Simple technique for simultaneous on-line estimation of biomass and acetate from base consumption and conductivity measurements in high-cell density cultures of Escherichia coli

In high-cell density cultures of Escherichia coli, the amount of ammonia required to maintain a constant pH was an effective on-line measure of biomass. When acetic acid formation occurred, biomass estimates were corrected by additional measurements of the culture conductivity. In the absence of acetic acid excretion, the culture conductivity decreased approximately linearly during the fed-batch phase of the high-cell density cultivation. Upward deviation of the culture conductivity from this trend was proportional to the acetate concentration in the medium. Thus, simultaneous determination of the acetic acid concentration and correction of biomass estimates from ammonia consumption were possible by conductivity measurements.     

Soft sensor control of metabolic fluxes in a recombinant Escherichia coli fed-batch cultivation producing green fluorescence protein

A soft sensor approach is described for controlling metabolic overflow from mixed-acid fermentation and glucose overflow metabolism in a fed-batch cultivation for production of recombinant green fluorescence protein (GFP) in Escherichia coli. The hardware part of the sensor consisted of a near-infrared in situ probe that monitored the E. coli biomass and an HPLC analyzer equipped with a filtration unit that measured the overflow metabolites. The computational part of the soft sensor used basic kinetic equations and summations for estimation of specific rates and total metabolite concentrations. Two control strategies for media feeding of the fed-batch cultivation were evaluated: (1) controlling the specific rates of overflow metabolism and mixed-acid fermentation metabolites at a fixed pre-set target values, and (2) controlling the concentration of the sum of these metabolites at a set level. The results indicate that the latter strategy was more efficient for maintaining a high titer and low variability of the produced recombinant GFP protein.     

Two-dimensional fluorescence spectroscopy: a novel approach for controlling fed-batch cultivations

In industrial fed-batch cultivations it is often necessary to control substrate concentrations at a low level to prevent the production of overflow metabolites and thus optimize the biomass yield. A new method for on-line monitoring and fed-batch control based on fluorescence measurements has been developed. Via instantaneous in situ measurements and multivariate data analysis a chemometric model has been established, which enables the rapid detection of ethanol production at aerobic Saccharomyces cerevisiae fed-batch cultivations. The glucose feed rate is controlled by predicting the metabolic state directly from the fluorescence intensities. Thus, ethanol production could be avoided completely while increasing the biomass yield accordingly. The robust instrumentation is suitable for industrial applications.     

Combined in silico modeling and metabolomics analysis to characterize fed-batch CHO cell culture

The increasing demand for recombinant therapeutic proteins highlights the need to constantly improve the efficiency and yield of these biopharmaceutical products from mammalian cells, which is fully achievable only through proper understanding of cellular functioning. Towards this end, the current study exploited a combined metabolomics and in silico modeling approach to gain a deeper insight into the cellular mechanisms of Chinese hamster ovary (CHO) fed-batch cultures. Initially, extracellular and intracellular metabolite profiling analysis shortlisted key metabolites associated with cell growth limitation within the energy, glutathione, and glycerophospholipid pathways that have distinct changes at the exponential-stationary transition phase of the cultures. In addition, biomass compositional analysis newly revealed different amino acid content in the CHO cells from other mammalian cells, indicating the significance of accurate protein composition data in metabolite balancing across required nutrient assimilation, metabolic utilization, and cell growth. Subsequent in silico modeling of CHO cells characterized internal metabolic behaviors attaining physiological changes during growth and non-growth phases, thereby allowing us to explore relevant pathways to growth limitation and identify major growth-limiting factors including the oxidative stress and depletion of lipid metabolites. Such key information on growth-related mechanisms derived from the current approach can potentially guide the development of new strategies to enhance CHO culture performance.     

Controlled fed-batch by tracking the maximal culture capacity

Fed-batch processes are well established in the biotech industry. The major reason to apply this technique is to avoid overflow metabolism and/or accumulation of toxic substrates. The basic idea of this approach is to control the physiological state of the culture, rather than just the typically exponential feed rate profile, by challenging a fed-batch cultivation repetitive in useful time intervals. The feed rate is reduced for a short period and culture responses are analysed in real time and on-line. Thus it is possible to get a positive response at the earliest detectable point of potential overfeeding. During the disturbance minute amounts of overflow metabolites will deplete simultaneously. This highly dynamic approach was applied successfully to industrially relevant production systems such as yeasts (Saccharomyces cerevisiae, Pichia pastoris) and bacteria (Escherichia coli).     

Macroscopic modelling of overflow metabolism and model based optimization of hybridoma cell fed-batch cultures

A macroscopic model that takes into account phenomena of overflow metabolism within glycolysis and glutaminolysis is proposed to simulate hybridoma HB-58 cell cultures. The model of central carbon metabolism is reduced to a set of macroscopic reactions. The macroscopic model describes three metabolism states: respiratory metabolism, overflow metabolism and critical metabolism. The model parameters and confidence intervals are obtained via a non linear least squares identification. It is validated with experimental data of fed-batch hybridoma cultures and successfully predicts the dynamics of cell growth and death, substrate consumption (glutamine and glucose) and metabolites production (lactate and ammonia). Based on a sensitivity analysis of the model outputs with respect to the parameters, a model reduction is proposed. Finally, the maximization of biomass productivity of hybridoma cell fed-batch cultures is analyzed. This model allows, on the one hand, quantitatively describing overflow metabolism in mammalian cell cultures and, on the other hand, will be valuable for monitoring and control of fed-batch cultures in order to optimize the process. This is illustrated in this contribution with the determination of optimal feeding profiles aiming at maximizing biomass productivity.     

Utility of an Escherichia coli strain engineered in the substrate uptake system for improved culture performance at high glucose and cell concentrations: an alternative to fed-batch cultures

Overflow metabolism is an undesirable characteristic of aerobic cultures of Escherichia coli. It results from elevated glucose consumption rates that cause a high substrate conversion to acetate, severely affecting cell physiology and bioprocess performance. Such phenomenon typically occurs in batch cultures under high glucose concentration. Fed-batch culture, where glucose uptake rate is controlled by external addition of glucose, is the classical bioprocessing alternative to prevent overflow metabolism. Despite its wide-spread use, fed-batch mode presents drawbacks that could be overcome by simpler batch cultures at high initial glucose concentration, only if overflow metabolism is effectively prevented. In this study, an E. coli strain (VH32) lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) with a modified glucose transport system was cultured at glucose concentrations of up to 100 g/L in batch mode, while expressing the recombinant green fluorescence protein (GFP). At the highest glucose concentration tested, acetate accumulated to a maximum of 13.6 g/L for the parental strain (W3110), whereas a maximum concentration of only 2 g/L was observed for VH32. Consequently, high cell and GFP concentrations of 52 and 8.2 g/L, respectively, were achieved in VH32 cultures at 100 g/L of glucose. In contrast, maximum biomass and GFP in W3110 cultures only reached 65 and 48%, respectively, of the values attained by the engineered strain. A comparison of this culture strategy against traditional fed-batch culture of W3110 is presented. This study shows that high cell and recombinant protein concentrations are attainable in simple batch cultures by circumventing overflow metabolism through metabolic engineering. This represents a novel and valuable alternative to classical bioprocessing approaches.     

Calorimetric control of fed-batch fermentations

Measurements of the heat produced by Saccharomyces cerevisiae CBS 426 were used in conjunction with elemental and enthalpic balances to control fed-batch fermentations. A proportional control using the ratio of carbon dioxide evolution rate to heat production resulted in high biomass yields and minimal ethanol production. For the special case where the carbon source contains no nitrogen, biomass production estimated from heat measurements agreed well with measured values. When the controller gain was set below the maximum specific cellular growth rate, stable control was achieved, even in response to large upsets in feed concentrations.     

Growth and energy metabolism in aerobic fed-batch cultures of Saccharomyces cerevisiae: simulation and model verification

A kinetic model of overflow metabolism in Saccharomyces cerevisiae was used for simulation of aerobic fed-batch cultivations. An inhibitory effect of ethanol on the maximum respiration of the yeast was observed in the experiments and included in the model. The model predicts respiration, biomass, and ethanol formation and the subsequent ethanol consumption, and was experimentally validated in fed-batch cultivations. Oscillating sugar feed with resulting oscillating carbon dioxide production did not influence the maximum respiration rate, which indicates that the pyruvate dehydrogenase complex is not involved as a bottleneck causing aerobic ethanol formation.     

On-line biomass monitoring by capacitance measurement.

An in-situ, steam-sterilizable capacitance probe was used to follow the biomass concentration on-line, in bioreactors from 20 to 2000 l total volume. Microbial cultures of Saccharomyces cerevisiae, Pichia pastoris and Streptomyces virginiae were grown in batch and fed-batch culture in both defined and complex media in order to demonstrate the wide dynamic operating range of the instrument. A linear correlation was found between the on-line capacitance measurement and the off-line measurements (optical density, OD620; packed mycelial volume, PMV; biomass concentration X, and colony forming units, CFU ml-1) for biomass concentrations (dry cell weight) up to 30 g l-1 (St. virginiae), 106 g l-1 (S. cerevisiae) and 89 g l-1 (P. pastoris). The on-line capacitance measurement was slightly influenced by variations in agitation speed and strong extraneous radio frequencies. A specific capacitance constant (Cs) was defined for all microbial cells which was dependent on cell viability and cell size. The Cs was easy to calculate using the on-line capacitance measurement and an off-line estimation of biomass concentration. The Biomass Monitor proved suitable for precise on-line monitoring of both homogeneous (uni-cellular) and heterogeneous (mycelial) cultures in bioreactors.     

Precultivation technique for studies of microorganisms exhibiting overflow metabolism

A precultivation technique for microorganisms exhibiting overflow metabolism is presented. It is based on a low initial medium volume in the fermenter. The medium feed contains all the nutrients, but is diluted with respect to sugar, the concentration of which determines the biomass concentration at the end of the preculture. By this method, effects of varying activity of the inocula from shake flask cultures are minimised and the metabolic state, i.e. oxido-reductive, oxidative growth on sugar plus overflow metabolite or oxidative growth on sugar alone, can be controlled at the start of the main fermentation.     

Fermentative capacity of dry active wine yeast requires a specific oxidative stress response during industrial biomass growth

Induction of the oxidative stress response has been described under many physiological conditions in Saccharomyces cerevisiae, including industrial fermentation for wine yeast biomass production where cells are grown through several batch and fed-batch cultures on molasses. Here, we investigate the influence of aeration on the expression changes of different gene markers for oxidative stress and compare the induction profiles to the accumulation of several intracellular metabolites in order to correlate the molecular response to physiological and metabolic changes. We also demonstrate that this specific oxidative response is relevant for wine yeast performance by construction of a genetically engineered wine yeast strain overexpressing the TRX2 gene that codifies a thioredoxin, one of the most important cellular defenses against oxidative damage. This modified strain displays an improved fermentative capacity and lower levels of oxidative cellular damages than its parental strain after dry biomass production.     

Microcalorimetric study on the growth and metabolism of microencapsulated microbial cell culture

The rate of heat output is one of the suitable measurements of metabolic activity of the organism or its parts, down to the cellular or even the sub-cellular levels. In this paper, microcalorimetry was first applied to study the metabolic activity of microbial in both alginate-polylysine-alginate and alginate-chitosan-alginate microencapsulated cultures as well as in free non-encapsulated culture. The organisms used for the measurements were Escherichia coli and Saccharomyces cerevisiae. As a result of this work, it was found that, despite E. coli cell in free non-encapsulated culture has the highest metabolic rate due to the highest value of heat output, the proliferation of the cells terminates quickly with a lowest biomass formed. And we found also an obviously longer stationary phase in microencapsulated culture. As far as S. cerevisiae was concerned, it was found that there was also the highest value of heat output in free non-encapsulated culture, but the cell density was lower than that in microencapsulated culture. On account of the microcalorimetric and metabolic measurements, it can be concluded that more substrate can be used to convert to biomass in microencapsulated culture which means a higher biomass yield existed.     

Predictive macroscopic modeling of cell growth,metabolism and monoclonal antibody production: Case study of a CHO fed-batch production

We describe a systematic approach to establish predictive models of CHO cell growth, cell metabolism and monoclonal antibody (mAb) formation during biopharmaceutical production. The prediction is based on a combination of an empirical metabolic model connecting extracellular metabolic fluxes with cellular growth and product formation with mixed Monod-inhibition type kinetics that we generalized to every possible external metabolite. We describe the maximum specific growth rate as a function of the integral viable cell density (IVCD). Moreover, we also take into account the accumulation of metabolites in intracellular pools that can influence cell growth. This is possible even without identification and quantification of these metabolites as illustrated with fed-batch cultures of Chinese Hamster Ovary (CHO) cells producing a mAb. The impact of cysteine and tryptophan on cell growth and cell productivity was assessed, and the resulting macroscopic model was successfully used to predict the impact of new, untested feeding strategies on cell growth and mAb production. This model combining piecewise linear relationships between metabolic rates, growth rate and production rate together with Monod-inhibition type models for cell growth did well in predicting cell culture performance in fed-batch cultures even outside the range of experimental data used for establishing the model. It could therefore also successfully be applied for in silico prediction of optimal operating conditions.     

Intensification of high cell-density cultivations of rE. coli for production of S. pneumoniae antigenic surface protein, PspA3, using model-based adaptive control

This work proposes an innovative methodology to control high density fed-batch cultures of E. coli, based on measurements of the concentration of dissolved oxygen and on estimations of the cellular specific growth rate (μ), of the yield of biomass/limiting substrate (Y (xs)) and of the maintenance coefficient (m). The underlying idea is to allow cells to grow according to their metabolic capacity, without the constraints inherent to pre-set growth rates. Cellular concentration was assessed on-line through a capacitance probe. Three configurations of the control system were compared: (1) pre-set value for the three control parameters; (2) continuously updating μ; (3) updating μ, Y (xs) and m. Implementation of an efficient noise filter for the signal of the capacitance probe was essential for a good performance of the control system. The third control strategy, within the framework of an adaptive model-based control, led to the best results, with biomass productivity reaching 9.2?g(DCW)/L/h.     

Alteration of mammalian cell metabolism by dynamic nutrient feeding

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10⁷ cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genome-scale analysis of Saccharomyces cerevisiae metabolism and ethanol production in fed-batch culture

A dynamic flux balance model based on a genome-scale metabolic network reconstruction is developed for in silico analysis of Saccharomyces cerevisiae metabolism and ethanol production in fed-batch culture. Metabolic engineering strategies previously identified for their enhanced steady-state biomass and/or ethanol yields are evaluated for fed-batch performance in glucose and glucose/xylose media. Dynamic analysis is shown to provide a single quantitative measure of fed-batch ethanol productivity that explicitly handles the possible tradeoff between the biomass and ethanol yields. Productivity optimization conducted to rank achievable fed-batch performance demonstrates that the genetic manipulation strategy and the fed-batch operating policy should be considered simultaneously. A library of candidate gene insertions is assembled and directly screened for their achievable ethanol productivity in fed-batch culture. A number of novel gene insertions with ethanol productivities identical to the best metabolic engineering strategies reported in previous studies are identified, thereby providing additional targets for experimental evaluation. The top performing gene insertions were substrate dependent, with the highest ranked insertions for glucose media yielding suboptimal performance in glucose/xylose media. The analysis results suggest that enhancements in biomass yield are most beneficial for the enhancement of fed-batch ethanol productivity by recombinant xylose utilizing yeast strains. We conclude that steady-state flux balance analysis is not sufficient to predict fed-batch performance and that the media, genetic manipulations, and fed-batch operating policy should be considered simultaneously to achieve optimal metabolite productivity.     

High-cell-density fermentation of recombinant Saccharomyces cerevisiae using glycerol

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 microm plasmid, pYES2 containing a GAL1 promoter for expression of the beta-galactosidase gene), the yeast was grown with glycerol as the substrate by fed-batch fermentation. The feeding strategy was based on an on-line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on-line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed-batch mode with pH control at 5.0 +/- 0.1 was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5-3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.     

Fed-batch mode in shake flasks by slow-release technique

Most industrial production processes are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild-type strain (DSM 70277) and a recombinant strain pC10-FMD (P(FMD)-GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH-drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow-release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38-0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6-MES mineral medium. In fed-batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420-fold improvement in fed-batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose.     

A simple feedback control of Escherichia coli growth for recombinant aldolase production in fed-batch mode

Fed-batch production of recombinant fuculose-1-phosphate aldolase (FucA) by Escherichia coli XL1 Blue MRF′ (pTrcfuc) has been automated by using a simple feedback specific growth rate control strategy. Non-induced continuous cultures were conducted in order to characterize substrate consumption and carbon dioxide production yields and rates. In fed-batch cultures, substrate feeding rate was adjusted using on-line biomass estimation based on exhaust gas analysis and macroscopic mass balances. Overexpression of recombinant protein induced by isopropyl-β-d-thiogalactopyranoside (IPTG) under trc promoter did not affect significantly the control of specific growth rate during 7 h after induction. Growth and protein production curves were parallel until high level of protein expression started to inhibit cell growth. The proposed specific growth rate control strategy has been successfully applied to both non-induced and induced fed-batch cultures that do not exhibit severe growth rate depression.