Real-time dissolved carbon dioxide monitoring II: Surface aeration intensification for efficient CO2 removal in shake flasks and mini-bioreactors leads to superior growth and recombinant protein yields

Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O₂ (DO) and dissolved CO₂ (dCO₂) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small-scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real-time monitoring of DO and dCO₂ in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO₂ at low level, which significantly improved not only biomass growth but also protein yield. We expect that implementing such controlled smart shake flasks can minimize the process development gap which currently exists in shake flask level and bioreactor level results.     

Hydrogen online monitoring based on thermal conductivity for anaerobic microorganisms

H₂-producing microorganisms are a promising source of sustainable biohydrogen. However, most H₂-producing microorganisms are anaerobes, which are difficult to cultivate and characterize. While several methods for measuring H₂ exist, common H₂ sensors often require oxygen, making them unsuitable for anaerobic processes. Other sensors can often not be operated at high gas humidity. Thus, we applied thermal conductivity (TC) sensors and developed a parallelized, online H₂ monitoring for time-efficient characterization of H₂ production by anaerobes. Since TC sensors are nonspecific for H₂, the cross-sensitivity of the sensors was evaluated regarding temperature, gas humidity, and CO₂ concentrations. The systems' measurement range was validated with two anaerobes: a high H₂-producer (Clostridium pasteurianum) and a low H₂-producer (Phocaeicola vulgatus). Online monitoring of H₂ production in shake flask cultivations was demonstrated, and H₂ transfer rates were derived. Combined with online CO₂ and pressure measurements, molar gas balances of the cultivations were closed, and an anaerobic respiration quotient was calculated. Thus, insight into the effect of medium components and inhibitory cultivation conditions on H₂ production with the model anaerobes was gained. The presented online H₂ monitoring method can accelerate the characterization of anaerobes for biohydrogen production and reveal metabolic changes without expensive equipment and offline analysis.     

FTIR and UV spectroscopy in real-time monitoring of S. cerevisiae cell culture

A combination of FTIR and UV spectroscopy is proposed as a novel technique for integrated real-time monitoring of metabolic activity and growth rates of cell cultures, required for systematic studies of cellular low-frequency (LF) electric and magnetic field (EMF) effects. As an example, we investigated simultaneous influence of periodic LF 3D EMFs on a culture of Saccharomyces cerevisiae (baker's yeast) cells. Amplitudes, frequencies and phases of the field components were the variable parameters. Electromagnetic fields were found to efficiently control the activity of the yeast cells, with the resulting CO₂ production rates, as monitored by FTIR spectroscopy, varying by at least one order of magnitude due to the field action. Additionally, population dynamics of the yeast cells was monitored by UV absorption of the yeast culture at λ_prob = 320 nm, and compared to the CO₂ production rates. The detected physiologically active frequencies are all below 1 kHz, namely, 800 Hz excitation was effective in reducing the metabolic rates and arresting cell proliferation, whereas 200 Hz excitation was active in accelerating both cell proliferation and overall metabolic rates. The proposed methods produce objective, reliable and quantitative real-time results within minutes and may be used in various tasks that could benefit from a rapid feedback they provide in the form of metabolic and growth rates. Amplitude and frequency dependences of the LF EMF effects from individual field components with different polarizations were recorded and qualitatively interpreted based on a simple model, describing ion diffusion through a membrane channel.     

Accelerated Death Kinetics of Aspergillus niger Spores under High-Pressure Carbonation

The death kinetics of Aspergillus niger spores under high-pressure carbonation were investigated with respect to the concentration of dissolved CO₂ (dCO₂) and treatment temperature. All of the inactivation followed first-order death kinetics. The D value (decimal reduction time, or the time required for a 1-log-cycle reduction in the microbial population) in the saline carbonated at 10 MPa was 0.16 min at 52°C. The log D values were linearly related to the treatment temperature and the concentration of dCO₂, but a significant interaction was observed between them.     

Allostatic Load Model Associated with Indoor Environmental Quality and Sick Building Syndrome among Office Workers

This study investigates whether indoor environmental quality (IEQ) influences allostatic load (AL) and whether AL can be a predictor for sick building syndrome (SBS). We also assessed and compared the associations between AL and SBS versus 8-hydroxydeoxyguanosine (8-OHdG) and SBS. A total of 115 office workers from 21 offices completed self-reported SBS questionnaires, and provided 11 biomarkers for their AL. Multiple linear regressions and logistic regression analysis were applied to examine the correlations between IEQ and AL or 8-OHdG and between AL or 8-OHdG and SBS, respectively. Our data revealed that the neuroendocrine system was correlated with CO₂, the difference between indoor and outdoor CO₂ levels (dCO₂), and the indoor-outdoor ratio of CO₂ (CO₂ I/O). Metabolic system effects were associated with illumination. The relationships between illumination, CO₂, dCO₂, CO₂ I/O and 8-OHdG were consistent with those and AL in specific systems. Furthermore, we found that risks for SBS syndromes were related with neuroendocrine and metabolic system of the AL. 8-OHdG was associated with eye dryness or irritation, eye tiredness and vomiting. We conclude that IEQ significantly influences AL and that AL can be a predictor for reporting SBS with information on system-specific effects.     

Towards a quantitative assessment of inorganic carbon cycling in photosynthetic microorganisms

Photosynthetic organisms developed various strategies to mitigate high light stress. For instance, aquatic organisms are able to spend excessive energy by exchanging dissolved CO₂ (dCO₂) and bicarbonate () with the environment. Simultaneous uptake and excretion of the two carbon species is referred to as inorganic carbon cycling. Often, inorganic carbon cycling is indicated by displacements of the extracellular dCO₂ signal from the equilibrium value after changing the light conditions. In this work, we additionally use (i) the extracellular pH signal, which requires non‐ or weakly‐buffered medium, and (ii) a dynamic model of carbonate chemistry in the aquatic environment to detect and quantitatively describe inorganic carbon cycling. Based on simulations and experiments in precisely controlled photobioreactors, we show that the magnitude of the observed dCO₂ displacement crucially depends on extracellular pH level and buffer concentration. Moreover, we find that the dCO₂ displacement can also be caused by simultaneous uptake of both dCO₂ and (no inorganic carbon cycling). In a next step, the dynamic model of carbonate chemistry allows for a quantitative assessment of cellular dCO₂, , and H⁺ exchange rates from the measured dCO₂ and pH signals. Limitations of the method are discussed.     

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kLa ratio as a direct indicator of gas stripping conditions

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO₂) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO₂ was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the k_La values of O₂ (k_La[O₂]) and CO₂ [k_La(CO₂)] in the SS bioreactors as a scale-up factor for dCO₂ reduction. The k_La ratios [k_La(CO₂)/k_La(O₂)] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the k_La ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO₂ trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This k_La ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The k_La ratios for the SUBs were evaluated and conditions that provided k_La ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the k_La ratio is a powerful scale-up factor useful to control dCO₂ during fed-batch cultures.     

An on-line technique for monitoring propionic acid fermentation

An on-line technique, based on measuring the increase in pressure due to CO₂ release in a closed air-tight reactor, was used to evaluate the fermentation of lactate by propionibacteria. The method was applied to batch cultures of Propionibacterium shermanii grown in yeast extract/sodium lactate medium containing lactate as a carbon source under micro-aerophilic conditions. Gas pressure evolution was compared both with substrate consumption and metabolites production and with acidification and growth. Linear relationships were found between gas pressure variation, lactate consumption and propionate and acetate production. The technique also enabled the evaluation of total CO₂ produced, by taking account of pressure, oxygen and pH measurements. These results tend to show that this simple and rapid method could be useful to monitor propionic acid bacteria growth.     

Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii

Hydrogen peroxide (H₂O₂) is recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H₂O₂ sensor that allows H₂O₂ monitoring in all major subcompartments of a Chlamydomonas cell. To this end, we used the Chlamydomonas Modular Cloning toolbox to target the hypersensitive H₂O₂ sensor reduction–oxidation sensitive green fluorescent protein2-Tsa2ΔC_R to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and endoplasmic reticulum (ER). The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H₂O₂ produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under nonphysiological conditions. Furthermore, in heat-stressed cells, we show that cytosolic H₂O₂ levels closely mirror temperature up- and downshifts and are independent from PET. Heat stress led to similar up- and downshifts of H₂O₂ levels in the nucleus and, more mildly, in mitochondria but not in the chloroplast. Our results thus suggest the establishment of steep intracellular H₂O₂ gradients under normal physiological conditions with limited diffusion into other compartments. We anticipate that these sensors will greatly facilitate future investigations of H₂O₂ biology in plant cells.

The establishment of a hypersensitive H₂O₂ sensor in six major compartments of the Chlamydomonas cell reveals steep intracellular H₂O₂ gradients under normal physiological conditions with limited diffusion into other compartments.     

Respiration activity monitoring system for any individual well of a 48-well microtiter plate

Background

Small-scale micro-bioreactors have become the cultivation vessel of choice during the first steps of bioprocess development. They combine high cultivation throughput with enhanced cost efficiency per cultivation. To gain the most possible information in the early phases of process development, online monitoring of important process parameters is highly advantageous. One of these important process parameters is the oxygen transfer rate (OTR). Measurement of the OTR, however, is only available for small-scale fermentations in shake flasks via the established RAMOS technology until now. A microtiter plate-based (MTP) μRAMOS device would enable significantly increased cultivation throughput and reduced resource consumption. Still, the requirements of miniaturization for valve and sensor solutions have prevented this transfer so far. This study reports the successful transfer of the established RAMOS technology from shake flasks to 48-well microtiter plates. The introduced μRAMOS device was validated by means of one bacterial, one plant cell suspension culture and two yeast cultures.

Results

A technical solution for the required miniaturized valve and sensor implementation for an MTP-based μRAMOS device is presented. A microfluidic cover contains in total 96 pneumatic valves and 48 optical fibers, providing two valves and one optical fiber for each well. To reduce costs, an optical multiplexer for eight oxygen measuring instruments and 48 optical fibers is introduced. This configuration still provides a reasonable number of measurements per time and well. The well-to-well deviation is investigated by 48 identical Escherichia coli cultivations showing standard deviations comparable to those of the shake flask RAMOS system. The yeast Hansenula polymorpha and parsley suspension culture were also investigated.

Conclusions

The introduced MTP-based μRAMOS device enables a sound and well resolved OTR monitoring for fast- and slow-growing organisms. It offers a quality similar to standard RAMOS in OTR determination combined with an easier handling. The experimental throughput is increased 6-fold and the media consumption per cultivation is decreased roughly 12.5-fold compared to the established eight shake flask RAMOS device.

     

Spatial and temporal variations of dissolved gases (CH4, CO2, and O2) in peat cores

Spatial and temporal variations in the concentrations of dissolved gases (CH₄, CO₂, and O₂) in peat cores were studied using membrane inlet mass spectrometry (MIMS). Variations in vertical gas profiles were observed between random peat cores taken from hollows on the same peat bog. Methane concentrations in profiles (0–30 cm) generally increased with depth and reached maximum values in the range of 200–450 m CH₄ below about 13-cm depth. In some profiles, a peak of dissolved methane was observed at 7-cm depth. Oxygen penetrated to approximately 2-cm depth in the hollows. The sampling probe was used to continuously monitor CH₄, CO₂, and O₂ concentrations at fixed depths in peat cores over periods of several days. The concentration of dissolved CO₂ and O₂ at 1-cm depth oscillated over a 24-h period with the maximum of CO₂ concentration corresponding with the minimum of 0₂. Diurnal variations in CO₂ but not CH₄ were measured at 15-cm depth; dissolved CO₂ levels decreased during daylight hours to a constant minimum concentration of 4.85 mm. This report also describes the application of MIMS for the measurement of gaseous diffusion rates in peat using an inert gas (argon); the value of D, the diffusion coefficient, was 2.07 × 10^–8 m² s^–1. Correspondence to: D. Lloyd     

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.     

Changes in the Carbonic Anhydrase Activity and the Rate of Photosynthetic O2 Evolution during the Cell Cycle of Chlorella ellipsoidea C-27

Rhythmical changes in carbonic anhydrase activity(CA) and inphotosynthesis were observed during the cell cycle of Chlorellaellipsoidea C-27 synchronized at various concentrations of dissolvedCO₂ (dCO₂ with a regime of 16 h of light and 8 h of darkness.At a constant low concentration of dCO₂ (11 {diaeresis}M), intracellularCA activity showed obvious fluctuations with a peak at 8 h afterthe initiation of illumination, while extracellular CA activity,located on the cell surface, showed only minor fluctuationsalthough the activity was as high as the maximum activity ofintracellular CA. In contrast, obvious changes in the activitiesof intra- and extracellular CA activities were not observedat a high concentration of dCO₂ (520 {diaeresis}M). The ratioof photosynthetic activity at limiting versus saturating concentrationsof dCO₂, which is indicative of the affinity of cells for CO₂,showed clear rhythmical changes during the cell cycle and theratio was higher in low-CO₂ cells than in high-CO₂ cells. Thechanges in the ratio seemed to reflect the changes in CA activity. When the cells that had been synchronized under high CO₂ conditionswere transferred to low CO₂ conditions at any given stage inthe cell cycle, CA activity was induced in every case but thecapacity for induction of CA was greater in young cells thanin mature cells. This result suggests that the capacity of cellsto induce CA over the course of the cell cycle is closely relatedto endogenous aging of the cell. (Received August 29, 1988; Accepted December 28, 1988)     

On-line soft sensing in upstream bioprocessing

This review provides an overview and a critical discussion of novel possibilities of applying soft sensors for on-line monitoring and control of industrial bioprocesses. Focus is on bio-product formation in the upstream process but also the integration with other parts of the process is addressed. The term soft sensor is used for the combination of analytical hardware data (from sensors, analytical devices, instruments and actuators) with mathematical models that create new real-time information about the process. In particular, the review assesses these possibilities from an industrial perspective, including sensor performance, information value and production economy. The capabilities of existing analytical on-line techniques are scrutinized in view of their usefulness in soft sensor setups and in relation to typical needs in bioprocessing in general. The review concludes with specific recommendations for further development of soft sensors for the monitoring and control of upstream bioprocessing.     

Design and Application of Highly Responsive Fluorescence Resonance Energy Transfer Biosensors for Detection of Sugar in Living Saccharomyces cerevisiae Cells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker₁-maltose-binding protein-linker₂-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 μM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).     

In-situ fluorescence monitoring of cyanobacteria: Laboratory-based quantification of species-specific measurement accuracy

In recent years, in-situ fluorometers have been extensively deployed to monitor cyanobacteria in near real-time. Acceptable accuracy can be achieved between measured pigments and cyanobacteria biovolume provided the cyanobacteria species are known. However, cellular photosynthetic pigment content and measurement interferences are site and species specific and can dramatically affect sensor reliability. We quantified the accuracy of an in-situ fluorometer compared with traditional methods using mono- and mixed cultures of four different cyanobacterial species. We found: (1) lower pigment content in cultures in stationary phase, (2) higher precision with the sensor compared to traditional pigment quantification methods of measuring phycocyanin and chlorophyll a, (3) species-specific relationships between sensor readings and measurements related to biovolume, (4) overestimation of pigments in mixed compared with mono cultures, (5) dissolved organic matter causing a loss in signal proportional to its degree of aromaticity, and (6) potential to quantify the degree of cell lysis with a fluorescent dissolved organic matter sensor. This study has provided important new information on the strengths and limitations of fluorescence sensors. The sensor readings can provide accurate biovolume quantification and species determination for a number of bloom-forming species when sensors are properly compensated and calibrated.     

Noninvasive measurement of dissolved oxygen in shake flasks

Shake flasks are ubiquitous in cell culture and fermentation. However, conventional devices for measuring oxygen concentrations are impractical in these systems. Thus, there is no definitive information on the oxygen supply of growing cells. Here we report the noninvasive, nonintrusive monitoring of dissolved oxygen (DO) in shake flasks using a low-cost optical sensor. The oxygen-sensitive element is a thin, luminescent patch affixed to the inside bottom of the flask. The sensitivity and accuracy of this device is maximal up to 60% DO, within the range that is critical to cell culture applications. By measuring actual oxygen levels every 1 or 5 min throughout the course of yeast and E. coli fermentations, we found that a modest increase in shaker speed and a decrease in culture volume slowed the onset of oxygen limitation and reduced its duration. This is the first time that in situ oxygen limitation is reported in shake flasks. The same data is unattainable with a Clark type electrode because the presence of the intrusive probe itself changes the actual conditions. Available fiber optic oxygen sensors require cumbersome external connections and recalibration when autoclaved.     

Multichannel automated chamber system for continuous monitoring of CO2 exchange between the agro-ecosystem or soil and the atmosphere

A multichannel automated chamber system was developed for continuous monitoring of CO₂ exchange at multiple points between agro-ecosystem or soil and atmosphere. This system consisted of an automated chamber subsystem with a CO₂ concentration analyzer and a data logging subsystem. Both subsystems were under the control of a programmable logic controller (PLC). The automated chamber subsystem contained 18 chambers (50 cm × 50 cm × 50 cm) and a compressor. The chamber lids were closed and can be automatically opened. During measurement, one of the 18 chambers was kept closed for three minutes for measuring and the other chambers were kept open to maintain the natural soil conditions to the maximum extent. Environmental variables were simultaneously measured using sensors and recorded by the data logger. The reliability of the multichannel automated chamber system was tested and the results showed that the turbulence of the fans had no significant effect on the CO₂ exchange. The changes in the air and the temperature of soil and soil moisture inside the chambers, caused by the enclosure of the chambers, were not significant. The net ecosystem CO₂ exchange for the wheat ecosystem was ?2.35 μmol·m^?2·s^>?1 and the soil respiration was 3.87 μmol·m^?2·s^>?1 in the wheat field, and 6.61 μmol·m^?2·s^>?1 in the apple orchard.     

Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast Saccharomyces cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allowed us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments under a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady-state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox systems have distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H₂O₂ bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol-rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.