Analysis of noise and bias in fermentation oxygen uptake rate data

The calculation of many derived fermentation variables such as the respiratory quotient (RQ) and mass transfer coefficient (K(L)a) requires the differences between the molar percentages of oxygen and carbon dioxide in the fermentor inlet and exit gas, called the %OUR and %CER. Noise and bias in %CER data is of order that in the exit gas carbon dioxide analysis. However, the relative amount of noise in the %OUR is one to two orders of magnitude greater than the noise in the raw oxygen analyses because the %OUR is calculated as a small difference between two large quantities. The noise in the %OUR is white with a Gaussian amplitude probability distribution of absolute standard deviation 0.0145. A chi-square filter of the %OUR data is shown to considerably improve the quality of the calculated RQ and K(L)a for a fermentation of Streptomyces clavuligerus. (c) 1992 John Wiley & Sons, Inc.     

Physiology and biochemistry of the pseudobranch: an unanswered question?

The structure and function of the pseudobranch has long interested scientists, but its overall role has remained a mystery. Previous studies have attributed respiratory, endocrine, osmoregulatory and sensory roles to the pseudobranch, and the present review concentrates on new findings. Perfusion experiments on the pseudobranch of the rainbow trout (Oncorhynchus mykiss) using both erythrocyte suspensions and Ringer solution have shown that this organ is able to generate values for the respiratory quotient (RQ) greater than 1.0. The release of carbon dioxide into the perfusate was found to be largely independent of flow between perfusion rates of 120-190 microl/min and could be inhibited by acetazolamide (10(-5) M), indicating a role for carbonic anhydrase. Noradrenaline (10(-5) M) had no effect on oxygen consumption or carbon dioxide release of the pseudobranch. The rate of carbon dioxide release was also dependent on the pH of the pre-pseudobranch perfusate, carbon dioxide release being reduced at lower perfusate pH values. Based on the glucose balance of the isolated saline-perfused rainbow trout pseudobranch and on the enzyme profiles for the rainbow trout, cod, swordfish and deep-water grenadier pseudobranch, it is suggested that the pentose phosphate shunt might be a source of carbon dioxide, yielding the high RQ values found for this organ. Most evidence now available indicates that the pseudobranch is integrally linked with the choroid rete and the supply of oxygen to the retina of the fish eye.     

Determination of the respiration quotient in mammalian cell culture in bicarbonate buffered media

The determination of the respiration quotient (RQ = CER/OUR) has not been used so far as a tool for understanding animal cell metabolism. This is due to problems in measuring the carbon dioxide evolution rate (CER) rather than the oxygen uptake rate (OUR). The determination of the CER is complicated by the use of bicarbonate in the medium. Using liquid and gas balances we have derived an equation for continuous culture to quantify the amount of CO(2) that comes from the bicarbonate in the feed. Under cell-free conditions, values predicted by this equation agree within 4% with the experimental results. In continuous culture using hybridoma cells, the CO(2) from the feed, as determined by an IR-gas analyzer, was found to represent a significant amount of the total measured CO(2) in the off-gas (50% in a suboptimal, and 30% in high-growth medium). Furthermore, the problem of CO(2) loss from the medium during medium preparation and storage was solved using both a theoretical and an experimental approach. RQ values in continuous culture were evaluated for two different growth media. Small but significant differences in RQ were measured, which were matched by differences in specific antibody rates and other metabolic quotients. In a medium with Primatone RL, an enzymatic hydrolysate of animal cell tissue that causes a more than twofold increase in cell density, the RQ was found to be 1.05, whereas in medium without Primatone RL (but containing amino acids equivalent in composition and concentration to Primatone RL) the RQ was found to be 0.97. We suggest the RQ to be a useful parameter for estimating the physiological state of cells. Its determination could be a suitable tool for both the on-line control of animal cell cultivations and the understanding of cell metabolism. (c) 1995 John Wiley & Sons, Inc.     

Determination of carbon dioxide evolution rate using on-line gas analysis during dynamic biodegradation experiments

Respirometry is a precious tool for determining the activity of microbial populations. The measurement of oxygen uptake rate is commonly used but cannot be applied in anoxic or anaerobic conditions or for insoluble substrate. Carbon dioxide production can be measured accurately by gas balance techniques, especially with an on-line infrared analyzer. Unfortunately, in dynamic systems, and hence in the case of short-term batch experiments, chemical and physical transfer limitations for carbon dioxide can be sufficient to make the observed carbon dioxide evolution rate (OCER) deduced from direct gas analysis very different from the biological carbon dioxide evolution rate (CER).To take these transfer phenomena into account and calculate the real CER, a mathematical model based on mass balance equations is proposed. In this work, the chemical equilibrium involving carbon dioxide and the measured pH evolution of the liquid medium are considered. The mass transfer from the liquid to the gas phase is described, and the response time of the analysis system is evaluated.Global mass transfer coefficients (K(L)a) for carbon dioxide and oxygen are determined and compared to one another, improving the choice of hydrodynamic hypotheses. The equations presented are found to give good predictions of the disturbance of gaseous responses during pH changes.Finally, the mathematical model developed associated with a laboratory-scale reactor, is used successfully to determine the CER in nonstationary conditions, during batch experiments performed with microorganisms coming from an activated sludge system. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 243-252, 1997.     

Determination of dissolved CO(2) concentration and CO(2) production rate of mammalian cell suspension culture based on off-gas measurement

The determination of dissolved CO(2) and HCO(3)(-) concentrations as well as the carbon dioxide production rate in mammalian cell suspension culture is attracting more and more attention since the effects on major cell properties, such as cell growth rate, product quality/production rate, intracellular pH and apoptosis, have been revealed. But the determination of these parameters by gas analysis is complicated by the solution/dissolution of carbon dioxide in the culture medium. This means that the carbon dioxide transfer rate (CTR; which can easily be calculated from off-gas measurement) is not necessarily equal to carbon dioxide production rate (CPR). In this paper, a mathematical method to utilize off-gas measurement and culture pH for cell suspension culture is presented. The method takes pH changes, buffer and medium characteristics that effect CO(2) mass transfer into account. These calculations, based on a profound set of equations, allow the determination of the respiratory activity of the cells, as well as the determination of dissolved CO(2), HCO(3)(-) and total dissolved carbonate. The method is illustrated by application to experimental data. The calculated dissolved CO(2) concentrations are compared with measurements from an electrochemical CO(2) probe.     

Computer-aided on-line monitoring of physiological variables in suspended cell cultures of Perilla frutescens in a bioreactor

A computer-aided on-line real-time monitoring system for plant cell bioprocesses was established and applied to the cultivation of Perilla frutescens plant cells in a bioreactor. This system calculated several informative process variables which were useful for the identification of the physiological states of the plant cells during cultivation. Some variables, such as the respiratory quotient (RQ), pH, and specific carbon dioxide evolution rate (SCER), could be used for the identification of the growing phase of cell cultures. The results also suggest that the oxygen uptake rate (OUR) and the specific OUR (SOUR) may depend on the accumulation of anthocyanin (a secondary metabolite) in P. frutescens cell cultures.     

A sensitive monitoring system for mammalian cell cultivation processes: a PAT approach

Biopharmaceuticals such as antibodies are produced in cultivated mammalian cells, which must be monitored to comply with good manufacturing practice. We, therefore, developed a fully automated system comprising a specific exhaust gas analyzer, inline analytics and a corresponding algorithm to precisely determine the oxygen uptake rate, carbon dioxide evolution rate, carbon dioxide transfer rate, transfer quotient and respiratory quotient without interrupting the ongoing cultivation, in order to assess its reproducibility. The system was verified using chemical simulation experiments and was able to measure the respiratory activity of hybridoma cells and DG44 cells (derived from Chinese hamster ovary cells) with satisfactory results at a minimum viable cell density of ~2.0 × 10⁵ cells ml^?1. The system was suitable for both batch and fed-batch cultivations in bubble-aerated and membrane-aerated reactors, with and without the control of pH and dissolved oxygen.     

A simple and modified manometric method for measuring oxygen uptake rate of plant cells in flask cultures

Summary A simple and convenient technique was developed based on the principle of Warburg manometric method to measure O₂ uptake rate (OUR) and CO₂ evolution rate (CER) of suspended cells in a shake flask culture. It was successfully applied to suspension cultures of rice (Oryza sativa) and Panax notoginseng cells, and some important bioprocess parameters, such as OUR, CER, respiratory quotient (RQ), specific OUR (SOUR) and specific CER (SCER), were quantitatively obtained. The measuring system is easy to operate, able to treat many samples simultaneously and is economical.     

Design and evaluation of control strategies for high cell density fermentations

A methodology for the design and evalution of bioprocess control strategies is presented. The strategies manage nutrient supply with demand and vary with the metabolic condition and phase of fermentation operation. Six carbon source addition strategies are based on different combinations of available measurements; they are described and evaluated under different operating conditions for yeast cultivation. It is concluded that a single control strategy is not the most appropriate under all possible operating conditions. An oxygen uptake rate-based control strategy performs better with a mean respiratory quotient (RQ) value less than 1.1 during an oxygen limitation than an ethanol control strategy which had a mean RQ of 14. The designed strategies and an approach of applying the strategy that best matches fermentation conditions consistently enables achievement of high cell densities 78.7 g DCW/L and yields 0.50 g DCW/g glucose as the mean values for three fermentations.     

Application of process mass spectroscopy to the detection of metabolic changes in plant tissue culture

There is a clear need in the area of plant cell culture for methods of on-line estimation of culture parameters. The introduction of plant cells into culture can result in a loss of their photoautotrophic character so that they are largely heterotrophic. As a result, fermentation off-gas analysis may not be confounded by photosynthetically-related O₂ production. In this study performance of a suspension culture of Syringa vulgaris, in a pneumatically agitated bioreactor of in-house design, was investigated. The effect of light on growth, carbohydrate metabolism and the respiratory quotient (RQ), determined by process mass spectroscopy, was studied. Yield coefficients for cells grown in the light and dark were similar although the patterns of carbohydrate uptake were quite different. Maximum biomass yields were higher in this bioreactor than normally observed in shake flasks. The RQ was dynamic during the course of the fermentation, peaking during the transition from the lag phase to the growth phase. It is suggested that the RQ may prove useful as an on-line parameter for monitoring transitions in cellular metabolism during plant cell culture fermentations.Abbreviations RQ respiratory quotient - v.v.m. volume of gas fed to fermenter per unit volume per minute - Y_X/S growth yield coefficient based on total carbohydrate     

Some comments on respiratory quotient (RQ) determination from the analysis of exit gas from a fermentor

Recently, the respiratory quotient (RQ) of microbes measured in situ in a fermentor by exit-gas analysis has been used successfully, for instance, in a fed-batch culture of baker's yeast as a criterion to control the feeding rate.(1-3) It is significant here to keep RQ values close to unity throughout; any deviations of RQ from unity give rise to deterioration of the cell growth yield.However easy it might be to keep RQ values around unity by controlling the feeding rate, the question of whether or not RQ values determined by gas analysis at the fermentor exit could generally represent those in vivo deserves attention. Indeed, for a fermentation carried out at an alkaline side, gas analysis would give RQ values that differ remarkably from true values because of the medium's "storage" of CO(2) released from microbes. The purpose of this communication is to make clear those factors that would affect true RQ values in the analysis of exit gas from a fermentor.     

Optimization of pro-urokinase secretion from recombinant Saccharomyces cerevisiae

Secretion of a nonglycosylated form of human pro-urokinase, also known as single-chain urinary plasminogen activator (scu-PA), from Saccharomyces cerevisiae is described. A "supersecreting" yeast strain harboring multiple copies of integrated plasmids was grown batchwise and at constant respiratory quotient (RQ) in 20-L fermenters. Because the promoters used to drive expression of the pro-urokinase genes are not tightly regulated, secretion into the culture supernatant was growth associated. Although the final cell density achieved in the perturbed-batch fermentation (45 g dry wt/L) was less than that observed in the RQ-controlled culture (77 g dry wt/L), the scu-PA titer in the perturbed-batch fermentation (1863 IU/mL) was nearly twice that attained at constant RQ (1108 IU/mL). The effects on cell growth and scu-PA titer of other process variables (pH, temperature, phosphate concentration, and medium composition) are also discussed.     

Studies on on-line bioreactor identification. IV. Utilization of pH measurements for product estimation

Relationships between the total rate of biomass growth and the rate of ammonia addition to a fermentor for pH control are presented. These equations make use of the concept of reaction invariants and provide the additional information needed for bioreactor identification. They are especially useful when the RQ measurement is not sufficient for this purpose, such as when sensitivities arise with the measured values of the respiratory quotient or when fermentation products are formed. The cases of batch, fed-batch and continuous fermentations, forming products with or without acidic/basic properties are considered. The derived relationships were successfully tested with nonbiological acid-base continuous flow reaction systems and subsequently applied to the identification of the continuous yeast fermentation of glucose to ethanol. Results of these experimental studies are also presented.     

Use of carbon isotope composition for characterization of microbial activity in arable soils

The effect of glucose on microbial mineralization of soil organic matter (SOM) was studied in arable soil specimens. The flows of carbon dioxide generated during this degradation were deduced from differences in the carbon isotope ratios of glucose (delta13C = -11.4 per mil) and SOM (delta13C = -27.01 per mil). The priming effect of glucose and respiratory quotient (RQ) were taken as indices of activation of SOM-consuming microbiota. The data on microbial mineralization of organic matter in soil, obtained in this study, show that addition of a readily consumable substance (glucose) to soil favors SOM degradation and increases the release of carbon dioxide from soil to atmosphere.     

Mass spectrometry: A tool for on-line monitoring of animal cell cultures

The magnetic sector mass spectrometer is able to detect oxygen uptake and carbon dioxide production rates from animal cell cultivations performed in 101 biorectors. Such data have not been available with the use of classic exhaust gas analysis applying paramagnetic analyzers and infra-red sensors due to the insensitivity of the apparatus available. In the course of the present work we were able to demonstrate, that the oxygen uptake rate correlates to the number of viable cells. Additionally oxygen uptake rates supplied on-line information about the actual physiology of the cells: When the rates changed during the cultivation process, this immediately indicated the occurrence of limitations of components in the medium. The information could be useful in timing key events, such as performing splits or harvesting the bioreactor.Abbreviations OUR oxygen uptake rate - CDPR carbon dioxide production rate - RQ respiratory quotient This publication is dedicated to the 65 ^th birthday of Prof. Dr. F. Wagner, University of Braunschweig.     

Estimating cell specific oxygen uptake and carbon dioxide production rates for mammalian cells in perfusion culture

We present robust methods for online estimation of cell specific oxygen uptake and carbon dioxide production rates (q(O2) and q(CO2), respectively) during perfusion cultivation of mammalian cells. Perfusion system gas and liquid phase mass balance expressions for oxygen and carbon dioxide were used to estimate q(O2), q(CO2) and the respiratory quotient (RQ) for Chinese hamster ovary (CHO) cells in perfusion culture over 12 steady states with varying dissolved oxygen (DO), pH, and temperature set points. Under standard conditions (DO = 50%, pH = 6.8, T = 36.5°C), q(O2) and q(CO2) ranges were 5.14-5.77 and 5.31-6.36 pmol/cell day, respectively, resulting in RQ values of 0.98-1.14. Changes to DO had a slight reducing effect on respiration rates with q(O2) and q(CO2) values of 4.64 and 5.47, respectively, at DO = 20% and 4.57 and 5.12 at DO = 100%. Respiration rates were lower at low pH with q(O2) and q(CO2) values of 4.07 and 4.15 pmol/cell day at pH = 6.6 and 4.98 and 5.36 pmol/cell day at pH = 7. Temperature also impacted respiration rates with respective q(O2) and q(CO2) values of 3.97 and 4.02 pmol/cell day at 30.5°C and 5.53 and 6.25 pmol/cell day at 37.5°C. Despite these changes in q(O2) and q(CO2) values, the RQ values in this study ranged from 0.98 to 1.23 suggesting that RQ was close to unity. Real-time q(O2) and q(CO2) estimates obtained using the approach presented in this study provide additional quantitative information on cell physiology both during bioprocess development and commercial biotherapeutic manufacturing.     

Use of Carbon Isotope Composition for Characterization of Microbial Activity in Arable Soils

The effect of glucose on microbial mineralization of soil organic matter (SOM) was studied in arable soil specimens. The fluxes of carbon dioxide generated during this degradation were deduced from differences in the carbon isotope abundance ratios of glucose δ¹³C = –11.4 per mil) and SOM δ¹³C = –27.01 per mil). The priming effect of glucose and respiratory quotient (RQ) were taken as indices of activation of SOM-consuming microbiota. The data on microbial mineralization of organic matter in soil obtained in this study show that the addition of a readily consumable substance (glucose) to soil favors SOM degradation and increases the release of carbon dioxide from soil to atmosphere.     

Heat production and lipid metabolism in broiler and layer chickens during embryonic development

We compared heat production (HP) and lipid metabolism in broiler and layer chickens (Gallus gallus) during embryonic development. To investigate HP and respiratory quotient (RQ), oxygen (O2) consumption and carbon dioxide (CO2) production were measured using an open-circuit calorimeter system. HP consistently had a tendency (P = 0.06) to be lower in broilers than in layers during embryonic development, and HP gradually decreased with developmental stage in both strains. RQ values of both strains were approximately 0.7 at every embryonic stage investigated. These results suggest that chicken embryos mainly use lipid for energy, and the RQ was significantly lower in broilers than in layers during embryonic development. Consumption of the yolk sac as a lipid source was faster in broilers than in layers. Plasma D-3-hydroxybutyrate (D3HB) and glycerol concentrations, associated with fatty acid oxidation, were lower in broiler than layer embryos. These results demonstrate that HP and lipid metabolism are different between the strains during embryonic development, and may be one factor for the growth difference between broiler and layer embryos.     

Metabolic transition step from ethanol consumption to sugar/ethanol

The metabolic pathway shift between only ethanol consumption to both sugar/ethanol consumption was measured by on-line analysis of respiratory quotient of a Saccharomyces cerevisiae. The experiments were carried out in a fed-batch culture under aerobic conditions. During the transition phase, respiratory quotient (RQ) profile shows that sugar can be metabolized through the fermentative pathway even to values of RQ lower than 1.     

Glucose metabolism in the yeast Schwanniomyces castellii: role of phosphorylation site I and an alternative respiratory pathway.

Glucose metabolism in a Crabtree-negative yeast, Schwanniomyces castellii, and a cytochrome b-deficient mutant of this strain was investigated in chemostat culture. The wild-type and mutant strains exhibited the same behavior. Oxidative metabolism was observed when the substrate uptake rate (qS) was low. Fermentative metabolites were excreted when the qS value was higher than 0.40 g.g-1.h-1, indicating the occurrence of a respirofermentative metabolism; however, the respiratory quotient (RQ) remained near 1. When fermentation occurred, the cytochrome pathway was repressed but not the salicylhydroxamic acid (SHAM)-sensitive pathway. The presence of an alternative SHAM-sensitive respiratory pathway and the presence of phosphorylation site I in all metabolic conditions explained the RQ value of 1 and accounted for high biomass yields in oxidative metabolism conditions (0.62 g.g-1 for the wild-type strain and 0.31 g.g-1 for the cytochrome b-deficient mutant strain).