Industrial-scale Bioreactors Research Articles

Simulation of dissolved CO2 gradients in a scale‐down system: A metabolic and transcriptional study of recombinant Escherichia coli

Deficient mixing in industrial-scale bioreactors is an important concern as it results in a heterogeneous environment that may affect microbial cell physiology. Dissolved carbon dioxide (dCO(2) ) fluctuations, which can occur in large-scale bioreactors, were simulated for the first time and their effects were evaluated on Escherichia coli expressing recombinant green fluorescent protein (GFP). The dCO(2) gradients were simulated by continuously circulating the medium between two vessels of a scale-down system to mimic mean circulation times (t(c) ) of 50, 170, and 375 s. Specific growth rate (μ) decreased by 11% and acetate concentration increased by 23% at the highest t(c) compared to reference cultures. An increase in the time needed for attaining maximum GFP concentration was also observed. The effect of dCO(2) fluctuations on the transcriptional levels of genes involved in the glutamate decarboxylase (gadA and gadC) and α-ketoglutarate dehydrogenase (sucA and sucB) were analyzed by quantitative real time PCR. Such genes are known to be highly over- or underexpressed at elevated constant dCO(2) . Expression levels of gadA and gadC increased up to 60% and 72%, and sucA decreased 1.8-fold in the culture performed at the highest t(c) . Only a minor decrease of sucB expression was observed at t(c) of 170 and 375 s. Although exposure to continuous high dCO(2) can affect culture performance, in this work it was shown that E. coli is able to rescue its metabolism in very short times when cells are intermittently returned to low dCO(2) conditions after being exposed to high dCO(2) .

Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics

Fourier transform infrared (FT-IR) spectroscopy combined with multivariate statistical analyses was investigated as a physicochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) and murine myeloma non-secreting 0 (NS0) cell lines. Medium samples were taken during culture of CHO and NS0 cells lines, which included both antibody-producing and non-producing cell lines, and analyzed by FT-IR spectroscopy. Principal components analysis (PCA) alone, and combined with discriminant function analysis (PC-DFA), were applied to normalized FT-IR spectroscopy datasets and showed a linear trend with respect to recombinant protein production. Loadings plots of the most significant spectral components showed a decrease in the C-O stretch from polysaccharides and an increase in the amide I band during culture, respectively, indicating a decrease in sugar concentration and an increase in protein concentration in the medium. Partial least squares regression (PLSR) analysis was used to predict antibody titers, and these regression models were able to predict antibody titers accurately with low error when compared to ELISA data. PLSR was also able to predict glucose and lactate amounts in the medium samples accurately. This work demonstrates that FT-IR spectroscopy has great potential as a tool for monitoring cell cultures for recombinant protein production and offers a starting point for the application of spectroscopic techniques for the on-line measurement of antibody production in industrial scale bioreactors.

Evaluation of a set of E. coli reporter strains as physiological tracer for estimating bioreactor hydrodynamic efficiency

A set of different green fluorescent protein (GFP) Escherichia coli reporter strains have been evaluated in mini- and stirred bioreactors operating in fed-batch mode with different degrees of perturbations in order to estimate their potential use as process-related stress biosensor. The mini-bioreactor platform comprises a set of parallel shake flasks operating in fed-batch mode. The advantage of this system is its high experimental throughput for the evaluation of the GFP synthesis capacity of our reporter strains. In the case of classical shake flask system, no significant evolution of GFP synthesis have been observed, considering the reduced microbial growth period allowed by the system, whereas in the case of fed-batch operated mini-bioreactors, evolution of GFP synthesis, as well as GFP distribution among the microbial population, has been observed for three preselected strains (prpoS, puspA and posmC::gfp). More interestingly, a binary mode of expression has been observed in the case of the cultures carried out with the reporter strains for which GFP synthesis is under the control of the rpoS promoter which is induced under carbon limitation conditions. However, the generation of controlled glucose perturbations is relatively limited in this system and, in a second step fully automated bioreactor with a sclae-down strategy has been used to correlate the response of a prpoS::gfp strains with extracellular glucose perturbations. In the case of the culture performed in perturbed bioreactor (glucose intermittent feeding or glucose addition at the level of the recycle loop of a two-compartment scale-down bioreactor), the slowdown of the GFP synthesis resulting in the observation of a binary repartition of GFP content among the microbial population, has been observed. This observation led to the conclusion that the prpoS::gfp can be used as a biosensor for the validation of a fed-batch profile in industrial-scale bioreactors.

Measurement of metabolic heat in a production-scale bioreactor by continuous and dynamic calorimetry

Measurement of metabolic heat has been attempted in an industrial-scale bioreactor using continuous and dynamic heat balance calorimetry. The contributions of individual heat sources influencing the temperature of the broth were evaluated and the magnitude of metabolic heat was calculated from general energy balance. Good correlations were obtained between oxygen uptake rate (OUR) and metabolic heat with heat yield under continuous and dynamic conditions with the values of Y Q/O =444 kJ/mole O 2 and Y Q/O =431 kJ/mole O 2 , respectively. There was also correlation between biomass production and heat generation. The value of heat yield, Y Q/X , was calculated to be 14546 kJ/kg; however, this was not constant and slightly decreased towards the end of fermentation. A similar value for heat yield was obtained under dynamic conditions and found to be 14667 kJ/kg. The consistency of experimental data was checked using carbon, electron, and heat balances. It was shown that reasonable accuracy could be achieved in an industrial environment. The difficult-to-measure variable biomass concentration was estimated using continuous metabolic heat flux, and good correlation was obtained between measured and estimated biomass concentrations. The results of this study suggest that heat balance around an industrial bioreactor can be simplified by accurately identifying individual heat sources as opposed to laboratory bioreactors where the contribution of each source can have significant impact. This reduces the number of measurements for accurate heat balance and makes use of heat balance feasible in a large scale.

The development and characterization of a high resolution bio-reaction calorimeter for weakly exothermic cultures

A unique bench-scale heat flux calorimeter is described, which is capable of detecting the low heat signal of an animal cell culture (a maximum of 200 mW/l is produced with a batch culture of SF9 insect cells). After the technical modifications that enhanced the sensitivity of a commercial heat flux reaction calorimeter, RC1 from Mettler-Toledo AG. Switzerland, the behavior of this system is analyzed here under biological operation conditions. Not only the heat flows provoked by the bioprocess parameters largely determine the final calorimetric resolution, but they are also more important than the culture heat signal (heat of reaction). Structural modifications aiming at the limitation of the non-biological heat flows are described such as insulating and thermostatic housings, and glass reactors with a different geometry. The current resolution of the RC1 is from ±4 to 12 mW/l compared to the former ±50 mW/l [Biotechnol. Bioeng. 57 (1998) 610], under usual animal cell culture conditions. This resolution is similar to that of microcalorimeters [Biotechnol. Bioeng. 58 (1998) 464; Thermochim. Acta 309 (1998) 63; Thermochim. Acta 332 (1999) 211; J. Biochem. Biophys. Methods 32 (1996) 191] with the advantage of an in situ measurement technique and a well-controlled culture (from the biological point of view). The monitoring sensitivity and control capacity of this instrument shows a clear potentiality for metabolic studies of low heat effect cell cultures and as a metabolic probe for industrial scale bioreactors’s control.

Penicillin Fermentation: Mechanisms and Models for Industrial-Scale Bioreactors

Even after many years of research and industrial practice, the production of penicillin G in fed-batch fermentation by Penicillium crysogenum continues to attract research interest. There are many reasons: the commercial and therapeutic importance of penicillin and its derivatives, the complexity of cell growth, and the impact of engineering variables, the last of which are significant in large bioreactors but are not yet fully understood. Extensive research has generated new information on the mechanisms of cellular reactions and morphological features of the mycelia and their role in the synthesis of the product. Given a choice of mechanisms, models of different degrees of complexity, for both cellular differentiation and bioreactor performance, have been proposed. The more complex models require and provide more information. They are also more difficult to evaluate and apply in automatic control systems for production-scale bioreactors. The present review considers the evolution of recent knowledge and models from this perspective

Oxygen availability and the growth of Escherichia coli W3110: A problem exacerbated by scale-up

By examining the effects of oxygen availability on the batch growth of Escherichia coli on glucose the present study seeks to determine the responses of growing cells to varying degrees of oxygen excess, limitation or starvation as might be experienced in an industrial-scale bioreactor. It was found that as the degree of oxygen limitation increases so too does the byproduct acetate production in addition to a concomitant decrease in the substrate based biomass yield coefficient and maximum specific growth rate. Similar, although not as severe, responses to excess oxygen growth conditions were also observed. This result supports the concept of oxygen being potentially toxic to growing organisms, resulting in, at the very least, a degree of inhibition.

Oxygen availability and growth of Escherichia coli W3110: Dynamic responses to limitation and starvation

It is well known that oxygen availability can have a profound effect upon the growth of an Escherichia coli culture with respect to acetate excretion or lower product yield. The current investigation seeks to determine the dynamic responses of steady state continuous cultures to abrupt changes in the oxygen supply. This should yield information regarding the behaviour of such cells as they circulate through areas of low and high oxygen availability in an industrial-scale bioreactor. It was found that a decoupling of catabolism and anabolism occurred following sudden switches both from and to oxygen limitation. It also appeared that the imposed growth rate had an effect on the speed of recovery of the system following any such changes.

The utilisation of glucose/acetate mixtures by Escherichia coli W3110 under aerobic growth conditions

Byproduct acetate is of major concern when considering the growth of Escherichia coli on glucose. Besides the fact that acetate production detracts from the overall yield, acetate itself is also a growth inhibitor. To further complicate matters, E. coli is capable of growth on acetate via the glyoxylate bypass. In an effort to evaluate the influence of acetate on the growth of E. coli, mixed glucose/acetate substrate batch and continuous cultivations have been carried out. The results have been examined in the context of their implications for industrial-scale bioreactors. It was found that acetate was growth inhibitory at relatively low concentrations <1.25 g/l. A batch culture supplied with both glucose and acetate utilised glucose in preference to acetate. Simultaneous utilisation of substrates was observed in continuous cultures at low imposed growth rates, i.e., below ca. 0.30 h−1.

Penicillin Fermentation: Mechanisms and Models for Industrial-Scale Bioreactors

ABSTRACT: Even after many years of research and industrial practice, the production of penicillin G in fed-batch fermentation by Penicillium crysogenum continues to attract research interest. There are many reasons: the commercial and therapeutic importance of penicillin and its derivatives, the complexity of cell growth, and the impact of engineering variables, the last of which are significant in large bioreactors but are not yet fully understood. Extensive research has generated new information on the mechanisms of cellular reactions and morphological features of the mycelia and their role in the synthesis of the product. Given a choice of mechanisms, models of different degrees of complexity, for both cellular differentiation and bioreactor performance, have been proposed. The more complex models require and provide more information. They are also more difficult to evaluate and apply in automatic control systems for production-scale bioreactors. The present review considers the evolution of recent knowledge and models from this perspective.

Survey of measurement techniques for gas–liquid mass transfer coefficient in bioreactors

In many fermentation processes, oxygen transfer is the rate limiting step. Correct measurement and subsequent estimation of the volumetric mass transfer coefficient is a crucial step in the design procedure of bioreactors. This article discusses some of the methods that are commonly used for the measurement of the mass transfer coefficient and their applicability for measurement in large scale bioreactors. It has been found that among the methods discussed here, the dynamic pressure method appears most useful for industrial scale bioreactors, with a small degree of approximations for gas–liquid mixing in the reactor and is suitable for large scale bioreactors with errors less than 10%, over the entire range of the operating conditions encountered in the fermentor operation.

Performance of industrial scale bioreactors with modified RUSHTON turbine agitators

AbstractThe data presented here with respect to the behaviour of industrial scale stirred tank bioreactors equipped with modified RUSHTON turbine agitators in the biosynthesis processes of antibiotics are valid for that case that the power consumption is the same as it is in standard RUSHTON turbine agitators.Each modified RUSHTON turbine agitator was obtained through the variation of the blade surface by adding perforations so that the ratio between the perforation surface area and the full surface area (or the surface fraction of the perforations) is 0.36.In the fermentations of Streptomyces aureofaciens, Streptomyces rimosus and Penicillium chrysogenum producing tetracycline, oxytetracyline and penicillin, respectively, in bioreactors equipped with modified RUSHTON turbine agitators, the relative antibiotic production is increased by more than 30% compared to standard bioreactors.

An expert system applied to the control of an industrial-scale bioreactor

Control of a fed-batch industrial-scale fermenter requires a large amount of “finger-tip feeling” on the part of operators, despite reasonably sophisticated instrumentation. The reason for this is that there are no applicable deterministic models giving aid to the control system in deciding the best operation strategies. This is a typical case of a heuristic experimental process, treating inexact knowledge that is excluded from deterministic and statistic modelling a priori. Essential parts of this problem can be solved by means of an expert system shell, capable of dealing with uncertain information, using the linguistic shell application. Knowledge was extracted from process records on fermentation batches producing baker's yeast on an industrial scale. A strategy for heuristically optimized yield and growth rate can be predicted using knowledge gained in previous runs and stored in the knowledge base of the expert system. The expert system, once built up, can also be used for experimental simulation of the effects of fundamental decision parameters: temperature, production rate, ethanol concentration and specific growth rate. The use of expert system consultations can be generally recommended as an efficient aid in fermentation control.

An Analysis of the Impact on Controlled Variables Selection on the Operation of Anaerobic Digestion Processes

Different adaptive nonlinear controller designs are evaluated for the operation of the anaerobic digestion process. The selected controlled variables are the Chemical Oxygen Demand (COD), propionate concentration, and dissolved hydrogen concentration. The manipulated variable is the dilution rate. Because the nonlinear control algorithms are designed based on the mass balance equations, distinct assumptions have to be made depending on the choice of the controlled variable. Although the obtained responses are model-specific, the methodology presented can be readily applied to an actual lab scale or industrial scale bioreactor. The study of the time response of the volatile fatty acids and dissolved hydrogen concentration, and of the methane flowrate obtained by controlling one variable at a time reveals the intrinsic behaviour of the balances between the assumed bacterial species and can be useful to investigate for instance the inhibition induced by a given product present in the reactor. The developed methodology demonstrates the usefulness of advanced control techniques to get a better understanding of the process under study even if the controller designs are based on incomplete knowledge.

Some effects of heat shocks on bacterial growth

In large industrial scale bioreactors process cultures are frequently exposed to supperoptimal temperatures for short intervals of time. Data concerning the physiologica effects of such exposures are scant. Here, experiments involving short term heat shocks on two bacteria, Klebsiella pneumoniae, a non-fastidious mesophile growing on glucose and Bacillus sp. NCIB 12522, a fastidious thermotolerant methylotroph growing on methanol, are described. Markedly different response patterns for the two bacteria are evident, clearly indicating not only the dangers of making generalizations with respect to the effects of superoptimal temperatures on growing bacterial cultures but also, the significance of scale related segregation phenomena such as wall growth.