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

A review with 55 refs. Oxygen is a key substrate in animal cell metab. 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 no. of viable cells. The measurement of OUR is difficult due to many different reasons. In particular, the very low specific consumption rate (0.2*10-12 mol cell h-1), the sensitivity of the cells to variations in dissolved oxygen concn. 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 solns. based on an oxygen balance on either the liq. phase or around the entire reactor, and with a variable or stable concn. of dissolved oxygen have been reported. The accuracy of the OUR measurements and the required anal. devices are very different from method to method. [on SciFinder (R)

Astaxanthin production by Phaffia rhodozyma

Astaxanthin is primarily used as a food supplement in the aquaculture industry, which over the past twenty years has experienced tremendous growth. A complex synthetic process produces almost all of the astaxanthin sold, and consequently, the selling price is very high. Thus a bioprodn. process of astaxanthin may be economically attractive, and in addn. would have the added advantage of being an \"all natural\" product. The yeast Phaffia rhodozyma is the only known yeast that simultaneously produces the carotenoid pigment astaxanthin and metabolizes simple carbohydrates. The wild strain produces low levels of the pigment, though through mutation/genetic modification economically attractive levels of astaxanthin are produced. Up until now, few studies have been conducted in continuous culture under precisely defined conditions. In this work, three P. rhodozyma strains (one wild and two mutants) have been characterized under stationary and dynamic growth conditions. Metabolic flux models are being developed to locate bottlenecks in the carotenoid biosynthesis pathway. [on SciFinder (R)

Comparison of different in situ product-removal methods to enhance the production of the aroma compound 2-phenylethanol

There is an increasing interest by the flavor industry for producing natural aroma compds. through biocatalysts. L-Phenylalanine is transformed by Saccharomyces cerevisiae into 2-phenylethanol, which has a rose-like odor. Unfortunately this product inhibits growth even at low concns. and therefore its max. concn. is 3.9 g/l. This problem of the inhibitory effect of the product on the cell can be tackled by different in-situ product removal (ISPR) techniques. The hydrophobicity of 2-phenylethanol differs from those of the precursor and culture medium. Therefore, extn. into a second org. phase seems to be an appropriate ISPR-technique. This method keeps the 2-phenylethanol concn. in the aq. reaction phase below an inhibitory level in order to sustain a high productivity of the system. In this work, three different integrated processes involving the solvent extn. of 2-phenylethanol from the fermn. broth are evaluated. The first case is a two-phase fermn., where a water-immiscible fatty acid, which shows neither a mol. nor a phase toxicity, is added directly into the reactor vessel. The inhibitory 2-phenylethanol preferably partitions in the org. phase and therefore its concn. in the aq. phase is reduced. In this way, the final concn. of 2-phenylethanol is increased up to 9.1 g/l. The second approach uses an ester as second water-immiscible phase that has more a favorable partitioning for 2-phenylethanol but reveals a phase toxicity towards the cells. Thus the yeast is immobilized in chitosan-alginate beads to protect them from the toxic extractant. The third integrated bioprocess, completely novel, uses alginate capsules that contain the extractant in the core. The capsules are added to the fermn. broth and ext. the inhibitory 2-phenylethanol while it is produced. In this way the encapsulated solvent is not in direct contact with the cells and can prevent the cells from the phase toxicity. Conclusion: Solvents that do not show a phase toxicity can be added directly into the reactor vessel, whereas solvents having a phase toxicity can be encapsulated in alginate capsules which prevent the direct contact of the cells with the solvent. [on SciFinder (R)

CHO immobilization in alginate/poly-l-lysine microcapsules: an understanding of potential and limitations

Microencapsulation offers a unique potential for high cell density, high productivity mammalian cell cultures. However, for successful exploitation there is the need for microcapsules of defined size, properties and mechanical stability. Four types of alginate/poly-l-Lysine microcapsules, containing recombinant CHO cells, have been investigated: (a) 800 μm liquid core microcapsules, (b) 500 μm liquid core microcapsules, (c) 880 μm liquid core microcapsules with a double PLL membrane and (d) 740 μm semi-liquid core microcapsules. With encapsulated cells a reduced growth rate was observed, however this was accompanied by a 2–3 fold higher specific production rate of the recombinant protein. Interestingly, the maximal intracapsular cell concentration was only 8.7 × 107 cell mL-1, corresponding to a colonization of 20% of the microcapsule volume. The low level of colonization is unlikely to be due to diffusional limitations since reduction of microcapsule size had no effect. Measurement of cell leaching and mechanical properties showed that liquid core microcapsules are not suitable for continuous long-term cultures (>1 month). By contrast semi-liquid core microcapsules were stable over long periods with a constant level of cell colonization (ϕ = 3%). This indicates that the alginate in the core plays a predominant role in determining the level of microcapsule colonization. This was confirmed by experiments showing reduced growth rates of batch suspension cultures of CHO cells in medium containing dissolved alginate. Removal of this alginate would therefore be expected to increase microcapsule colonization