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.     

Development of a perfusion fed bioreactor for embryonic stem cell-derived cardiomyocyte generation: oxygen-mediated enhancement of cardiomyocyte output

Cell transplantation is emerging as a promising new approach to replace scarred, nonfunctional myocardium in a diseased heart. At present, however, generating the numbers of donor cardiomyocytes required to develop and test animal models is a major limitation. Embryonic stem (ES) cells may be a promising source for therapeutic applications, potentially providing sufficient numbers of functionally relevant cells for transplantation into a variety of organs. We developed a single-step bioprocess for ES cell-derived cardiomyocyte production that enables both medium perfusion and direct monitoring and control of dissolved oxygen. Implementation of the bioprocess required combining methods to prevent ES cell aggregation (hydrogel encapsulation) and to purify for cardiomyocytes from the heterogeneous cell populations (genetic selection), with medium perfusion in a controlled bioreactor environment. We used this bioprocess to investigate the effects of oxygen on cardiomyocyte generation. Parallel vessels (250 mL culture volume) were run under normoxic (20% oxygen tension) or hypoxic (4% oxygen tension) conditions. After 14 days of differentiation (including 5 days of selection), the cardiomyocyte yield per input ES cell achieved in hypoxic vessels was 3.77 +/- 0.13, higher than has previously been reported. We have developed a bioprocess that improves the efficiency of ES cell-derived cardiomyocyte production, and allows the investigation of bioprocess parameters on ES cell-derived cardiomyogenesis. Using this system we have demonstrated that medium oxygen tension is a culture parameter that can be manipulated to improve cardiomyocyte yield.     

The automatic maintenance of low dissolved oxygen using a photobacterial oxygen sensor for the study of microaerobiosis

K avanagh , E. & H ill , S. 1990. The automatic maintenance of low dissolved oxygen using a photobacterial oxygen sensor for the study of microaerobiosis. Journal of Applied Bacteriology 69 , 539–549.
Conventional galvanic and polarographic oxygen electrodes are not sensitive enough to investigate microaerobic phenomena such as nitrogen fixation. Two sensors are available for this application. They are the oxygen-dependent changes in either the absorption spectrum of leghaemoglobin, or the light emission by photo-bacteria. We have coupled the latter to the automatic feedback control of agitation for the maintenance of predetermined low dissolved oxygen concentrations (DOCs) in bacterial suspensions. This method is inexpensive, provides accurate control of DOC, and the photobacterium is easily propagated. We have used this equipment to determine the optimum DOC for microaerobic nitrogenase activity in the facultative anaerobe Klebsiella pneumoniae .     

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.     

Low-cost microbioreactor for high-throughput bioprocessing

The design of a microbioreactor is described. An optical sensing system was used for continuous measurements of pH, dissolved oxygen, and optical density in a 2 mL working volume. The K(L)a of the microbioreactor was evaluated under different conditions. An Escherichia coli fermentation in both the microbioreactor and a standard 1 L bioreactor showed similar pH, dissolved oxygen, and optical density profiles.%The low cost of the microbioreactor, detection system, and the small volume of the fermentation broth provide a basis for development of a multiple-bioreactor system for high-throughput bioprocess optimization.     

On-line infrared spectroscopy for bioprocess monitoring

One of the major aims of bioprocess engineering is the real-time monitoring of important process variables. This is the basis of precise process control and is essential for high productivity as well as the exact documentation of the overall production process. Infrared spectroscopy is a powerful analytical technique to analyze a wide variety of organic compounds. Thus, infrared sensors are ideal instruments for bioprocess monitoring. The sensors are non-invasive, have no time delay due to sensor response times, and have no influence on the bioprocess itself. No sampling is necessary, and several components can be analyzed simultaneously. In general, the direct monitoring of substrates, products, metabolites, as well as the biomass itself is possible. In this review article, insights are provided into the different applications of infrared spectroscopy for bioprocess monitoring and the complex data interpretation. Different analytical techniques are presented as well as example applications in different areas.     

Downscaling screening cultures in a multifunctional bioreactor array-on-a-chip for speeding up optimization of yeast-based lactic acid bioproduction

A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench-scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time-consuming, and labor-intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab-scale culture strategies. We present a multifunctional microfluidic device containing 12 individual bioreactors (V_t = 15 µl) in a 26 mm × 76 mm area with in-line biosensing of dissolved oxygen and biomass concentration. Following initial device characterization, the bioreactor lab-on-a-chip was used in a proof-of-principle study to identify the most productive cell line for lactic acid production out of two engineered yeast strains, evaluating whether it could reduce the time needed for collecting meaningful data compared with shake flasks cultures. Results of the study showed significant difference in the strains' productivity within 3 hr of operation exhibiting a 4- to 6-fold higher lactic acid production, thus pointing at the potential of microfluidic technology as effective screening tool for fast and parallelizable industrial bioprocess development.     

Process adapted calibration improves fluorometric pH sensor precision in sophisticated fermentation processes

Miniaturization and automation have become increasingly popular in bioprocess development in recent years, enabling rapid high‐throughput screening and optimization of process conditions. In addition, advances in the bioprocessing industry have led to increasingly complex process designs, such as pH and temperature shifts, in microbial fed‐batch fermentations for optimal soluble protein expression in a range of hosts. However, in order to develop an accurate scale‐down model for bioprocess screening and optimization, small‐scale bioreactors must be able to accurately reproduce these complex process designs. Monitoring methods, such as fluorometric‐based pH sensors, provide elegant solutions for the miniaturization of bioreactors, however, previous research suggests that the intrinsic fluorescence of biomass alters the sigmoidal calibration curve of fluorometric pH sensors, leading to inaccurate pH control. In this article, we present results investigating the impact of biomass on the accuracy of a commercially available fluorometric pH sensor. Subsequently, we present our calibration methodology for more precise online measurement and provide recommendations for improved pH control in sophisticated fermentation processes.     

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.     

A completely noninvasive method of dissolved oxygen monitoring in disposable small‐scale cell culture vessels based on diffusion through permeable vessel walls

Disposable cell culture vessels are extensively used at small scales for process optimization and validation, but they lack monitoring capabilities. Optical sensors that can be easily adapted for use in small‐scale vessels are commercially available for pH, dissolved oxygen (DO), and dissolved carbon dioxide (DCO₂). However, their use has been limited due to the contamination and compatibility issues. We have developed a novel solution to these problems for DO monitoring. Oxygen diffusion through permeable vessel wall can be exploited for noninvasive monitoring. An optical oxygen sensor can be placed outside the oxygen permeable vessel wall thereby allowing oxygen diffusing through the vessel wall to be detected by the sensor. This way the sensor stays separate from the cell culture and there are no concerns about contaminants or leachants. Here we implement this method for two cell culture devices: polystyrene‐made T‐75 tissue culture flask and fluorinated ethylene propylene (FEP)‐made Vuelife^® cell culture bag. Additionally, mammalian and microbial cell cultures were performed in Vuelife^® cell culture bags, proving that a sensor placed outside can be used to track changes in cell cultures. This approach toward noninvasive monitoring will help in integrating cell culture vessels with sensors in a seamless manner. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:172–177, 2014     

Evaluation of oxygen transfer rates in stirred-tank bioreactors for clinical manufacturing

Several methods are available for determining the volumetric oxygen transfer coefficient in bioreactors, though their application in industrial bioprocess has been limited. To be practically useful, mass transfer measurements made in nonfermenting systems must be consistent with observed microbial respiration rates. This report details a procedure for quantifying the relationship between agitation frequency and oxygen transfer rate that was applied in stirred-tank bioreactors used for clinical biologics manufacturing. The intrinsic delay in dissolved oxygen (DO) measurement was evaluated by shifting the bioreactor pressure and fitting a first-order mathematical model to the DO response. The dynamic method was coupled with the DO lag results to determine the oxygen transfer rate in Water for Injection (WFI) and a complete culture medium. A range of agitation frequencies was investigated at a fixed air sparge flow rate, replicating operating conditions used in Pichia pastoris fermentation. Oxygen transfer rates determined by this method were in excellent agreement with off-gas calculations from cultivation of the organism (P = 0.1). Fermentation of Escherichia coli at different operating parameters also produced respiration rates that agreed with the corresponding dynamic method results in WFI (P = 0.02). The consistency of the dynamic method results with the off-gas data suggests that compensation for the delay in DO measurement can be combined with dynamic gassing to provide a practical, viable model of bioreactor oxygen transfer under conditions of microbial fermentation.     

Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T‐75 flask

Routine cell culture is done in small‐scale disposable vessels (typically 0.1–100 mL volumes) in academia and industry. Despite their wide use in bioprocess development (i.e., process optimization and process validation), miniature process scouting devices (PSDs) are considered “black boxes” because they are generally not equipped with sensors. In this study, we show that on‐line monitoring of dissolved oxygen (DO) and pH in a T‐75 flask‐based PSD can be achieved during cell passaging and that this information can be linked to different cellular metabolic states. In this case, on‐line monitoring of DO and pH show three distinctive metabolic regions in passages 1–18, 19–28, 29–54 and in particular, the shift in the pH curve, the specific oxygen uptake rate (q_O2), and the lactate production rate to the oxygen consumption rate yield (Y_Lac/ox) confirm the existence of these distinctive metabolic regions. These findings are particularly useful because they show that sensor equipped PSDs can help to monitor cell culture behavior after thaw, in pre‐ and seed culture prior to scale‐up and in development/optimization studies. Such routine monitoring will help to develop more consistent cell culture techniques. Biotechnol. Bioeng. 2010;105: 1040–1047. © 2009 Wiley Periodicals, Inc.     

A multikinetic model approach to predict gluconic acid production in an airlift bioreactor

This paper uses a multikinetic approach to predict gluconic acid (GA) production performance in a 4.5 L airlift bioreactor (ALBR). The mathematical model consists of a set of simultaneous firstorder ordinary differential equations obtained from material balances of cell biomass, GA, glucose, and dissolved oxygen. Multikinetic models, namely, logistic and contois equations constitute kinetic part of the main model. The main model also takes into account the hydrodynamic and mass transfer parameters. These equations were solved using ODE solver of MATLAB v6.5 software. The mathematical model was validated with the experimental data available in the literature and is used to predict the effect of change in initial biomass and air sparging rate on the GA production. It is concluded that the mathematical model incorporated with multikinetic approach would be more efficient to predict the change in operating parameters on overall bioprocess of GA production in an ALBR.     

Observability analysis of biochemical process models as a valuable tool for the development of mechanistic soft sensors

By enabling the estimation of difficult‐to‐measure target variables using available indirect measurements, mechanistic soft sensors have become important tools for various bioprocess monitoring and control scenarios. Despite promising higher process efficiencies and increased process understanding, widespread application of soft sensors has been stalled by uncertainty about the feasibility and reliability of their estimations given present process analytical constraints. Observability analysis can provide an indication of the possibility and reliability of soft sensor estimations by analyzing the structural properties of first‐principle (mechanistic) models. In addition, it can provide a criteria for selection of suitable measurement methods with respect to their information content; thereby leading to successful implementation of soft sensors in bioprocess development and manufacturing environments. We demonstrate the utility of observability analysis for two classes of upstream bioprocesses: the processes involving growth and ethanol formation by Saccharomyces cerevisiae and the process of penicillin production by Penicillium chrysogenum. Results obtained from laboratory‐scale cultivations in addition to in‐silico experiments enable a comparison of theoretical aspects of observability analysis and the real‐life performance of soft sensors. By taking the expected error of measurements provided to the soft sensor into account, an innovative scaling approach facilitates a higher degree of comparability of observability results among various measurement configurations and process conditions. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1703–1715, 2015     

Kinetics of lactose fermentation using a recombinant Saccharomyces cerevisiae strain

This work presents a multi-route, non-structural kinetic model for interpretation of ethanol fermentation of lactose using a recombinant flocculent Saccharomyces cerevisiae strain expressing both the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces lactis. In this model, the values of different metabolic pathways are calculated applying a modified Monod equation rate in which the growth rate is proportional to the concentration of a key enzyme controlling the single metabolic pathway. In this study, three main metabolic routes for S. cerevisiae are considered: oxidation of lactose, reduction of lactose (producing ethanol), and oxidation of ethanol. The main bioprocess variables determined experimentally were lactose, ethanol, biomass, and dissolved oxygen concentrations. Parameters of the proposed kinetic model were established by fitting the experimental data obtained in a small lab-scale fermentor with the initial lactose concentrations ranging from 5 g/dm3 to 50 g/dm3. A very good agreement between experimental data and simulated profiles of the main variables (lactose, ethanol, biomass, and dissolved oxygen concentrations) was achieved.     

Biosensors for process monitoring

Summary A short review about the biosensor research activities for bioprocess monitoring in the F.R.G. after its reunification is given. The principles of biosensor applications are presented. In situ sensors and sensors based on the principles of flow injection analysis are studied. Some applications of a four-channel enzyme thermistor, bio-field effect transistors, and immunoanalysis systems for real process monitoring are presented.     

Online medium-throughput respirometry-based OTR measurements in magnetically stirred cultures

Intensified bioprocess development requires parallelized medium- to high-throughput experimentation with high on- and offline data density across all early scales of the development trajectory from microtiter plate via shake flask to lab-scale reactor. We developed a widespread measurement principle for intermediate scales, respirometry, into a parallelized oxygen transfer rate measurement device that could accurately record common process development-relevant effects such as acetate formation, diauxic growth, and nutrient limitations. The device was further equipped with dissolved oxygen measurement capability and sampling ports that allowed repetitive monoseptic sample withdrawal without disturbing the cultivation. Optimization of the operating parameters lead to k(L) a values of up to 160 h(-1) and corresponding oxygen transfer rates of 1 g L(-1) h(-1) for cultivation volumes of 50 mL.     

Design and performance of a 24-station high throughput microbioreactor

Two prototype 24-unit microbioreactors are presented and reviewed for their relative merits. The first used a standard 24-well plate as the template, while the second consisted of 24-discrete units. Both systems used non-invasive optical sensors to monitor pH and dissolved oxygen. The systems were used to cultivate Escherichia coli. Both designs had their merits and the results obtained are presented. In addition, dissolved oxygen control was demonstrated at the milliliter scale and 24 simultaneously monitored fermentations were successfully carried out. These results demonstrated high quality high throughput bioprocessing and provide important insights into operational parameters at small scale.     

On-line state estimation of biomass based on acid production in Zymomonas mobilis cultures

The concentrations of biomass, substrate and product are very important state variables of almost every bioprocess and generally unable to be measured directly in?situ due to the lack of reliable sensors. In this paper, an adaptive observer of the biomass concentration is proposed for an anaerobic fermentation process where only the measurement of the acid product is available on-line. The observer was tested to be effective by several experiments under various operating conditions. In this experimental system, an auto-sampling device was connected between the bioreactor for the fermentation of Zymomonas mobilis and a HPLC so that the concentrations of glucose and ethanol could be directly measured through such implementation.