Determination of yeast cell viability: viable count vs ATP-based bioluminescence assay

During fed-batch cultivation to produce heterologous protein with yeast cells, cell viability should be determined quickly. Indeed, the early arrest of cell proliferation is a phenomenon often observed in auxotrophic yeast strains such as Saccharomyces cerevisiae BY4741 and ascribable to stressful environmental conditions arising in fed-batch systems. Choosing a cell viability assay can be a challenging task because the growth arrest is not easily attributable to one of the many mechanisms of cell death. In this work, two techniques used to assay cell viability have been compared: the viable cell count on agar plate and the bioluminescence assay based on the luciferase reaction to measure the amount of ATP from viable cells. To compare the methods, either the maximum specific velocity of growth or its specific death rate (kd) at 50 and 53 ◦C of BY4741 strain, were determined. In the first case, both the viable count and the bioluminescence assay gave the same results, showing that the amount of ATP in exponentially growing cells correlates with cell viability. On the contrary, during thermal inactivation the kd value obtained via bioluminescence, resulted always smaller than that obtained by viable cell count even though it was always possible to correlate the kd value obtained by bioluminescence with that obtained by viable count, through a factor. Apparently, in the operative condition examined, cell death either did not lead to the loss of membrane integrity, nor allow the endogenous ATPases to destroy any remaining ATP; thus the ATP levels did not fall precipitously. In the light of the results obtained, due to the ease of use, high sensitivity and action in real-time, ATP-based bioluminescence assay is the natural candidate to replace the cell viable count method which contrarily requires many replicates and extende

Gelatin immobilized growing yeast cells: changes in the glycosylation level of external invertase and cell wall composition

Free and gelatin immobilized yeast cells from two different strains were analyzed by means of SDS-PAGE as regards: i) the glycosylation levels of external invertase before and after treatment with endo-beta-N-acetylglycosaminidase (Endo H), ii) the pattern of mannoproteins released from the cell wall after extraction with SDS, beta-mercaptoethanol and Zymoliase digestion. The results obtain suggest that the composition and structure of the cell wall is modified when cell growth takes place in the state of immobilization

A general kinetic and mass transfer model to simulate the baker's yeast growth in bioreactors

In this paper a general cybernetic model has been developed to describe the growth of baker's yeast in every type of reactor (batch, fed-batch, continuous). The model, which takes into account also the mass-transfer oxygen limitation, has been tested on literature continuous runs performed at different aerating gas composition. The results obtained show that the model can describe the growth of S. cerevisiae also under conditions of inefficient aeration, so it should be useful to optimise and modeling industrial bioreactors

Rhodanese-BSA polymers entrapped in insolubilised gelatin gels: properties and behaviour in continuous systems

The enzymatic oxidation of cephalosporin C to glutaryl-7-amminocephalosporanic acid (glutaryl-7-ACA) was carried out utilizing permeabilized whole cells of the yeast Trigonopsis variabilis entrapped in Ca-alginate beads. The biomass, cultured in a rich medium containing D,L methionine and harvested after 72 h of growth, exhibited high levels of D-aminoacid oxidase activity. Prior to use, thewho,lke cells were permeabilized with four freeze-thawing cycles and immobilized in polysaccharide matrices, such as Ca-alginate and K- carrageenan, and in an insolubilised gelatin gel. The best results in terms of activity yield and storage stability were obtained with cells entrapped in Ca-alginate beads. These cells were utilized for glutaryl-7-ACA production in a continuous stirred batch reactor (CSTR) and in a packed bed reactor working in a plug flow reactor (PFR), using 50 mm Cephalosporin C as substrate. The performances of the two systems were compared. The overall on a void volume basis) were 1.63 g and 255 mg of glutaryl-7-ACA h-1 in the PFR and in CSTR, respectively

Effect of auxotrophies on yeast performance in aerated fed-batch reactor

A systematic investigation on the effects of auxotrophies on the performance of yeast in aerated fed-batch reactor was carried out. Six isogenic strains from the CEN.PK family of Saccharomyces cerevisiae,one prototroph and five auxotrophs, were grown in aerated fed-batch reactor using the same operative conditions and a proper nutritional supplementation. The performance of the strains, in terms of final biomass decreased with increasing the number of auxotrophies. Auxotrophy for leucine exerted a profound negative effect on the performance of the strains. Accumulation of reactive oxygen species (ROS) in the cells of the strain carrying four auxotrophies and its significant viability loss, were indicative of an oxidative stress response induced by exposure of cells to the environmental conditions. The mathematical model was fundamental to highlight how the carbon flux, depending on the number and type of auxotrophies, was diverted towards the production of increasingly large quantities of energy for maintenance

Relation between growth dynamics and diffusional limitations in Saccharomyces cerevisiae cells growing as entrapped in insolubilized gel

Flow-cytometric analysis was employed to investigate growth dynamics of a yeast cell population immobilised in an insolubilised gelatin gel by means of the quantitative determination of the average protein content per cell. This analysis was carried out on both the immobilised cell population considered as a whole and the subpopulations colonising the gelatin matrix at different depths. The results show that growth of the gelatin-immobilised yeast population was affected by the existence of a gradient of nutrient concentrations through the matrix and are in agreement with the unsteady-state diffusion model employed for the description of glucose transfer in the gel

D-amino acid oxidase from Trigonopsis variabilis: immobilisation of whole cells in natural polymeric gels for glutaryl-7-aminocephalosporanic acid production

The enzymatic oxidation of cephalosporin C to glutaryl-7-amminocephalosporanic acid (glutaryl-7-ACA) was carried out utilizing permeabilized whole cells of the yeast Trigonopsis variabilis entrapped in Ca-alginate beads. The biomass, cultured in a rich medium containing D,L methionine and harvested after 72 h of growth, exhibited high levels of D-aminoacid oxidase activity. Prior to use, thewho,lke cells were permeabilized with four freeze-thawing cycles and immobilized in polysaccharide matrices, such as Ca-alginate and K- carrageenan, and in an insolubilised gelatin gel. The best results in terms of activity yield and storage stability were obtained with cells entrapped in Ca-alginate beads. These cells were utilized for glutaryl-7-ACA production in a continuous stirred batch reactor (CSTR) and in a packed bed reactor working in a plug flow reactor (PFR), using 50 mm Cephalosporin C as substrate. The performances of the two systems were compared. The overall on a void volume basis) were 1.63 g and 255 mg of glutaryl-7-ACA h-1 in the PFR and in CSTR, respectively

Effect of auxotrophies on yeast growth in aerated fed-batch reactor

Mutant and deletion strains of the yeast Saccharomyces cerevisiae having one/several auxotrophies are largely used in the development of recombinant strains for heterologous protein production because they ensure maintenance of plasmids with selectable markers. The production is usually carried out by culturing the recombinant strain in aerated fed-batch, where sugar limitation achieves high yields of biomass and product. In a previous work, it was evidenced that growth of the auxotrophic S. cerevisiae BY4741 (MATa, ura30, leu20, met150, his31) engineered for human IL-1 production, and employed in aerated fed-batch, early arrested even in the presence of a correct nutritional complementation (being specific nutrients for genetically uncomplemented auxotrophies provided in no growthlimiting amounts). It was assumed that this behaviour may depend on the high number of auxotrophies, since the prototrophic S288C, from which BY4741 derives, showed a typical performance under the same cultivation mode. Therefore, a systematic investigation on the effect of auxotrophies on yeast growth in aerated fed-batch was carried out. Four isogenic strains of the CEN.PK family, with a progressively increasing number of auxotrophies (from one to four) were assayed under fed-batch conditions and a proper nutritional complementation. Feeding to the reactor was exponentially increased imposing a specific growth rate below the critical one. The behaviour of the auxotrophic strains was compared with that of the isogenic prototrophic strain. by evaluating the capacity to keep the specific growth rate chosen. A clear correlation among optimum growth and number of auxotrophies has been found. Furthermore we have investigated the possible effect of the type of auxotrophy (ura- or leu-) on the strain performance, monitoring as well cell viability of each strain. The study is a contribution to know the phenotypic effects of auxotrophies in yeast and can have implications for biotechnological applications

Immobilization of Saccharomyces cerevisiae cells by adhesion to polymeric matrices obtained by radiation-induced polymerization

An immobilization method based on the spontaneous adhesion of invertase-active cells of yeast (Saccharomyces cerevisiae) to tuff granules was described by Parascandola, Scardi, and Tartaglione. Compared with gel entrapment, immobilization by adhesion is much more simple and free from diffusional limitations. However, adhesion is a rather complicated process involving surface interactions between microbial cells and the so-called substratum, that is, the solid support to which they attach. Because there are still many unanswered questions about the mechanism of adhesion, the selection of suitable substrata for a given microbial species can be made only empirically. Thus, to find substrata better than tuff or insolubilized gelatin, polymeric hydrogels that were obtained by radiation-induced polymerization below 0 °C and that were employed successfully for immobilizing enzymes, cells and antibodies were considered. A dozen of such polymer matrices with different hydrophilicities were synthesized and assayed as possible substrata for S. cereviisae cells used in continuous ethanol production

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

<p>Abstract</p> <p>Background</p> <p><it>Saccharomyces cerevisiae </it>BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1<it>β </it>(IL1<it>β</it>), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1<it>β</it>] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the <it>S. cerevisiae </it>mutant BY4741 Δ<it>yca1 </it>[PIR4-IL1<it>β</it>], carrying the deletion of the <it>YCA1 </it>gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Δ<it>yca1 </it>mutant strain.</p> <p>Results</p> <p>Our results show that the concentrations of ACA in the feeding solution, corresponding to those routinely used in the literature, are limiting for the growth of <it>S. cerevisiae </it>BY4741 [PIR4-IL1<it>β</it>] in fed-batch reactor. Even in the presence of a proper ACA supplementation, <it>S. cerevisiae </it>BY4741 [PIR4-IL1<it>β</it>] did not achieve a high cell density. The Δ<it>yca1 </it>deletion did not have a beneficial effect on the overall performance of the strain, but it had a clear effect on its viability, which was not impaired during fed-batch operations, as shown by the <it>k</it>_{<it>d </it>}value (0.0045 h^-1), negligible if compared to that of the parental strain (0.028 h^-1). However, independently of their robustness, both the parental and the Δ<it>yca1 </it>mutant ceased to grow early during fed-batch runs, both strains using most of the available carbon source for maintenance, rather than for further proliferation. The mathematical model used evidenced that the energy demand for maintenance was even higher in the case of the Δ<it>yca1 </it>mutant, accounting for the growth arrest observed despite the fact that cell viability remained comparatively high.</p> <p>Conclusions</p> <p>The paper points out the relevance of a proper ACA formulation for the outcome of a fed-batch reactor growth carried out with <it>S. cerevisiae </it>BY4741 [PIR4-IL1<it>β</it>] strain and shows the sensitivity of this commonly used auxotrophic strain to aerated fed-batch operations. A Δ<it>yca1 </it>disruption was able to reduce the loss of viability, but not to improve the overall performance of the process. A mathematical model has been developed that is able to describe the behaviour of both the parental and mutant producer strain during fed-batch runs, and evidence the role played by the energy demand for maintenance in the outcome of the process.</p