Ian Marison

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.

Characterization of an encapsulation device for the production of monodisperse alginate beads for cell immobilization

An encapsulation device, designed on the basis of the laminar jet break-up technique, is characterized for cell immobilization with different types of alginate. The principle of operation of the completely sterilizable encapsulator, together with techniques for the continuous production of beads from 250 microm to 1 mm in diameter, with a size distribution below 5%, at a flow rate of 1-15 mL/min, is described. A modification of the device, to incorporate an electrostatic potential between the alginate droplets and an internal electrode, results in enhanced monodispersity with no adverse effects on cell viability. The maximum cell loading capacity of the beads strongly depends on the nozzle diameter as well as the cells used. For the yeast Phaffia rhodozyma, it is possible to generate 700 microm alginate beads with an initial cell concentration of 1 x 10(8) cells/mL of alginate whereas only 1 x 10(6) cells/ml could be entrapped within 400 microm beads. The alginate beads have been characterized with respect to mechanical resistance and size distribution immediately after production and as a function of storage conditions. The beads remain stable in the presence of acetic acid, hydrochloric acid, water, basic water, and sodium ions. The latter stability applies when the ratio of sodium: calcium ions is less than 1/5. Complexing agents such as sodium citrate result in the rapid solubilization of the beads due to calcium removal. The presence of cells does not affect the mechanical resistance of the beads. Finally, the mechanical resistance of alginate beads can be doubled by treatment with 5-10 kDa chitosan, resulting in reduced leaching of cells.

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)]

Extractive bioconversion of 2-phenylethanol from L-phenylalanine by "Saccharomyces cerevisiae"

The bioconversion of l‐phenylalanine (l‐Phe) to 2‐phenylethanol (PEA) by the yeast Saccharomyces cerevisiae is limited by the toxicity of the product. PEA extraction by a separate organic phase in the fermenter is the ideal in situ product recovery (ISPR) technique to enhance productivity. Oleic acid was chosen as organic phase for two‐phase fed‐batch cultures, although it interfered to some extent with yeast viability. There was a synergistic inhibitory impact toward S. cerevisiae in the presence of PEA, and therefore a maximal PEA concentration in the aqueous phase of only 2.1 g/L was achieved, compared to 3.8 g/L for a normal fed‐batch culture. However, the overall PEA concentration in the fermenter was increased to 12.6 g/L, because the PEA concentration in the oleic phase attained a value of 24 g/L. Thus, an average volumetric PEA production rate of 0.26 g L−1 h−1 and a maximal volumetric PEA production rate of 0.47 g L−1 h−1 were achieved in the two‐phase fed‐batch culture. As ethanol inhibition had to be avoided, the production rates were limited by the intrinsic oxidative capacity of S. cerevisiae. In addition, the high viscosity of the two‐phase system lowered the kla, and therefore also the productivity. Thus, if a specific ISPR technique is planned, it consequently has to be remembered that the productivity of this bioconversion process is also quickly limited by the kla of the fermenter at high cell densities.

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.

Separation of binary mixtures by thermostatic sweeping gas membrane distillation: II. Experimental results with aqueous formic acid solutions

Aqueous solutions of formic acid have been experimentally investigated in a modified sweeping gas membrane distillation (SGMD) configuration. A thermostated sweeping gas was used in order to enhance the mass transfer performance. A new tubular module was designed and built for this purpose. The effects of the relevant process parameters on the permeate flux and selectivity have been studied. Experiments with pure water and pure formic acid were used to estimate certain parameters in the model. From these mass transfer coefficients, the fluxes and selectivity for aqueous formic acid mixtures have been calculated using the mathematical model previously described [J. Membr. Sci., 2001, in press]. The model predictions were compared with the experimental data and a good agreement between both flux values were obtained.

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.

Microencapsulation using vibrating technology

For over a half a century now, microencapsulation has played a very important role in many industries and in the recent decades, this versatile technology has been applied to numerous biotechnology and medical processes. However, successful application in these areas requires a methodology which has the capability to produce mono-dispersed, homogenous-shaped capsules, with a narrow size distribution, using a short production time. The manufacture of capsules using vibrating technology has gained significant interest mainly due to its simplistic approach to produce homogenous microcapsules with the desired characteristics for biotechnological and medical processes. However, certain limitations still exist for this methodology, which include the inability to manufacture microcapsules at large quantities and/or using highly viscous polymers. In this review, a detailed description of the theoretical and practical aspects behind the production of different types of alginate-based microcapsules, for application in biotechnological and medical processes, using vibrating technology, is given.

Inhibition aspects of the bioconversion of l-phenylalanine to 2-phenylethanol by Saccharomyces cerevisiae

The inhibitory impacts of the bioconversion of L-phenylalanine (L-Phe) to 2-phenylethanol (PEA), a very important natural aroma compd., on the metab. of Saccharomyces cerevisiae Giv 2009 were investigated in batch and chemostat cultures. The bioconversion was found to be completely growth assocd. and lead to a maximal final PEA concn. of 3.8 g/l of PEA. This was attained in a fed-batch procedure on glucose in order to prevent the formation of ethanol, which generally reduced the final achievable PEA concn. by its synergistic inhibitory effect. Chemostat cultures revealed that the bioconversion uncoupled the catabolism from anabolism of S. cerevisiae esp. under oxidative growth conditions and thereby reduced the crit. diln. rate Dcrit. In addn., higher specific oxygen uptake rates qO2 were found in the presence of the bioconversion at oxidative growth than the maximal respiratory capacity qO2max found in continuous cultures without bioconversion. [on SciFinder (R)]

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.