtiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes. It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time. The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system.

     

A photobioreactor system for precision cultivation of photoautotrophic microorganisms and for high-content analysis of suspension dynamics

Small-scale photobioreactors for cultivation of photoautotrophic microbes are required for precise characterization of the growth parameters of wild-type and engineered strains of these organisms, for their screening, and for optimization of culture conditions. Here, we describe the design and use of a flat-cuvette photobioreactor that allows accurate control of culture irradiance, temperature, pH, and gas composition combined with real-time monitoring by a built-in fluorometer and densitometer. The high-power LED light source generates precise irradiance levels that are programmed by user-designed protocols. The irradiance, temperature, and gas composition may be static or dynamically modulated, while optical density and pH may be stabilized in turbidostat and pH-stat modes, respectively. We demonstrate that the instrument is able to detect minute variations of growth caused, for example, by sudden dilution or by circadian rhythms. The sensitivity of the instrument is sufficient to monitor suspension optical density as low as 10(-2). This newly designed photobioreactor can significantly contribute to the study and use of photoautotrophic microbes in systems biology and biotechnology.     

The baffled microtiter plate: Increased oxygen transfer and improved online monitoring in small scale fermentations

Most experiments in screening and process development are performed in shaken bioreactors. Today, microtiter plates are the preferred vessels for small‐scale microbial cultivations in high throughput, even though they have never been optimized for this purpose. To interpret the experimental results correctly and to obtain a base for a meaningful scale‐up, sufficient oxygen supply to the culture liquid is crucial. For shaken bioreactors this problem can generally be addressed by the introduction of baffles. Therefore, the focus of this study is to investigate how baffling and the well geometry affect the maximum oxygen transfer capacity (OTR_max) in microtiter plates. On a 48‐well plate scale, 30 different cross‐section geometries of a well were studied. It could be shown that the introduction of baffles into the common circular cylinder of a microtiter plate well doubles the maximum oxygen transfer capacity, resulting in values above 100 mmol/L/h (k_La > 600 1/h). To also guarantee a high volume for microbial cultivation, it is important to maximize the filling volume, applicable during orbital shaking. Additionally, the liquid height at the well bottom was examined, which is a decisive parameter for online‐monitoring systems such as the BioLector. This technology performs fiber‐optical measurements through the well bottom, therefore requires a constant liquid height at all shaking frequencies. Ultimately, a six‐petal flower‐shaped well geometry was shown to be the optimal solution taking into account all aforementioned criteria. With its favorable culture conditions and the possibility for unrestricted online monitoring, this novel microtiter plate is an efficient tool to gain meaningful results for interpreting and scaling‐up experiments in clone screening and bioprocess development. Biotechnol. Bioeng. 2009;103: 1118–1128. © 2009 Wiley Periodicals, Inc.     

Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates

Background  

An advanced version of a recently reported high-throughput fermentation system with online measurement, called BioLector, and its validation is presented. The technology combines high-throughput screening and high-information content by applying online monitoring of scattered light and fluorescence intensities in continuously shaken microtiter plates. Various examples in calibration of the optical measurements, clone and media screening and promoter characterization are given.     

Screening for enzyme activity in turbid suspensions with scattered light

New screening techniques for improved enzyme variants in turbid media are urgently required in many industries such as the detergent and food industry. Here, a new method is presented to measure enzyme activity in different types of substrate suspensions. This method allows a semiquantitative determination of protease activity using native protein substrates. Unlike conventional techniques for measurement of enzyme activity, the BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of samples in microtiter plates. The BioLector technique is hereby used to monitor the hydrolysis of an insoluble protein substrate by measuring the decrease of scattered light. The kinetic parameters for the enzyme reaction (V(max,app) and K(m,app)) are determined from the scattered light curves. Moreover, the influence of pH on the protease activity is investigated. The optimal pH value for protease activity was determined to be between pH 8 to 11 and the activities of five subtilisin serine proteases with variations in the amino acid sequence were compared. The presented method enables proteases from genetically modified strains to be easily characterized and compared. Moreover, this method can be applied to other enzyme systems that catalyze various reactions such as cellulose decomposition.     

A Dual Read-Out Assay to Evaluate the Potency of Compounds Active against Mycobacterium tuberculosis

Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis. There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days’ growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2-a]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors.     

Advances in shaking technologies

Shaking bioreactors are the most frequently used reactor system for screening and process optimization on a small scale. Their success can be attributed to their simple and functional design, which make shaking systems suitable for a large number of cost-efficient parallel experiments. Recently reported findings for oxygen transfer, power input, out-of-phase operation, hydromechanical stress and mixing in shaken bioreactors are summarized in this article. Novel monitoring techniques for the control of culture conditions in shake flasks and microtiter plates are described. The methods for characterizing culture conditions and the novel online measurement techniques that are summarized in this article can be utilized to tap the full potential of shaking reactor systems.     

Real-time monitoring of shake flask fermentation and off gas using triple disposable noninvasive optical sensors

Bioprocess development is a data-driven process requiring a large number of experiments to be conducted under varying conditions. Small-scale upstream bioprocess development is often performed in shake flasks because they are inexpensive and can be operated in parallel. However, shake flasks are often not equipped to accurately monitor critical process parameters such as pH, dissolved oxygen, and CO2 concentrations. Therefore, there is no definitive information on oxygen supply of growing cells, CO2 formation, and pH changes. Here we describe several shake flask fermentations where all three parameters are monitored by disposable noninvasive optical sensors. The sensitive element of these sensors is a thin, luminescent patch affixed inside the flask. Small electronic devices for excitation and fluorescence detection are positioned outside the shake flask for noninvasive monitoring. By measuring the process parameters throughout the course of the E. coli fermentations, we obtain information that is not routinely available in shake flask fermentations. For example, for cultures with only a few millimeters liquid depth, oxygen limitation can occur at relatively low agitation speeds. Under certain conditions oscillations in dissolved oxygen can occur. An increase in shaker speed and a decrease in culture volume can increase the oxygen availability and reduce the duration of oxygen limitation.     

Effect of pH on the growth cycle of HeLa cells in batch suspension culture without oxygen control

HeLa cells have been cultured at different controlled pH levels in batch suspension culture with open gas phase. Continuous monitoring of the dissolved oxygen tension throughout the culture cycle reveals a characteristic pattern of variation which correlates with various aspects of growth and carbohydrate metabolism. The effect of pH, size, and physiological state of the inoculum on this pattern have been determined, and the results are discussed in terms of a theoretical respiratory rate equation.     

Micro 3D cell culture systems for cellular behavior studies: Culture matrices,devices, substrates,and in‐situ sensing methods

Microfabricated systems equipped with 3D cell culture devices and in‐situ cellular biosensing tools can be a powerful bionanotechnology platform to investigate a variety of biomedical applications. Various construction substrates such as plastics, glass, and paper are used for microstructures. When selecting a construction substrate, a key consideration is a porous microenvironment that allows for spheroid growth and mimics the extracellular matrix (ECM) of cell aggregates. Various bio‐functionalized hydrogels are ideal candidates that mimic the natural ECM for 3D cell culture. When selecting an optimal and appropriate microfabrication method, both the intended use of the system and the characteristics and restrictions of the target cells should be carefully considered. For highly sensitive and near‐cell surface detection of excreted cellular compounds, SERS‐based microsystems capable of dual modal imaging have the potential to be powerful tools; however, the development of optical reporters and nanoprobes remains a key challenge. We expect that the microsystems capable of both 3D cell culture and cellular response monitoring would serve as excellent tools to provide fundamental cellular behavior information for various biomedical applications such as metastasis, wound healing, high throughput screening, tissue engineering, regenerative medicine, and drug discovery and development.     

Quasi-continuous combined scattered light and fluorescence measurements: a novel measurement technique for shaken microtiter plates

A novel quasi-continuous on-line measuring technique for shaken microtiter plates is presented. Light scattering as well as intracellular and/or protein fluorescence (e.g. NADH, YFP) is measured during the shaking procedure, thus allowing a process monitoring of 96 different simultaneous cultures in a microtiter plate. In contrast to existing measurement techniques, the shaking process does not have to be stopped to take the measurements, thus avoiding the corresponding interruption of the cultures' oxygen supply and any unpredictable effects on the cultures. Experiments were conducted with E. coli in LB, TB, and MOPS minimal medium and V. natriegens in modified LB and TB media. Intensity curves of scattered light and NADH fluorescence were used to distinguish different lag phases, growth velocities, or inoculation densities. Data from this new method corresponded well to the off-line measured optical densities and to the oxygen transfer rates of cultures run in simultaneously conducted shake flask experiments at equivalent oxygen transfer capacities. With the aid of yellow fluorescence protein fused to interleukin-6 the optimal induction time of an expressing E. coli strain could be determined by on-line monitoring of product formation. Thus, this measuring technique enables the researcher to evaluate and to discriminate different cultures on a screening level and to improve screening conditions, process development and scale-up.     

Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes

Mitochondrial dysfunction has been associated with a variety of currently marketed therapeutics and has also been implicated in many disease states. Alterations in the rate of oxygen consumption are an informative indicator of mitochondrial dysfunction, but the use of such assays has been limited by the constraints of traditional measurement approaches. Here, we present a high-throughput, fluorescence-based methodology for the analysis of mitochondrial oxygen consumption using a phosphorescent oxygen-sensitive probe, standard microtitre plates and plate reader detection. The protocol describes the isolation of mitochondria from animal tissue, initial establishment and optimization of the oxygen consumption assay, subsequent screening of compounds for mitochondrial toxicity (uncoupling and inhibition), data analysis and generation of dose-response curves. It allows dozens of compounds (or hundreds of assay points) to be analyzed in a single day, and can be further up-scaled, automated and adapted for other enzyme- and cell-based screening applications.     

An image-based assay for high throughput screening of Giardia lamblia

Giardia lamblia is a protozoan parasite that causes widespread gastrointestinal illness. Drugs to treat giardiasis are limited, but efforts to discover new anti-giardial compounds are constrained by the lack of a facile system for cell culture and inhibitor testing. We achieved robust and reproducible growth of G. lamblia in 384-well tissue culture plates in a modified TYI-S-33 medium. A high throughput assay for the screening of potential anti-giardial compounds was developed utilizing the WB strain of G. lamblia and automated optical detection of parasites after growth with tested inhibitors. We screened a library of 1600 known bioactive molecules and identified 12 compounds that inhibited growth of G. lamblia at low- or sub-micromolar concentrations. Our high throughput assay should facilitate evaluation of available chemical libraries for novel drugs to treat giardiasis.