Production of savinase and population viability of Bacillus clausii during high-cell-density fed-batch cultivations

The growth and product formation of a Savinase-producing Bacillus clausii were investigated in high-cell-density fed-batch cultivations with both linear and exponential feed profiles. The highest specific productivity of Savinase was observed shortly after the end of the initial batch phase for all feed profiles applied and, in addition, there was a time-dependent decrease in specific productivity. The specific glucose uptake rate increased with time for constant specific growth rate indicating that the maintenance requirements increased with time, possibly due to a decreasing K(+) concentration. The physiological state of the cells was monitored during the cultivations using a flow cytometry assay based on the permeability of the cell membrane to propidium iodide. In the latter parts of the fed-batch cultures with a linear feed profile, a large portion of the cell population was found to have a permeable membrane, indicating a large percentage of dead cells. By assuming that only cells with a nonpermeable membrane contributed to growth and product formation, the physiological properties of this subpopulation were calculated.     

Experience in using an ethanol sensor to control molasses feed-rates in baker's yeast production

An ethanol sensor has been tested for feed-rate control of baker's yeast prouction. The yeast was grown on molasses in an 8 dm³ fed-batch reactor up to a cell concentration of 60–70 kg/m³. Studies were made on three levels: reliability of the sensor system, characterisation of the control problem, and evaluation of ethanol-controlled cultivations in terms of yield and production rate. Arguments are given for the conceptual advantages of ethanol control compared to other methods of substrate control. It is also shown that ethanol control allows for a simple regulator structure. In fact, a PID regulator, with constant parameters, was used around an exponential dosage scheme. Tuning of the regulator parameters was performed by using simulation on a simplified model of the process. Several cultivations have been carried out. Results from four comparable cultivations are given in detail, and the experience from many others is summarized.     

On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells

Changes in the nutrient availability of mammalian cell cultures are reflected in the β-dispersion parameter characteristic frequency (f _C ) and the on-line dual frequency permittivity signal. Multi-frequency permittivity measurements were therefore evaluated in fed-batch cultivations of two different CHO cell lines. Similar responses to nutrient depletions and discontinuous feed additions were monitored in different cultivation phases and experimental setups. Sudden increases in permittivity and f _C occurred when feed additions were conducted. A constant or declining permittivity value in combination with a decrease in f _C indicated nutrient limitations. f _C correlated well with changes in oxygen uptake rate when cell diameter remained constant, indicating that metabolic activity is reflected in the value of f _C . When significant cell size changes occurred during the cultivations, the analysis of the β-dispersion parameters was rendered complex. For the application of our findings in other systems it will be hence required to conduct additional off-line measurements. Based on these results, it is hypothesized that multi-frequency permittivity measurements can give information on the intracellular or physiological state in fed-batch mode. Similar observations were made when using different cell lines and feeding strategies, indicating that the findings are transferable to other cell lines and systems. The results should lead to an improved understanding of routine fed-batch processes. Additional studies are, however, required to explore how these observations can be used for fed-batch process development and optimization.     

Clavulanic acid degradation in Streptomyces clavuligerus fed-batch cultivations

Clavulanic acid (CA) is an important antibiotic that is produced by Streptomyces clavuligerus. CA is unstable and product degradation has turned out to have a major impact on product titers in fed-batch cultivations. Three different types of experiments have been used to elucidate CA degradation under fed-batch cultivation conditions. First, the influence of individual medium compounds was examined. Second, degradation was monitored during the exponential growth phase in batch cultivations. Third, CA degradation was studied in the supernatant of samples taken during a fed-batch. In addition, data from six fed-batch cultivations were studied to derive information about CA degradation during the production phase. These cultivations were based on a mineral medium, containing glycerol, glutamate, ammonium, and phosphate as the main nutrients. The ammonium concentration had a large influence on the degradation rate constant. In addition, either changes in the substrate availability or high concentrations of ammonium or glycerol cause a major increase in the degradation rate constant. Finally, a linear and a fuzzy logic model were made to predict CA degradation rates in these fed-batches.     

Simulation of penicillin production in fed-batch cultivations using a morphologically structured model

A mathematical model is formulated to describe trends in biomass and penicillin formation as well as substrate consumption for fed-batch cultivations. The biomass is structured into three morphological compartments, and glucose and corn steep liquor are considered as substrates for growth. Penicillin formation is assumed to take place in the subapical compartment and in the growing region of the hyphal compartment. Furthermore, it is inhibited by glucose. Model parameters are estimated using an evolutionary algorithm and fitting the model to a standard fed-batch cultivation. The model is validated on experimental data from three different fed-batch cultivations, including two repeated fed-batch cultivations. The model predictions show good agreement with the measurements of biomass and pencillin concentrations for all fed-batch cultivations. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 593-604, 1997.     

Adaptive,model-based control by the Open-Loop-Feedback-Optimal (OLFO) controller for the effective fed-batch cultivation of hybridoma cells

Although fed-batch suspension culture of animal cells continues to be of industrial importance for the large scale production of pharmaceutical products, existing control concepts are still insufficient. Changes in cell metabolism during cultivation and between similar cultivations, the complexity of the cell metabolism, and the lack of on-line state variables restrict the transfer of available control strategies established in bioprocess engineering. A process control strategy designed to achieve optimized process control must account for all these difficulties and fit sophisticated requirements toward adaptability and flexibility. The combination of a fed-batch process and an Open-Loop-Feedback-Optimal (OLFO) control provides a new approach for cell culture process control that couples an efficient cultivation concept to a capable process control strategy. The application of an adaptive, model-based OLFO controller to a hybridoma cultivation and experimental results are presented.     

Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His₆ tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L⁻¹ could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process.     

Monitoring population dynamics of the thermophilic Bacillus licheniformis CCMI 1034 in batch and continuous cultures using multi-parameter flow cytometry

Multi-parameter flow cytometry was used to monitor the population dynamics of Bacillus licheniformis continuous cultivations and the physiological responses to a starvation period and a glucose pulse. Using a mixture of two specific fluorescent stains, DiOC6(3) (3,3'-dihexylocarbocyanine iodide), and PI (propidium iodide), flow cytometric analysis revealed cell physiological heterogeneity. Four sub-populations of cells could be easily identified based on their differential fluorescent staining, these correspond to healthy cells (A) stained with DiOC6(3); cells or spores with a depolarised cytoplasmic membrane (B), no staining; cells with a permeabilised depolarised cytoplasmic membrane (C), stained with PI; and permeablised cells with a disrupted cytoplasmic membrane 'ghost cells' (D), stained with both DiOC6(3) and PI. Transmission electron micrographs of cells starved of energy showed different cell lysis process stages, highlighting 'ghost cells' which were associated with the double stained sub-population. It was shown, at the individual cell level, that there was a progressive inherent fluctuation in physiological heterogeneity in response to changing environmental conditions. All four sub-populations were shown to be present during glucose-limited continuous cultures, revealing a higher physiological stress level when compared with a glucose pulsed batch. A starvation period (batch without additional nutrients) increased the number of cells in certain sub-populations (cells with depolarised cytoplasmic membranes and cells with permeabilised depolarised cytoplasmic membranes), indicating that such stress may be caused by glucose limitation. Such information could be used to enhance process efficiency.     

Phenomenological Background and Some Preliminary Trials of Automated Substrate Supply in pH-Stat Modal Fed-Batch Culture Using a Setpoint of High Limit

First, by considering all possible combination of methanol (as a carbon-energy source), peptone (as an organic carbon-nitrogen source), and ammonium sulfate (as an inorganic nitrogen source), five batch cultures of a methanol-assimilating bacterium, Protomonas extorquens, were done to elucidate the cause(s) of pH variations during the microbial cultivations. The batch cultures have been classified into five types in terms of stoichiometric equations of cell growth which involve the elements, C, H, O, and N. The equations explained the pH variation (drop and rise) of the batch cultures on the basis of the consumption and liberation of ammonium ion. Then, six fed-batch cultures using a setpoint of high limit were done by feeding either methanol only or methanol plus peptone. Growth rates could be controlled by the amount of substrate(s) fed per pulse. Supplying peptone in addition to methanol enhanced cell growth. Characteristic differences between pH-stat modal fed-batch cultures using a low limit and those using a high limit, and advantages of the pH-stat modal fed-batch culture using a setpoint of high limit are discussed.     

A new microfluidic concept for parallel operated milliliter-scale stirred tank bioreactors

Parallel miniaturized stirred tank bioreactors are an efficient tool for "high-throughput bioprocess design." As most industrial bioprocesses are pH-controlled and/or are operated in a fed-batch mode, an exact scale-down of these reactions with continuous dosing of fluids into the miniaturized bioreactors is highly desirable. Here, we present the development, characterization, and application of a novel concept for a highly integrated microfluidic device for a bioreaction block with 48 parallel milliliter-scale stirred tank reactors (V = 12 mL). The device consists of an autoclavable fluidic section to dispense up to three liquids individually per reactor. The fluidic section contains 144 membrane pumps, which are magnetically driven by a clamped-on actuator section. The micropumps are designed to dose 1.6 μL per pump lift. Each micropump enables a continuous addition of liquid with a flow rate of up to 3 mL h(-1) . Viscous liquids up to a viscosity of 8.2 mPa s (corresponds to a 60% v/v glycerine solution) can be pumped without changes in the flow rates. Thus, nearly all feeding solutions can be delivered, which are commonly used in bioprocesses. The functionality of the first prototype of this microfluidic device was demonstrated by double-sided pH-controlled cultivations of Saccharomyces cerevisiae based on signals of fluorimetric sensors embedded at the bottom of the bioreactors. Furthermore, fed-batch cultivations with constant and exponential feeding profiles were successfully performed. Thus, the presented novel microfluidic device will be a useful tool for parallel and, thus, efficient optimization of controlled fed-batch bioprocesses in small-scale stirred tank bioreactors. This can help to reduce bioprocess development times drastically.     

Chemometric analysis of in-line multi-wavelength fluorescence measurements obtained during cultivations with a lipase producing Aspergillus oryzae strain

The filamentous fungus, Aspergillus oryzae, was cultivated in batch and fed-batch cultivations in order to investigate the use of multi-wavelength fluorescence for monitoring course of events during filamentous fungi cultivations. The A. oryzae strain applied expressed a fungal lipase from Thermomyces lanuginosus. Spectra of multi-wavelength fluorescence were collected every 5 min with the BioView system (DELTA, Denmark) and both explorative and predictive models, correlating the fluorescence data with cell mass and lipase activity, were built. During the cultivations, A. oryzae displayed dispersed hyphal growth and under these conditions no fouling of the multi-wavelength fluorescence sensor was observed. The scores of a parallel factor analysis (PARAFAC) model, based on the fluorescence spectra, gave clear evidence of, for example, the on-set of the feeding phase. The predictive models, estimating the cell mass, showed correlations between 0.73 and 0.97 with root mean square error of cross validation (RMSECV) values between 1.48 and 0.77 g . kg(-1). A model estimating the lipase activity was also constructed for the fed-batch cultivations with a correlation of 0.93. The results presented here clearly show that multi-wavelength fluorescence is a useful tool for monitoring fed-batch cultivations of filamentous fungi.     

Assessment of the performance of a fed-batch cultivation from the preculture quality using an electronic nose

An electronic nose, a gas-phase multisensor system, was used to monitor precultivations of a recombinant tryptophan-producing Escherichia coli strain. The electronic nose signals showed a high correlation toward the main stages of the precultivations, namely, exponential growth, oxygen-limited growth, and glucose depletion. Principal component analysis (PCA) of the electronic nose signals was performed and shown to be useful for monitoring preculture progression. More importantly, PCA also allowed a qualitative assessment of the preculture performance during subsequent fed-batch cultivations. The electronic nose signals from the precultures showed, furthermore, a high correlation to the time of phosphate limitation and the tryptophan yield coefficient of the subsequent fed-batch cultivations, which allowed an accurate prediction of these process variables using partial least squares (PLS). The results demonstrate on data from 12 cultivations how the electronic nose can be a useful tool for the assessment of inoculum quality, thereby providing means of reducing batch-to-batch variation and increasing the productivity of bioprocesses.     

Simple and efficient control of CHO cell cultures

Cell cultures must tightly be kept under control in order to guarantee a sufficiently small variability in the protein product quality. A simple and efficient technique for CHO-cell cultures is presented that allows keeping the viable cell count X(v) and the specific growth rate μ of the cells on predefined trajectories. As X(v) and μ cannot directly be measured online, they are controlled indirectly via the total mass of oxygen consumed. Online values of the latter can precisely be estimated from off gas analysis, i.e. from the O? volume ratio measured in the vent line and air flow rate measurements. In glutamine-limited fed-batch cultivations, the glutamine feed rate can be manipulated in such a way that the viable cell density and the specific growth rate are kept on predefined profiles for nearly the entire cultivation time. The viability of the cells is not affected by the closed loop control actions. The technique was validated with CHO-cells cultured in a 2.5-L fully instrumented stirred tank bioreactor. It is shown that the controller is able to run the process exactly on predefined tracks with a high batch-to-batch reproducibility. By means of six fed-batch cultivations of CHO cells it was shown that a remarkable reproducibility of viable cell concentration could be achieved throughout 140 h cultivation time.     

Intracellular pH-based controlled cultivation of yeast cells: II. cultivation methodology

Intracellular pH (pH(i)) was measured on-line in a bioreactor using a fluorescent pH(i) indicator, 9-aminoacridine, and controlled fed-batch cultivations of yeast cells based on pH(i) (FB-pH(i)) were performed. In FB-pH(i) cultivations, automated glucose additions were made to the culture in response to culture pH(i). The average ethanol (an-aerobic product) yield was significantly lower [0.12 g g(-1) glucose in fed-batch pH(i) cultivations with 100 ppm glucose additions (FB-pH(i)-100 cultivation) vs. 0.48 g g(-1) glucose in batch] and cell yield was higher (0.54 g g(-1) glucose in FB-pH(i)-100 cultivation vs. 0.3 g g(-1) glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pH(i) from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pH(i), performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB-pH(i) cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB-pH(i) cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations. (c) 1993 John Wiley & Sons, Inc.     

Protein release and foaming in Escherichia coli cultures grown in minimal medium

Protein release was studied in Escherichia coli cultivations in minimal medium under different conditions. The energy source concentration was oscillating either due to the cultivation technique or due to an applied on/off feed rate concept in fed-batch cultivations. It was found that the magnitude of protein release was dependent on the cultivation technique and the strain. The use of batch technique resulted in highest specific rate of protein release compared to fed-batch cultivations. No dependence of protein release on oscillating glucose concentration could be distinguished with oscillating periods of minutes of carbon starvation. Proteins released by cells acted as foaming agents and caused stabilisation of foam, during cultivation of Escherichia coli grown in minimal medium. Since the total cell protein was reflected in the medium the protein release is considered to be caused by cell lysis. However, only a few dominating proteins were present in the foam. The work was supported by grants from the Nordic Programme on Bioprocess Engineering under the auspices of NI, the Nordic Fund for Technology and Industrial Development; and from NUTEK, the Swedish National Board for Industrial and Technical Development.     

High-level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of Escherichia coli using lactose as inducer

Expression of a recombinant pyranose oxidase (P2O) from the basidiomycete Trametes ochracea has been increased 10-fold in shaking flask cultures of Escherichia coli BL21(DE3) harboring plasmid pSE33 by optimizing the composition of the culture medium using an experimental design approach. Inexpensive lactose was used as a medium component and inducer of expression of the P2O gene, which is under the control of a trc promoter. The expression system was studied in detail in batch and fed-batch cultivations with the aim to improve the expression level of active recombinant protein and to minimize the formation of inclusion bodies. In batch cultivations, the highest specific P2O activity of 0.9 U (mg of soluble protein)(-1) was measured in oxygen-limited cultures grown at 25 degrees C. The highest overall volumetric productivity of 33 mg of active P2O per liter and hour (corresponding to 345U (L h)(-1)) has been determined in a high-density fed-batch process with a feed-forward exponential feeding strategy. During the fed-batch process, lactose was added intermittently to the culture. A final biomass concentration of 33 g L(-1) (based on cell dry weight) was obtained. Compared to shaking flask cultures in not optimized culture media, the overall volumetric P2O productivity has been improved by a factor of 110 using the fed-batch strategy and the optimized culture medium. Recombinant P2O was expressed in the cytoplasm with 9% of the total soluble protein being active P2O. In terms of physical and enzyme kinetic properties, the purified recombinant P2O was found to be similar to the previously published data of P2O isolated from its original host.     

Calorimetric control of fed-batch cultures of Saccharomyces cerevisiae

Calorimetry has been used to control the glucose feeding in fed-batch cultures of S. cerevisiae in order to avoid ethanol formation and maintain a fully respiratory metabolism. Comparisons between batch and fed-batch cultivations showed that the former had a much lower growth yield. The growth yields for fed-batch cultivations were more than 30% higher than for batch cultures. However, energy balance calculations showed that a large part of the increase could be explained by the evaporation of ethanol during batch cultivations. When the growth yields obtained from the batch cultures were corrected for the evaporation of ethanol, the increase in growth yield for fed-batch cultures was about 10%.     

In situ removal and purification of biosurfactants by automated surface enrichment

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, was cultivated in a 5-L stirred and aerated bioreactor under different dissolved oxygen tensions (0%, 5%, and 30% of saturation) for evaluation of the influence of oxygen on cell growth as well as on the production of the main antigenic component of the vaccine against erysipelas, a 64–69 kDa protein (SpaA). The microorganism presented different growth profiles for different aeration conditions. However, at the end of the batch cultivations, similar cell concentrations were obtained under the studied conditions. In order to maximize biomass titers and antigen production, the microorganism was cultivated in fed-batch operation mode under aerobic conditions. Under this condition, there was a fivefold increase in biomass production in comparison to the results attained in batch cultivations. To follow up antigen expression, samples collected during batch cultivations were concentrated and treated with choline for antigen extraction. Antigen expression was then assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by murine immunization tests. It was observed a direct influence of oxygen availability upon antigen expression, which is favored in the presence of oxygen. Analysis of the samples collected throughout the fed-batch process also revealed that antigen production is growth associated.     

Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting

With the increased awareness of the problems associated with the growth dependent analysis of bacterial populations, direct optical detection methods such as flow cytometry have enjoyed increased popularity over the last few years. Among the analyses discussed here are: (1) Bacterial discrimination from other particles on the basis of nucleic acid staining, using sample disaggregation to provide fast reliable enumeration while minimizing data artefacts due to post sampling growth; (2) Determination of basic cell functions such as reproductive ability, metabolic activity and membrane integrity, to characterise the physiological state or degree of viability of bacteria; and (3) The use of single cell sorting onto agar plates, microscope slides or into multi-well plates to correlate viability as determined by cell growth with fluorescent labelling techniques. Simultaneous staining with different fluorochromes provides an extremely powerful way to demonstrate culture heterogeneity, and also to understand the functional differences revealed by each stain in practical applications. Analysis of bacterial fermentations showed a considerable drop (20%) in membrane potential and integrity during the latter stages of small scale (5L), well mixed fed-batch fermentations. These changes, not found in either batch or continuous culture fermentations, are probably due to the severe, steadily increasing stress associated with glucose limitation during the fed-batch process, suggesting 'on-line' flow cytometry could improve process control. Heat injured cells can already show up to 4 log of differences in recovery in different pre-enrichment media, thus contributing to the problem of viable but non-culturable cells (VBNC's). Cytometric cell sorting demonstrated decreasing recovery with increasing loss of membrane function. However, a new medium protecting the cells from intracellular and extracellular causes of oxidative stress improved recovery considerably. Actively respiring cells showed much higher recovery improvement than the other populations, demonstrating for the first time the contribution of oxidative respiration to intracellular causes of damage as a key part of the VBNC problem. Finally, absolute and relative frequencies of one species in a complex population were determined using immunofluorescent labelling in combination with the analysis of cell function. The detail and precision of multiparameter flow cytometric measurements of cell function at the single cell level now raise questions regarding the validity of classical, growth dependent viability assessment methods.