Urs von Stockar

The importance of ammonia in mammalian cell culture

Ammonia has been reported to be toxic and inhibitory for mammalian cell cultures for many years. Reduction of growth rates and maximal cell densities in batch cultures, changes in metabolic rates, perturbation of protein processing and virus replication have been reported. However, cellular mechanisms of ammonia toxicity are still the subject of controversy and are presented here. The physical and chemical characteristics of ammonia and ammonium are important, with the former capable of readily diffusing across cellular membranes and the latter competing with other cations for active transport by means of carrier proteins. The main source of the ammonia which accumulates in cell cultures is glutamine, which plays an important role in the metabolism of rapidly growing cells. Strategies to overcome toxic ammonia accumulation include substitution of glutamine by glutamate or other amino acids, nutrient control, i.e., controlled addition of glutamine at low concentrations, or removal of ammonia or ammonium from the culture medium by means of ion-exchange resins, ion-exchange membranes, gas-permeable membranes or electrodialysis.

In situ product removal (ISPR) in whole cell biotechnology during the last twenty years

This review sums up the activity in the field of in situ product removal in whole cell bioprocesses over the last 20 years. It gives a complete summary of ISPR operations with microbial cells and cites a series of interesting ISPR applications in plant and animal cell technology. All the ISPR projects with microbial cells are categorized according to their products, their ISPR techniques, and their applied configurations of the ISPR set-up. Research on ISPR application has primarily increased in the field of microbial production of aromas and organic acids such lactic acid over the last ten years. Apart from the field of de novo formation of bioproducts, ISPR is increasingly applied to microbial bioconversion processes. However, despite of the large number of microbial whole cell ISPR projects (approximately 250), very few processes have been transferred to an industrial scale. The proposed processes have mostly been too complex and consequently not cost effective. Therefore, this review emphasizes that the planning of a successful whole cell ISPR process should not only consider the choice of ISPR technique according to the physicochemical properties of the product, but also the potential configuration of the whole process set-up. Furthermore, additional process aspects, biological and legal constraint need to be considered from the very beginning for the design of an ISPR project. Finally, future trends of new, modified or improved ISPR techniques are given.

Thermodynamic considerations in constructing energy balances for cellular growth

A review with 48 refs. The topics discussed include mass and energy balances for open and non-steady state systems, calcn. of reaction enthalpies, and enthalpy balance in aerobic and anaerobic growth. [on SciFinder (R)]

Physiologic studies with the sulfate-reducing bacterium Desulfovibrio desulfuricans: evaluation for use in a biofuel cell.

The growth kinetics of the sulfate-reducing bacteria Desulfovibrio desulfuricans Essex 6 was investigated under various conditions for potential use in a microbial fuel cell that recovers electrons generated from the reduction of sulfate to hydrogen sulfide. Hydrogen sulfide was found to inhibit growth and decrease both the growth yields and the sulfate-specific reduction rate. Hydrogen sulfide inhibition was direct, reversible, and not due to limitation by iron deficiency. A high initial lactate concentration also retarded bacterial growth, reduced the specific sulfate reduction rates, and gave variable biomass growth yields. This effect resulted from a bottleneck in the lactate oxidation pathway which induced the production of the secondary product butanol. The use of pyruvate as a carbon source was more advantageous than lactate in terms of growth rate and biomass growth yields, with only a slight decrease in the rate of specific sulfate reduction. For equal biomass, a slightly higher current density was generated from lactate than pyruvate, but pyruvate required nearly 40% less sulfate.

The relationship between elemental composition and heat of combustion of microbial biomass

SummaryFour models taken from the literature, which permit calculation of heats of combustion from elemental analysis, are evaluated from a theoretical point of view. In order to obtain experimental values of heats of combustion with a higher degree of accuracy than those being available in the literature, an improved sample preparation technique based on lyophilisation of microbial biomass has been developed. Heats of combustion were determined by direct measurement in a calorimeter and compared to calculated values from each of the literature models. Gieses formula turned out to predict heats of combustion the most accurately. The enthalpy content of the bacteria investigated (23.13±0.52 kJ/g) differs from that of yeasts (21.21±0.47 kJ/g) in a significant manner.

Measurement of volumetric (OUR) and determination of specific (qO2) oxygen uptake rates in animal cell cultures

Oxygen is a key substrate in animal cell metabolism. It has been reported that the oxygen uptake rate (OUR) is a good indicator of cellular activity, and even under some conditions, a good indicator of the number of viable cells. The measurement of OUR is difficult due to many different reasons. In particular, the very low specific consumption rate (0.2 x 10(-12) mol cell h-1), the sensitivity of the cells to variations in dissolved oxygen concentration and the difficulty to provide oxygen without damaging the cells are problems which must be taken into account for the development of OUR measurement methods. Different solutions based on an oxygen balance on either the liquid phase or around the entire reactor, and with a variable or stable concentration of dissolved oxygen have been reported. The accuracy of the OUR measurements and the required analytical devices are very different from method to method.

Antigen Binding Properties of Purified Immunoglobulin A and Reconstituted Secretory Immunoglobulin A Antibodies

The hybridoma cell line ZAC3 expresses Vibrio cholerae lipopolysaccharide (LPS)-specific mouse IgA molecules as a heterogeneous population of monomeric (IgAm), dimeric (IgAd), and polymeric (IgAp) forms. We describe a gentle method combining ultrafiltration, ion-exchange chromatography, and size exclusion chromatography for the simultaneous and qualitative separation of the three molecular forms. Milligram quantities of purified IgA molecules were recovered allowing for direct comparison of the biological properties of the three forms. LPS binding specificity was tested after purification; IgAd and IgAp were found to bind strongly to LPS whereas IgAm did not. Secretory IgA (sIgA) could be reconstituted in vitro by combining recombinant secretory component (rSC) and purified IgAd or IgAp, but not IgAm. Surface plasmon resonance-based binding experiments using LPS monolayers indicated that purified reconstituted sIgA and IgA molecules recognize LPS with identical affinity (KA 1.0 × 108 M−1). Thus, this very sensitive assay provides the first evidence that the function of SC in sIgA complex is not to modify the affinity for the antigen. KA falls to 6.6 × 105 M−1 when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is critical for recognition by the antibody.

A new method for on-line measurement of the volumetric oxygen uptake rate in membrane aerated animal cell cultures

Oxygen is a key substrate in animal cell metabolism and its consumption is thus a parameter of great interest for bioprocess monitoring and control. A system for measuring it based on an oxygen balance on the liquid phase was developed. The use of a gas-permeable membrane offered the possibility to provide the required quantity of oxygen into the culture, while avoiding problems of foaming or shear stress generally linked to sparging. This aeration system allowed moreover to keep a known and constant k(L)a value through cultures up to 400 h. Oxygen uptake rate (OUR) was measured on-line with a very good accuracy of +/-5%, and the specific OUR for a CHO cell line was determined during batch (growth phase) and continuous culture as, respectively, equal to 2. 85x10(-13) and 2.54x10(-13) mol O(2) cell(-1) h(-1). It was also shown that OUR continuous monitoring gives actually more information about the metabolic state of the culture than the cell concentration itself, especially during transition phases like the end of the growth phase in a batch culture.

Systematic errors in data evaluation due to ethanol stripping and water vaporization.

Systematic errors due to the neglect of water and/or ethanol partition between liquid and gaseous phases are discussed for bioreactors equipped with or without a condenser. Both water vapor and ethanol vapor are present in the off-gas leaving the condenser. Presence of residual water vapor largely influences the gas measurements by dilution. As a consequence, the oxygen consumption rate can be overestimated by a factor of 3 if calculations are not corrected for water vapor content or if no additional device is implemented after the condenser to completely dry the off-gases. The mass balance and partition equations predict that the condenser has only a small effect on reduction of the ethanol vapor content of the off-gas. The reason is the high ethanol concentration of the condensate droplets on the condenser wall in contact with the off-gases. Model predictions as well as experimental results show that ethanol evaporation represents a large fraction of the ethanol production rate and influences greatly the elemental recoveries. For a reactor working at 30 degrees C without condensation of the vapors and for a volumetric aeration rate of 0.63vvm, stripping of ethanol resulted in a gaseous dilution rate of 0.016 h-1 for ethanol. The dilution rate by stripping was reduced to 0.014 h-1 when a condenser at 12 degrees C was implemented. The fraction of ethanol that is stripped is mainly dependent on the ratio D/vvm (liquid to gaseous flow rates), and the effect is only slightly influenced by low condenser temperature. The evaporation of ethanol may account for more than 20% of the ethanol formation rate. Therefore, the condenser does not succeed to reflux all ethanol to the reactor broth. In terms of a unit operation, ethanol vapor can be efficiently reduced by absorption instead of condensation. To demonstrate the feasibility, a simple modification of the reactor was tested for continuous cultures: the feed port was changed from the top-plate to the top of the condenser, which was used as an absorption column. Ethanol stripping was reduced by a factor of 4 as compared to the condensation setup (at 12 degrees C): it accounted for 2% of the ethanol production rate as compared to 8.2% at D = 0.19 h-1 and 0.63vvm.

Influence of nutritional factors on the nature, yield, and composition of exopolysaccharides produced by Gluconacetobacter xylinus I-2281.

ABSTRACT The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G. xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing G. xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrases activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.