Metabolite Production Rates Research Articles

Vertical stratification and stability of biogeochemical processes in the deep saline waters of Lake Vanda, Antarctica

AbstractLake Vanda is a permanently ice‐covered lake in the McMurdo Dry Valleys of Antarctica. Its bottom waters remain stratified year‐round because of a strong salinity‐driven density gradient. We have assessed spatial patterns in and relationships between major biogeochemical processes in the water column of Lake Vanda. Samples were collected in the austral summers of 2008 and 2011 to measure concentrations of metabolites associated with a suite of biogeochemical processes across the deep salinity gradient. The shapes of the resulting geochemical profiles were consistent between 2008 and 2011. Metabolite production and consumption rates were estimated using a reactive transport model based on the assumption that vertical diffusion was the only active physical transport process. We validated this model for nitrification by using stable isotope incubations to show that this process was only active at depths predicted by the model. No nitrification activity was observed at 68 m depth in spite of overlapping oxygen and ammonium gradients. We attribute this lack of activity to the competitive inhibition of ammonia monooxygenase by methane. Net nitrous oxide and nitrate consumption were observed in the oxic water column, providing evidence of aerobic denitrification. The depth of maximum net oxygen production did not coincide with the deep chlorophyll maxima (at 59.3, 63, and 68.2 m) measured in the same profile. Finally, the integrated sulfide oxidation rate was high compared with other oxidation processes, indicating that sulfide was an important electron donor for the water column microbial community.

Real-time ALT and LDH activities determined in viable precision-cut mouse liver slices using hyperpolarized [1-13 C]pyruvate-Implications for studies on biopsied liver tissues.

Precision-cut liver slices (PCLS) are widely used in liver research as they provide a liver model with all liver cell types in their natural architecture. The purpose of this study was to demonstrate the use of PCLS for hyperpolarized metabolic investigation in a mouse model, for potential future application in liver biopsy cores. Fresh normal liver was harvested from six mice. 500μm PCLS were prepared and placed in a 10mm NMR tube in an NMR spectrometer and perfused continuously. 31 P spectra were acquired to evaluate the presence of adenosine triphosphate (ATP) and validate viability in all samples. Hyperpolarized [1-13 C]pyruvate was flushed into the NMR tube in the spectrometer. Consecutive 13 C NMR spectra were acquired immediately after the injection using both non-selective (five injections, two livers) and selective RF excitation (six injections, three livers). The 31 P spectra showed the characteristic signals of ATP, confirming the viability of the PCLS for more than 2.5h in the spectrometer. After each of the [1-13 C]pyruvate injections, both [1-13 C]lactate and [1-13 C]alanine signals were detected. Selective RF excitation aimed at both [1-13 C]lactate and [1-13 C]alanine enabled better visualization and quantification of the metabolic activity. Using this acquisition approach only the newly formed metabolites are observed upon excitation, and their intensities relative to those of hyperpolarized pyruvate enable quantification of metabolite production rates. This rate of lactate and alanine production appeared to be constant throughout the measurement time, with alanine production about 2.3 times higher than lactate. In summary, the viability of PCLS in an NMR spectrometer was demonstrated and hyperpolarized [1-13 C]pyruvate metabolism was recorded. This study opens up the possibility of evaluating alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities in human liver biopsies, while preserving the tissue architecture and viability. In healthy, well-perfused liver slices the ratio of ALT to LDH activity is about 2.3.

Comprehensive Secondary Metabolite Profiling Toward Delineating the Solid and Submerged-State Fermentation of Aspergillus oryzae KCCM 12698.

Aspergillus oryzae has been commonly used to make koji, meju, and soy sauce in traditional food fermentation industries. However, the metabolic behaviors of A. oryzae during fermentation in various culture environments are largely uncharacterized. Thus, we performed time resolved (0, 4, 8, 12, 16 day) secondary metabolite profiling for A. oryzae KCCM 12698 cultivated on malt extract agar and broth (MEA and MEB) under solid-state fermentation (SSF) and submerged fermentation (SmF) conditions using the ultrahigh performance liquid chromatography-linear trap quadrupole-ion trap-mass spectrometry (UHPLC-LTQ-IT-MS/MS) followed by multivariate analyses. We observed the relatively higher proportions of coumarins and oxylipins in SSF, whereas the terpenoids were abundant in SmF. Moreover, we investigated the antimicrobial efficacy of metabolites that were extracted from SSF and SmF. The SSF extracts showed higher antimicrobial activities as compared to SmF, with higher production rates of bioactive secondary metabolites viz., ketone-citreoisocoumarin, pentahydroxy-anthraquinone, hexylitaconic acid, oxylipins, and saturated fatty acids. The current study provides the underpinnings of a metabolomic framework regarding the growth and bioactive compound production for A. oryzae under the primarily employed industrial cultivation states. Furthermore, the study holds the potentials for rapid screening and MS-characterization of metabolites helpful in determining the consumer safety implications of fermented foods involving Koji mold.

Design of stable metabolic networks.

In this work, we propose eigenvalue optimization combined with Lyapunov theory concepts to ensure stability of the Embden-Meyerhof-Parnas pathway, the pentose-phosphate pathway, the phosphotransferase system and fermentation reactions of Escherichia coli. We address the design of a metabolic network for the maximization of different metabolite production rates. The first case study focuses on serine production, based on a model that consists of 18 differential equations corresponding to dynamic mass balances for extracellular glucose and intracellular metabolites, and thirty kinetic rate expressions. A second case study addresses the design problem to maximize ethanol production, based on a dynamic model that involves mass balances for 25 metabolites and 38 kinetic rate equations. The nonlinear optimization problem including stability constraints has been solved with reduced space Successive Quadratic Programming techniques. Numerical results provide useful insights on the stability properties of the studied kinetic models.

Modeling Substrate Utilization, Metabolite Production, and Uranium Immobilization in Shewanella oneidensis Biofilms

In this study, we developed a two-dimensional mathematical model to predict substrate utilization and metabolite production rates in Shewanella oneidensis MR-1 biofilm in the presence and absence of uranium (U). In our model, lactate and fumarate are used as the electron donor and the electron acceptor, respectively. The model includes the production of extracellular polymeric substances (EPS). The EPS bound to the cell surface and distributed in the biofilm were considered bound EPS (bEPS) and loosely associated EPS (laEPS), respectively. COMSOL® Multiphysics finite element analysis software was used to solve the model numerically (model file provided in the Supplementary Material). The input variables of the model were the lactate, fumarate, cell and EPS concentrations, half saturation constant for fumarate, and diffusion coefficients of the substrates and metabolites. To estimate unknown parameters and calibrate the model, we used a custom designed biofilm reactor placed inside a nuclear magnetic resonance (NMR) microimaging and spectroscopy system and measured substrate utilization and metabolite production rates. From these data we estimated the yield coefficients, maximum substrate utilization rate, half saturation constant for lactate, stoichiometric ratio of fumarate and acetate to lactate and stoichiometric ratio of succinate to fumarate. These parameters are critical to predicting the activity of biofilms and are not available in the literature. Lastly, the model was used to predict uranium immobilization in S. oneidensis MR-1 biofilms by considering reduction and adsorption processes in the cells and in the EPS. We found that the majority of immobilization was due to cells, and that EPS was less efficient at immobilizing U. Furthermore, most of the immobilization occurred within the top 10 μm of the biofilm. To the best of our knowledge, this research is one of the first biofilm immobilization mathematical models based on experimental observation. It has the ability to predict the relative contributions to U immobilization of laEPS, bEPS, and cells.

Influence of fluid dynamic conditions on 1,3‐propanediol production from glycerol by Shimwellia blattae: carbon flux and cell response

ABSTRACTBACKGROUNDThe study of fluid dynamic conditions is essential in the development and the scaling‐up of bioprocesses. If the working volume is large, different turbulence regimes could appear during the fermentation, affecting the metabolic state of the overall culture. In anaerobic systems, increasing the stirrer speed can induce hydrodynamic stress, increasing shear stress, and its consequences can negatively affect the cell viability, reducing the yield or the productivity of the process.RESULTSA kinetic model was employed for describing the time course of biomass growth rate and the different metabolite production rates. An increase in agitation from 100 to 500 rpm enhanced 1,3‐propanediol (PDO) production and biomass growth rate. However, when stirrer speed was increased from 600 to 1000 rpm, the shear stress had a negative effect, decreasing the maximum PDO concentration reached and the glycerol uptake rate. Cellular response to shear stress conditions was also observed by the time course viable cells and cell images.CONCLUSIONSThe production of PDO from raw glycerol by S. blattae is favored by increasing the stirrer speed to 500 rpm. Cell viability and integrity are compromised by hydrodynamic stress under aggressive agitation conditions (>600 rpm). © 2017 Society of Chemical Industry

A single metabolite production by Escherichia coli BW25113 and its pflA.cra mutant cultivated under microaerobic conditions using glycerol or glucose as a carbon source.

Abundant, low prices and a highly reduced nature make glycerol to be an ideal feedstock for the production of reduced biochemicals and biofuels. Escherichia coli has been paid much attention as the platform of microbial cell factories due to its high growth rate (giving higher metabolite production rate) and the capability of utilizing a wide range of carbon sources. However, one of the drawbacks of using E. coli as a platform is its mixed metabolite formation under anaerobic conditions. In the present study, it was shown that ethanol could be exclusively produced from glycerol by the wild type E. coli, while d-lactic acid could be exclusively produced from glucose by pflA.cra mutant, where the glucose uptake rate could be increased by this mutant as compared to the wild type strain. It was also shown that the growth rate is significantly reduced in pflA.cra mutant for the case of using glycerol as a carbon source due to redox imbalance. The metabolic regulation mechanisms behind the fermentation characteristic were clarified to some extent.

Triptolide Induces hepatotoxicity via inhibition of CYP450s in Rat liver microsomes

BackgroundTriptolide (TP), an active constituent of Tripterygium wilfordii, possesses numerous pharmacological activities. However, its effects on cytochrome P450 enzymes (CYP450s) in rats remain unexplored.MethodsIn this study, the effects of triptolide on the six main CYP450 isoforms (1A2, 2C9, 2C19, 2D6, 2E1, and 3A) were investigated both in vivo and in vitro. We monitored the body weight, survival proportions, liver index, changes in pathology, and biochemical index upon TP administration, in vivo. Using a cocktail probe of CYP450 isoform-specific substrates and their metabolites, we then carried out in vitro enzymatic studies in liver microsomal incubation systems via ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Finally, we verified our results at the messenger ribonucleic acid (mRNA) and protein level through quantitative real-time polymerase chain reaction (RT-qPCR), western blotting, and immunohistochemical detection.ResultsThe in vivo toxicity study confirmed that Sprague-Dawley (SD) rats exhibited dose-dependent hepatotoxicity after intragastric administration of TP [200, 400, and 600 μg/(kg.day)] for 28 days. In case of the CYP450 isoforms 3A, 2C9, 2C19, and 2E1, the in vitro metabolic study demonstrated a decrease in the substrate metabolic rate, metabolite production rate, and Vmax, with an increase in the Km value, compared with that observed in the control group. Additionally, a TP dose-dependent decrease in the mRNA levels was observed in the four major isoforms of CYP3A subfamily (3A1/3A23, 3A2, 3A9, and 3A62) and CYP2C9. A similar effect was also observed with respect to the protein levels of CYP2C19 and CYP2E1.ConclusionsThis study suggests that TP can cause hepatotoxicity by reducing the substrate affinity, activity, and expression at the transcriptional and protein levels of the CYP450 isoforms 3A, 2C9, 2C19, and 2E1. TP also has the potential to cause pharmacokinetic drug interactions when co-administered with drugs metabolized by these four isoforms. However, further clinical studies are needed to evaluate the significance of this interaction.

Gauging circadian variation in ketamine metabolism by real-time breath analysis.

The time-of-day of drug application is an important factor in maximizing efficacy and minimizing toxicity. Real-time in vivo mass spectrometric breath analysis of mice was deployed to investigate time-of-day variation in ketamine metabolism. Different production rates of ketamine metabolites, including the recently described anti-depressant hydroxynorketamine, were found in opposite circadian phases. Thus, breath analysis has potential as a rapid and 3Rs (Replacement, Reduction and Refinement) conforming screening method to estimate the time-dependence of drug metabolism.

Segmented linear modeling of CHO fed-batch culture and its application to large scale production.

ABSTRACTWe describe a systematic approach to model CHO metabolism during biopharmaceutical production across a wide range of cell culture conditions. To this end, we applied the metabolic steady state concept. We analyzed and modeled the production rates of metabolites as a function of the specific growth rate. First, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. For this, the smoothed derivative of the metabolic rates with respect to the growth rate were used followed by hierarchical clustering of the obtained partition. We then applied a piecewise regression to the metabolic rates with the previously determined number of phases. This allowed identifying the growth rates at which the cells underwent a metabolic shift. The resulting model with piecewise linear relationships between metabolic rates and the growth rate did well describe cellular metabolism in the fed‐batch cultures. Using the model structure and parameter values from a small‐scale cell culture (2 L) training dataset, it was possible to predict metabolic rates of new fed‐batch cultures just using the experimental specific growth rates. Such prediction was successful both at the laboratory scale with 2 L bioreactors but also at the production scale of 2000 L. This type of modeling provides a flexible framework to set a solid foundation for metabolic flux analysis and mechanistic type of modeling. Biotechnol. Bioeng. 2017;114: 785–797. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Physiological responses to acid stress by Saccharomyces cerevisiae when applying high initial cell density.

High initial cell density is used to increase volumetric productivity and shorten production time in lignocellulosic hydrolysate fermentation. Comparison of physiological parameters in high initial cell density cultivation of Saccharomyces cerevisiae in the presence of acetic, formic, levulinic and cinnamic acids demonstrated general and acid-specific responses of cells. All the acids studied impaired growth and inhibited glycolytic flux, and caused oxidative stress and accumulation of trehalose. However, trehalose may play a role other than protecting yeast cells from acid-induced oxidative stress. Unlike the other acids, cinnamic acid did not cause depletion of cellular ATP, but abolished the growth of yeast on ethanol. Compared with low initial cell density, increasing initial cell density reduced the lag phase and improved the bioconversion yield of cinnamic acid during acid adaptation. In addition, yeast cells were able to grow at elevated concentrations of acid, probable due to the increase in phenotypic cell-to-cell heterogeneity in large inoculum size. Furthermore, the specific growth rate and the specific rates of glucose consumption and metabolite production were significantly lower than at low initial cell density, which was a result of the accumulation of a large fraction of cells that persisted in a viable but non-proliferating state.

ChemInform Abstract: Challenges in the Heterologous Production of Antibiotics in Streptomyces

AbstractReview: 62 refs.

Cortisol metabolism, postnatal depression and weight changes in the first 12 months postpartum.

Postnatal depression correlates with postpartum weight retention, and dysregulated cortisol metabolism is evident in depressed individuals. Cortisol metabolism, BMI and metabolic phenotype are robustly associated, but the role of cortisol metabolism in postnatal mental health and weight loss has never been examined. A longitudinal observation. Forty nine healthy women with uncomplicated pregnancy. BMI and urinary steroid metabolites at 1 week and 1, 3, 6 and 12 months postpartum. Validated urinary steroid metabolite ratios were measured to determine the activities of 11β-hydroxysteroid dehydrogenases (11β-HSD) that interconvert inactive cortisone and active cortisol and the 5α-reductases that clear cortisol to its inactive metabolites. Postnatal depression symptoms were measured at 1, 6 and 12 months. Low 5α-reductase activity was associated with greater weight loss across the first year, independent of demographics, breastfeeding and depression. Postpartum BMI change was unrelated to postnatal depression at any time. Symptoms of postnatal depression were related to higher cortisol metabolite production at 12 months, independent of demographics and breastfeeding. Greatest weight loss in the postpartum year was associated with lower conversion of cortisone to cortisol and lower conversion of cortisol to its metabolites, supporting previous work that demonstrates the facilitative role of lower 5α-reductase and 11β-HSD-1 in weight loss. Greater depression symptoms were associated with higher cortisol metabolite production rates. Whilst weight and mental health are both associated with dysregulation of the HPA axis, there may be different pathways towards depressed and obese phenotypes in healthy postpartum samples.

A methodology for building a macroscopic FBA-based dynamical simulator of cell cultures through flux variability analysis

A dynamical FBA-based simulator of hybridoma cell fed-batch cultures predicting the dynamics of biomass growth, substrate consumption (glucose and glutamine), metabolites production (lactate, ammonium and alanine) and associate intracellular metabolism based on a simplified metabolic network is proposed. A preliminary comparison between the range of admissible flux distribution obtained based on all available measurements (the two substrates uptake rates and the three metabolite production rates) and based on only part of them (only the two substrates uptake rates) is performed to deal with the usual problem of system underdetermination in constraint-based modeling context. This comparative flux variability analysis allows the objective identification of some additional constraints (to be used in the final FBA-based simulator) so as to obtain similar admissible flux intervals in both cases. Moreover, the proposed approach legitimates the cost criterion used for the linear optimization, i.e. cell growth maximization. This methodology is validated on experimental data of two fed-batch cultures and cross validated on a batch culture of hybridoma cells HB-58. The flux distribution results are in agreement with overflow metabolism description available in literature.

Challenges in the Heterologous Production of Antibiotics in Streptomyces.

The fast growing genome databases provide us with a large number of so far unknown secondary metabolite biosynthetic gene clusters. A key method to study these gene clusters is their heterologous expression in an engineered host strain. Gene clusters derived from actinomycetes are usually expressed in a Streptomyces host strain to identify and investigate the corresponding compounds. However, heterologous expression is often accompanied with some challenges affecting the production rates of secondary metabolites. The first step is therefore the selection of a suitable expression vector and host strain. Once production has been established, there are several possibilities to improve compound yields either by media screens, by overexpression of regulatory or transport genes or by introduction of constitutive or inducible promoters. A surely important, but hitherto little studied factor is also the regulation of a heterologously expressed gene cluster by its host strain. This review gives a short overview on the chances and challenges provided by heterologous production of secondary metabolites in Streptomyces.

A rapid, sensitive, simple plate assay for detection of microbial alginate lyase activity

Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2–3min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also worked well for screening and identification of alginate lyase producers and non-producers from environmental samples on common laboratory media. They did this by clearly showing the presence or absence of clearance zones around the microbial colonies grown. This new method is rapid, efficient, and could easily be performed for screening a large number of microbial cultures. This is the first report on the use of Gram's iodine for the detection of alginate lyase production by microorganisms using plate assay.

Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations.

As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in rational bioreactor design and in the development of novel culture media for hESC maintenance and expansion.

SAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin.

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin-targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.

Tissue-Specific Metabolism of Benzo[a]pyrene in Rainbow Trout (Oncorhynchus mykiss): A Comparison between the Liver and Immune Organs

Polycyclic aromatic hydrocarbons (PAHs) are immunotoxicants in fish. In mammals, phase I metabolites are believed to be critically involved in the immunotoxicity of PAHs. This mechanism has been suggested for fish as well. The present study investigates the capacity of immune organs (head kidney, spleen) of rainbow trout, Oncorhynchus mykiss, to metabolize the prototypic PAH, benzo[a]pyrene (BaP). To this end, we analyzed 1) the induction of enzymatic capacity measured as 7-ethoxyresorufin-O-deethylase (EROD) activity in immune organs compared with liver, 2) the organ profiles of BaP metabolites generated in vivo, and 3) rates of microsomal BaP metabolite production in vitro. All measurements were done for control fish and for fish treated with an intraperitoneal injection of 15 mg BaP/kg body weight. In exposed trout, the liver, head kidney, and spleen contained similar levels of BaP, whereas EROD induction differed significantly between the organs, with liver showing the highest induction factor (132.8×), followed by head kidney (38.4×) and spleen (1.4×). Likewise, rates of microsomal metabolite formation experienced the highest induction in the liver of BaP-exposed trout, followed by the head kidney and spleen. Microsomes from control fish displayed tissue-specific differences in metabolite production. In contrast, in BaP-exposed trout, microsomes of all organs produced the potentially immunotoxic BaP-7,8-dihydrodiol as the main metabolite. The findings from this study show that PAHs, like BaP, are distributed into immune organs of fish and provide the first evidence that immune organs possess inducible PAH metabolism leading to in situ production of potentially immunotoxic PAH metabolites.

Transfer of an adherent Vero cell culture method between two different rocking motion type bioreactors with respect to cell growth and metabolic rates

Rapid and simple cell and virus cultivation can currently be carried out using disposable bioreactors. The CELL-tainer ® (CELLution Biotech BV) disposable bioreactor is a rocking-type bioreactor which not only has vertical movement but horizontal displacement as well. Due to this two-directional movement relatively high mass-transfer capacities can be reached when compared with conventional rocking motion-type bioreactors. Using the design of experiments (DoE) approach we have developed models for the mixing times in both the CELL-tainer ® and the BIOSTAT ® CultiBag RM (Sartorius Stedim Biotech) bioreactor (standard rocking motion-type). The conditions for cultivation of Vero cells in the CELL-tainer ® bioreactor were chosen based on comparable mixing times. Vero cells growing adherent to Cytodex 1 microcarriers were cultivated in the CELL-tainer ® and in the BIOSTAT ® CultiBag RM. Both bioreactors were controlled with regard to temperature, pH and % dissolved oxygen. Vero cell growth in both bioreactors was comparable with respect to the growth characteristics and main metabolite production and consumption rates. Additionally, polio virus production in both bioreactors was shown to be similar.