Shake Bioreactor Research Articles

Improving product quality and productivity of an antibody-based biotherapeutic using inverted frustoconical shaking bioreactors.

The Chinese hamster ovarian (CHO) cells serve as a common choice in biopharmaceutical production, traditionally cultivated in stirred tank bioreactors (STRs). Nevertheless, the pursuit of improved protein quality and production output for commercial purposes demand exploration into new bioreactor types. In this context, inverted frustoconical shaking bioreactors (IFSB) present unique physical properties distinct from STRs. This study aims to compare the production processes of an antibody-based biotherapeutic in both bioreactor types, to enhance production flexibility. The findings indicate that, when compared to STRs, IFSB demonstrates the capability to produce an antibody-based biotherapeutic with either comparable or enhanced bioprocess performance and product quality. IFSB reduces shear damage to cells, enhances viable cell density (VCD), and improves cell state at a 5-L scale. Consequently, this leads to increased protein expression (3.70g/L vs 2.56g/L) and improved protein quality, as evidenced by a reduction in acidic variants from 27.0% to 21.5%. Scaling up the culture utilizing the Froude constant and superficial gas velocity ensures stable operation, effective mixing, and gas transfer. The IFSB maintains a high VCD and cell viability at both 50-L and 500-L scales. Product expression levels range from 3.0 to 3.6g/L, accompanied by an improved acidic variants attribute of 20.6%-22.7%. The IFSB exhibits superior productivity and product quality, underscoring its potential for incorporation into the manufacturing process for antibody-based biotherapeutics. These results establish the foundation for IFSB to become a viable option in producing antibody-based biotherapeutics for clinical and manufacturing applications.

“One ring to rule them all”: The melt‐down of oxygen transfer limitations in shaken bioreactors

“One ring to rule them all”: The melt‐down of oxygen transfer limitations in shaken bioreactors

Enhanced Phenazine-1-Carboxamide Production in Pseudomonas chlororaphis H5△fleQ△relA through Fermentation Optimization

Phenazine-1-carboxamide (PCN) is effective to control many plant pathogens, and improving PCN production would be of great significance in promoting its development as a biopesticide. This study was conducted to improve the PCN production of Pseudomonas chlororaphis H5△fleQ△relA through fermentation optimization in both shake flask and bioreactor. The PCN production of H5△fleQ△relA was improved from 2.75 ± 0.23 g/L to 5.51 ± 0.17 g/L by medium optimization in shake flask using Plackett-Burman design, the path of steepest ascent experiment and central composite design. Then, PCN production reached 8.58 ± 0.25 g/L through optimizing pH in 1 L bioreactor. After pH optimization, the transcriptional levels of ccoO_2 and ccoQ_2 genes related to microbial aerobic respiration were significantly upregulated, and the relative abundance of 3-oxo-C14-HSL was significantly enhanced 15-fold, and these changes were vital for cell activity and metabolites production. Furthermore, the PCN production reached 9.58 ± 0.57 g/L after optimization of the fed-batch fermentation strategy in 1 L bioreactor. Finally, the fermentation scale-up of the optimal medium and optimal feeding strategy were conducted in 30 L bioreactor at the optimal pH, and their PCN production reached 9.17 g/L and 9.62 g/L respectively, which were comparable to that in 1 L bioreactor. In this study, the high PCN production was achieved from the shake-flask fermentation to 30 L bioreactor, and the optimal feeding strategy improved PCN production in bioreactor without increasing total glycerol compared with in shake flask. It provides promising pathways for the optimization of processes for the production of other phenazines.

Effect of baffle structure on flow field characteristics of orbitally shaken bioreactor.

Disposable orbitally shaken bioreactors have been widely used for mammalian cell culture in suspension. Three kinds of baffle structures: vertical baffle, inclined baffle and horizontal baffle were designed in this work. The flow fields of the shaking bioreactor with different baffle structures were simulated, and the turbulence, dissolved oxygen and shear strain rate of the bioreactor were analyzed. The results showed that the quasi-steady-state flow patterns of the unbaffled shaking bioreactors were broken for the bioreactors with the strengthening effects of baffles. The mixing and the oxygen volumetric mass transfer coefficient (kLa) (simulated results) were improved significantly, and the shear strain rates were also increased greatly for the baffle bioreactors. The shear strain rates of the baffle bioreactors were mainly in the range of 0-20s-1, and they were still low enough for CHO cell cultures.

Resin composite blocks for dental CAD/CAM applications reduce biofilm formation in vitro

ObjectivesModern dentistry is increasingly focusing on digital procedures, including CAD/CAM technologies. New materials have to resist in a demanding environment that includes secondary caries occurrence. The current study hypothesized that the microbiological behavior of different RBCs for CAD/CAM applications is better than that of their counterparts for direct restorations due to differences in the surface characteristics. MethodsBoth direct and CAD/CAM RBCs were tested. Specimens were obtained from each group, polished, cleaned, stored in artificial saliva (1w), then sterilized under UV (24h). Specimens’ surface was assessed using profilometry, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction; resin/filler content was assessed using thermogravimetry. After pre-incubation with sterile human saliva (24h), the microbiological behavior of the materials was assessed using four models: Streptococcus mutans adherence (2h), S. mutans biofilm formation in an orbital shaking bioreactor (24h), S. mutans biofilm formation in a continuous-flow bioreactor simulating shear forces (24h), and mixed-plaque formation in the bioreactor (24h). The viable biomass adhering to the specimens’ surfaces was measured using a tetrazolium dye-based test. Statistical analysis included verification of normality of distribution and homoscedasticity, then Oneway ANOVA and Tukey's test (α=5%). ResultsWhen using the bioreactor setup, CAD/CAM RBCs generally yielded lower S. mutans and mixed-plaque biofilm formation compared to direct RBCs. This difference was not evidenced in the first two microbiological models. Differences in manufacturing and curing processes rather than in materials’ surface roughness and composition could explain these results. SignificanceCAD/CAM RBCs are promising materials from a microbiological point of view, featuring reduced biofilm formation on their surfaces when shear conditions similar to in vivo ones are present.

Disruption or reduced expression of the orotidine-5\u2032-decarboxylase gene pyrG increases citric acid production: a new discovery during recyclable genome editing in Aspergillus niger

BackgroundAspergillus niger is a filamentous fungus used for the majority of global citric acid production. Recent developments in genome editing now enable biotechnologists to engineer and optimize A. niger. Currently, however, genetic-leads for maximizing citric acid titers in industrial A. niger isolates is limited.ResultsIn this study, we try to engineer two citric acid A. niger production isolates, WT-D and D353, to serve as platform strains for future high-throughput genome engineering. Consequently, we used genome editing to simultaneously disrupt genes encoding the orotidine-5′-decarboxylase (pyrG) and non-homologous end-joining component (kusA) to enable use of the pyrG selection/counter selection system, and to elevate homologous recombination rates, respectively. During routine screening of these pyrG mutant strains, we unexpectedly observed a 2.17-fold increase in citric acid production when compared to the progenitor controls, indicating that inhibition of uridine/pyrimidine synthesis may increase citric acid titers. In order to further test this hypothesis, the pyrG gene was placed under the control of a tetracycline titratable cassette, which confirmed that reduced expression of this gene elevated citric acid titers in both shake flask and bioreactor fermentation. Subsequently, we conducted intracellular metabolomics analysis, which demonstrated that pyrG disruption enhanced the glycolysis flux and significantly improved abundance of citrate and its precursors.ConclusionsIn this study, we deliver two citric acid producing isolates which are amenable to high throughput genetic manipulation due to pyrG/kusA deletion. Strikingly, we demonstrate for the first time that A. niger pyrG is a promising genetic lead for generating citric acid hyper-producing strains. Our data support the hypothesis that uridine/pyrimidine biosynthetic pathway offer future avenues for strain engineering efforts.

Orbitally Shaken Single-Use Bioreactor for Animal Cell Cultivation: Fed-Batch and Perfusion Mode.

Increasing the cultivation volume from small to large scale can be a rather complex and challenging process when the method of aeration and mixing is different between scales. Orbitally shaken bioreactors (OSBs) utilize the same hydrodynamic principles that define the success of smaller-scale cultures, which are developed on an orbitally shaken platform, and can simplify scale-up. Here we describe the basic working principles of scale-up in terms of the volumetric oxygen transfer coefficient (kLa) and mixing time and how to define these parameters experimentally. The scale-up process from an Erlenmeyer flask shaken on an orbital platform to an orbitally shaken single-use bioreactor (SB10-X, 12 L) is described in terms of both fed-batch and perfusion-based processes. The fed-batch process utilizes a recombinant variant of the mammalian cell line, Chinese hamster ovary (CHO), to express a biosimilar of a therapeutic monoclonal antibody. The perfusion-based process utilizes either an alternating tangential flow filtration (ATF) or a tangential flow filtration (TFF) system for cell retention to cultivate an avian cell line, AGE1.CR.pIX, for the propagation of influenza A virus, H1N1, in high cell density. Based on two example cell cultivations, processes outline the advantages that come with using an orbitally shaken bioreactor for scaling-up a process. The described methods are also applicable to other suspension cell lines.

Numerical simulation of scaling-up an inverted frusto-conical shaking bioreactor with low shear stress for mammalian cell suspension culture.

Shear stress is one of the key factors affecting the large-scale culture of mammalian cells. In this study, numerical simulation based on computational fluid dynamics was used to conduct a flow-field analysis of 7, 50, 200, and 1200 L inverted frusto-conical shaking bioreactors. The results show that the shear rate, specific mass transfer area (a), and volumetric oxygen mass transfer coefficient (kLa) gradually decreased as the scale of the bioreactor increased. Through application of BHK21 and CHO cells in 7, 200, and 1200 L bioreactors, it was found that the cell density and antibody expression level increased as the volume of the bioreactor increased. Moreover, the antibody expression level in a 1200L bioreactor was nearly 30% and 35% higher than that of 7 and 200L bioreactors, respectively. The results demonstrate that the environment with a larger volume is more suitable for the growth and antibody expression of CHO cells, indicating shear stress might be the most critical factor affecting the scale-up of mammalian cells.

Microtiter miniature shaken bioreactor system as a scale-down model for process development of production of therapeutic alpha-interferon2b by recombinant Escherichia coli

BackgroundDemand for high-throughput bioprocessing has dramatically increased especially in the biopharmaceutical industry because the technologies are of vital importance to process optimization and media development. This can be efficiently boosted by using microtiter plate (MTP) cultivation setup embedded into an automated liquid-handling system. The objective of this study was to establish an automated microscale method for upstream and downstream bioprocessing of α-IFN2b production by recombinant Escherichia coli. The extraction performance of α-IFN2b by osmotic shock using two different systems, automated microscale platform and manual extraction in MTP was compared.ResultsThe amount of α-IFN2b extracted using automated microscale platform (49.2 μg/L) was comparable to manual osmotic shock method (48.8 μg/L), but the standard deviation was 2 times lower as compared to manual osmotic shock method. Fermentation parameters in MTP involving inoculum size, agitation speed, working volume and induction profiling revealed that the fermentation conditions for the highest production of α-IFN2b (85.5 μg/L) was attained at inoculum size of 8%, working volume of 40% and agitation speed of 1000 rpm with induction at 4 h after the inoculation.ConclusionAlthough the findings at MTP scale did not show perfect scalable results as compared to shake flask culture, but microscale technique development would serve as a convenient and low-cost solution in process optimization for recombinant protein.

Biodegradation of Polyaromatic Hydrocarbons by Acclimatized Mixed Culture Using Shake Flask and Roller Bioreactors

The ability of acclimatized mixed culture from sewage waste sludge was tested to biodegrade (PAHs): naphthalene and phenanthrene each with a concentration of 300 mg/L. Sewage sludge was selected as an inexpensive source of mixed culture of microorganisms, usually available in large quantities in wastewater treatment plants. Two types of reactors were employed in the investigation: shake flask and roller bioreactors. Complete biodegradation of naphthalene and phenanthrene was achieved in the shake flask bioreactor after 13 and 14 days of treatment, respectively. The corresponding durations in the roller bioreactor were 11 and 12 days. The obtained results show that the said culture is capable of consuming PAHs as energy and carbon source and have a promising application in bioremediation of PAH contaminated environments. The biodegradation of naphthalene was enhanced when using the roller bioreactor compared to its biodegradation in the shake flask bioreactor. The microorganisms’ specific growth rate was raised from 0.014 to 0.022 h-1 due to the enhanced mixing in the roller bioreactor. No enhancement was observed for phenanthrene biodegradation when using the roller bioreactor: the microorganisms’ specific growth rate was equal to 0.016 h-1 for the shake flask bioreactor compared to 0.012 h-1 for the roller bioreactor. Logistic models were employed for the description of the microorganisms’ growth and the PAHs degradation in both shake flask and roller bioreactor. Additionally, second order inhibition model was used to describe the possible inhibitions. The results obtained from the models well-matched the biodegradation experimental data with R2 of more than 97%.

The production and antiprotozoal activity of abietane diterpenes in Salvia austriaca hairy roots grown in shake flasks and bioreactor

ABSTRACTThe biomass and concentration of bioactive quinone methide-type diterpenes in hairy roots of Salvia austriaca were determined and compared with levels of these metabolites in roots of field-grown plants. The cultures were maintained in shake flasks and a nutrient sprinkle bioreactor. Diterpene production was more efficient in the shake flask root culture than the bioreactor one. Biomass and diterpene production within the shake flask culture was evaluated using Schenk and Hildebrandt (SH), Gamborg (B5), and woody plant medium (WPM), with both full- and half-strength macro and micronutrient concentrations (1/2 SH, 1/2 B5, and 1/2 WPM). Among the tested media, SH medium proved to be most effective for biomass and diterpene production. In this medium, the transformed roots accumulated the levels of taxodone (3.89 mg g−1 DW; equivalent to 63.3 mg L−1), taxodione (1.15 mg g−1 DW; equivalent to 17.4 mg L−1), 15-deoxy-fuerstione (2.15 mg g−1 DW; equivalent to 32.5 mg L−1), and 7-(2′-oxohexyl)-taxodione (0.076 mg g−1 DW; equivalent to 1.1 mg L−1). Three diterpenes were also detected in the roots of S. austriaca intact plants, but their concentrations were lower than those in hairy root culture. No 7-(2′-oxohexyl)-taxodione was found in the roots of field-grown plants. The hairy roots were able to maintain high metabolite levels even for 6 years of cultivation. Taxodone, taxodione, 15-deoxy-fuerstione, and 7-(2′-oxohexyl)-taxodione were tested for in vitro activity against Trypanosoma brucei rhodesiense, T. cruzi, and Plasmodium falciparum and their cytotoxicity was determined using L6 cells. Among these compounds, taxodione was the most active against T. brucei rhodesiense [IC50 = 0.05 µM with high selectivity, selectivity index (SI) = 38]. Taxodione was found to inhibit the growth of P. falciparum and T. cruzi by 50% at respective concentrations of 1.9 and 7.1 µM (SI values of 1.0 and 0.27). Other diterpenoids demonstrated weaker activity against tested parasites (IC50 values ranging from 0.62 to 194.7 µM) and lower selectivity (SI value ranged from 0.4 to 5.0).

Appraisal of fluid flow in a shaken bioreactor with conical bottom at different operating conditions

The flow dynamics in cylindrical shaken bioreactors of different conical bottom geometries is investigated by means of phase-resolved particle image velocimetry. Amid the variety of shaken vessels used for bioprocess applications, a cylindrical bioreactor with a conical bottom geometry was selected to assess its potential application in three-dimensional cultures, and improve solid suspension in shaken systems. This work builds upon the study of Weheliye et al. (2013. AIChE J. 59, 344) for a flat bottom with the objective to evaluate the effects of conical shaped bottoms of different heights on the fluid flow under different operating conditions with water being the working fluid. The results provide evidence that the presence of the conical bottom affects the transition from laminar to turbulent flow documented by Weheliye et al. (2013. AIChE J. 59, 344). The conical bottom with the greatest height investigated extended significantly the range of speeds over which flow transition occurs, with high intensity vortical structures spanning over the entire height of the bioreactor at lower speeds than those reported for a flat bottom geometry. This combined with the observed higher levels of kinetic energy should provide more efficient mechanisms for solid suspension.

MORPHOGENIC PATTERNS OF PLANTS MICROPROPAGATED IN LIQUID MEDIUM IN SHAKEN FLASKS OR LARGE-SCALE BIOREACTOR CULTURES

ABSTRACT Plant propagation in vitro through axillary and/or adventitious buds is at present labor intensive, which contributes to the high cost of plant production. Liquid cultures used to scale up mass propagation often affect adversely shoot morphogenesis. Abnormal shoot morphogenesis due to continuous submergence and vigorous aeration in the liquid medium results in vitreous shoots which poorly survive transplanting ex vitro. Nephrolepis, Philodendron, and Gladiolus bud explants were cultured in liquid medium in shaken cultures or bioreactors. Ferns propagated in liquid medium in shaken cultures produced meristemoid clusters, while leafy shoots developed in agar. In bioreactors the biomass was two to three times higher than in shaken cultures. Meristemoid clusters separated and inoculated to pretransplanting hardening agar medium developed normal shoots. In Gladiolus, shoot morphogenesis was controlled by the use of growth retardants, which also enhanced bud and protocorm production. In the presence of...

Removal of reactive orange 16 by immobilized Coprinus plicatilis in the batch shaking bioreactor

Coprinus plicatilis, white rot fungi, capable of removing Reactive Orange 16 (Remazol Brillant Orange 3R) was immobilized in calcium alginate gel beads and used for decolorization of RO16 in aqueous in the Batch Shaking Bioreactor. The optimum conditions for immobilization of the microorganism, such as alginate and calcium ion concentration, initial cell amout, hardening time and bead size, were determined with a view to improving the RO16 removal rate. The characteristics of RO16 decolorization by immobilized fungal cells were investigated. The repeated use of immobilized cells for RO16 decolorization was performed and the results revealed that the bioactivity of immobilized cells was stable over 120 hours in the repeated batch cultivation RO16 removal. At the end of decolorization process, the metabolites of the dye decolorization produced by immobilized C. plicatilis, were analyzed via GC-MS. It was concluded that the removal of the dye by immobilized C. plicatilis was achieved.

Characterization of Interfacial Hydrodynamics in a Single Cell of Shaken Microtiter Plate Bioreactors Applying Computational Fluid Dynamics Technique

Development of orbital shaking technology for enhanced mixing with lower mechanical demand has been receiving significant attention since the advent of advanced mixing schemes. Amongst shaken bioreactors,microtiter plates play significant role both for research and industrial purposes due to their capability of handling tiny amount of liquid in parallel experimentations. Detailed understanding of complicated Flow hydrodynamics thus seems to be considered a continual effort, as it is responsible for efficient gas-liquid mass transfer. Computational fluid dynamics (CFD) technique is shown to be a suitable numerical method in particular for discovering concealed flow facts, which can reliably predict the unsteady state flow within individual plate cells. In this study, CFD is employed to account for gas-liquid interface shape determination to reflect details of hydrodynamics for a novel geometrically modified microtiterplate, which would address diverse unknown flow characteristics vital for accurate mass transfer calculations.

Combined TGE-SGE Expression of Novel PAI-1-Resistant t-PA in CHO DG44 Cells Using Orbitally Shaking Disposable Bioreactors

An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncatedmutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncatedmutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.

KLa as a predictor for probe-independent mammalian cell bioprocesses in orbitally shaken bioreactors

Background Orbitally shaken flasks are commonly used at an early stage of bioprocess development with mammalian cells. In contrast to large-scale stirred-tank bioreactors, shaken flasks are usually operated in probe-independent bioprocesses, i.e. without strictly controlling the pH or dissolved oxygen concentration (DO). As a consequence, gas transfer issues are thought to limit the effectiveness of orbitally shaken flasks and bioreactors (OSRs). To define optimal operating conditions for probe-independent bioprocesses in OSRs, we tested the effects of the mass transfer coefficient of oxygen (kLa) on mammalian cell growth, recombinant protein production, and environmental conditions of the culture (pH, DO).

Disposable bioreactors: from process development to production

Single-use bioreactors are commonly used for seeding stainless steel bioreactors or for producing material. The profitability of these equipments has been well demonstrated on more that decade. But few data on its scalability have been published. In 2010-2011, Merck Serono Biodevelopment performed a study in order to evaluate the performance of several disposable bioreactors. As different technologies and scales were available, this study compared the performance of several types of mixing in single-use bioreactors for process development and pilot scale production. In addition, the evaluation was performed for both seeding applications and for clinical material production. Thus the feature of this study is the comparison of 3 to 50L disposable bioreactors with internal mixing system or external agitation (Table ​(Table1).1). In order to compare their performance with traditional bioreactors, gas flow rate applied in disposable bioreactors was scaled-down from seeding and production stainless steel bioreactors. Furthermore the ratio working volume on total volume and the power input applied to the 3L disposable bioreactor were similar to those in glass bioreactors. Table 1 Description of the different disposable bioreactors assessed. In order to compare the 5 different types of disposable bioreactors, a fed-batch process producing a highly glycosylated molecule was performed. The cell growth during the amplification phase was similar reflected by the doubling time of the cells. It was similar between traditional bioreactors (21h in 250L stainless steel seeding bioreactor) and disposable bioreactors (from 17h in the Nucleo and the Orbital to 19h in the STR) The quality of the molecule together with the molecule titer and the cell growth were compared in the 5 single use technologies during a production process. The process performance was also compared in 250L and 1.25kL bioreactors and to a 3.6L glass development bioreactor. The overall production process performance was similar in traditional and disposable bioreactors (Figure ​(Figure11). Figure 1 Quantity of highly glycosylated molecule produced in different bioreactors This study was completed by a characterization of liquid/liquid and gas/liquid transfers inside each disposable bioreactor in order to estimate their potential in terms of cell culture. These cells did not require a lot of oxygen. A low kLa (0.44h–1 in the Nucleo) was sufficient to run this process. The air-flow rates applied in the orbital shaking bioreactor during the process were defined according to Merck Serono Biodevelopment and supplier experience. These flow rates should have been decreased as kLa was higher (3.6h-1) than in traditional bioreactors (2.6 h-1). Mixing time was measured in phosphate saline buffer at the maximum working volume and the agitation speed applied during production process. Mixing time was measured by injecting concentrated sodium hydroxide from the top of the bag and from the bottom of the bag. The Nucleo and the CellReady 3L system have classical probes, while the Orbital, the RM and the STR bioreactors have optical probes. Optical probes were more stable because they took a measure every five seconds. Classical probes were less stable and showed variations of around 0.03 pH unit even a few minutes after injection in the Nucleo. Nevertheless, mixing times on all disposable bioreactors (from 15s in CellReady 3L to 100s in Orbital) were higher than in traditional bioreactors (12s in 3.6L glass bioreactor to 28s in 1250L stainless steel bioreactor). For the STR, the mixing time was close to one minute, which is the recommended maximum for mammalian cell culture. The Orbital and the Nucleo had a mixing time below two minutes. Howerver, after sodium hydroxide injection, pH measured below or close to the final pH value. The disposable bioreactor which has a mixing time closest to traditional bioreactors is the CellReady 3L. Finally, an evaluation grid was applied to choose the best disposable bioreactor. All these comparisons allowed Merck Serono Biodevelopment to come to a conclusion about disposable bioreactor use and on the scalability (up and down) of these disposable systems. It also enabled us to highlight critical points for disposable bioreactor implementation such as tubing size and length, use of disposable or reusable probes or factory.

Study of Oxygen Transfer Process of Starch Wastewater in the Shaking Bioreactor

The oxygen transfer process of starch wastewater in the shaking bioreactor was studied. A biological oxygen consumption system was simulated by the sodium sulfite solution. The effects of liquid volume ratio in different volume shake flasks, rotational speed and temperature on the volumetric oxygen transfer coefficient were investigated. The results show that the volumetric oxygen transfer coefficient increased with the increasing rotational speed and temperature, while decreased with the increasing volume of shaking flasks.

Determination of CO2 sensitivity of micro-organisms in shaken bioreactors. II. Novel online monitoring method

In the present study, a new online monitoring method for the determination of the CO₂ sensitivity of micro-organisms, based on the values of the respiration factors [OTR (oxygen transfer rate) and CTR (carbon dioxide transfer rate)], obtained by using the RAMOS (respiratory activity monitoring system) device considering a variety of aeration rates in the measuring flask, is investigated. Based on the data of the OTR, obtained by RAMOS under a variety of specific aeration rates, the proposed new method was developed as an online monitoring method for CO₂ sensitivity of micro-organisms in shaken bioreactors. A maximum accumulated CO₂ concentration of 12% was derived in applied methods, provided that the cultivation system is carried out under optimal conditions. Additionally, to predict these conditions, an unsteady-state gas transfer model in shaken bioreactors would be very advantageous. The data of OTR obtained using the RAMOS device were analysed and recalculated by a programme considering the calibration factor (Cf). The major advantage of the new method is the possibility to determine the metabolic activity, regardless of manual sampling.