Padraig K. Walsh

Effect of cell morphology on dead-end filtration of the dimorphic yeast Kluyveromyces marxianus Var. marxianus NRRLy2415

The dead‐end filtration characteristics of the dimorphic yeast Kluyveromyces marxianus var. marxianus (formerly fragilis) NRRLy2415 were investigated for a range of mean cell morphologies, ranging from predominantly yeast‐like to predominantly filamentous. Semiautomated image analysis was used to measure the mean cell specific surface area, Sv, and the mean ratio of cell length to equivalent cylindrical diameter, Ldm, in each broth. The method of Ju and Ho (Biotechnol. Bioeng. 1988, 32, 95–99) was used to show that for broths with Ldm values between 1.72 and 10.03, the voidage of cell pellets formed by centrifugation increased with increasing Ldm. In the pressure range 30–180 kPa, the specific filter cake resistance, α, was found to be related to pressure, ΔP, through the equation α = α0(1 + kcΔP) . The dependence of α0/Sv2 on Ldm was found to be qualitatively consistent with the pellet voidage data and the Carman−Kozeny equation. Considerably better agreement with the experimental data was obtained when the Kozeny constant, K, was treated as variable and related to Ldm through the equation K = 4.83 + 7.08 log10 Ldm. The cake compressibility constant, kc, was found to increase with increasing Ldm, a phenomenon consistent with the wide range of voidages that can be displayed by beds of long cylinders.

Experimental techniques for quantifying the cake mass, the cake and membrane resistances and the specific cake resistance during crossflow filtration of microbial suspensions

Abstract Simple techniques are reported for (i) measuring the cake mass formed during crossflow filtration of microbial cells, (ii) recovering and measuring the cake mass after crossflow filtration, and (iii) measuring the specific cake resistance during crossflow filtration. The first technique is based on measuring the decline in cell concentration in the feed reservoir during total recycle operation. Cell concentrations are measured on a dry basis and converted to wet weight concentrations using the previously measured wet-to-dry weight ratio of the cells. The second technique involves a series of flushing steps at zero transmembrane pressure followed by wet weight analysis of the suspension formed by the recovered cake. In the third technique, two types of specific cake resistance are defined; an apparent value, α app , and the true value, α true . These are related by the expression α app = α true +( R m − R m0 )/ m where R m − R m0 is the flow resistance due to membrane fouling (as distinct from cake formation), and m is the cake mass per unit area. A methodology for decoupling the effects of cake formation and membrane fouling during crossflow filtration is presented, thus, providing a way of monitoring the time evolution of α true . The techniques described were used to investigate the crossflow filtration of the polymorphic microorganism Kluyveromyces marxianus var. marxianus NRRLy2415 in tubular ceramic membranes. This microorganism displays a range of morphologies ranging from simple ovoid yeast to branched filamentous forms. The approach of measuring the cake and membrane resistances, as well as the specific cake resistance, provides a wealth of information, which greatly adds to our understanding of the mechanism of flux decline in complex biological suspensions.

Growth patterns of Saccharomyces cerevisiae microcolonies in alginate and carrageenan gel particles: Effect of physical and chemical properties of gels

Abstract When Saccharomyces cerevisiae cells were inoculated at low density (1 · 103–1.5 · 105 cells [g gel]−1) in alginate gel beads and cylinders, cells grew in the form of distinct microcolonies throughout the gel matrix. Alginate gel beads and cylinders, formed by external gelation with Ca2+, gave rise to microcolonies which became elongated and lens-shaped, with their major axes aligned with the gel surface. The aspect ratio (major axis/minor axis length) of the microcolonies and the local concentration of alginate increased with increasing distance from the center of the gel particles. In contrast, spherical microcolonies were observed in alginate cylinders formed by internal gelation and no significant local concentration gradients of alginate were detected in these gels. Nonspherical microcolonies were also observed in carrageenan gel beads. However, the colonies were irregularly shaped, and their major axes demonstrated no preferential alignment.

Cell growth patterns in immobilization matrices

Within an immobilized cell matrix, mass transfer limitations on substrate delivery or product removal can often lead to a wide range of local chemical environments. As immobilized living cell populations actively grow and adapt to their surroundings, these mass transfer effects often lead to strong, time-dependent spatial variations in substrate concentration and biomass densities and growth rates. This review focuses on the methods that have been devised, both experimentally and theoretically, to study the non-uniform growth patterns that arise in the mass transfer limited environment of an immobilization matrix, with particular attention being paid to cell growth in polysaccharide gels.

Morphological characterization of the dimorphic yeast Kluyveromyces marxianus var. marxianus NRRLy2415 by semi-automated image analysis

A semiautomatic image analysis method has been developed to characterize the morphology of the dimorphic yeast Kluyveromyces marxianus var. marxianus (formerly fragilis) NRRLy2415 undergoing alcoholic fermentation of cheese whey permeate. The method is capable of separating cells into six defined categories, varying from simple ovoid yeast cells to branched mycelial cells. A sample size of 300 cells was found to be sufficient to obtain a statistically significant categorization. The processing time for a sample was found to be approximately 90 min. In addition to qualitative characterization, the method permits the measurement of geometric properties such as the width, length, and volume of individual cells or clusters of cells. When the cells analyzed by the automatic method were categorized on a manual basis, the error level in the automatic routine was found to be less than 3%.

Characterising the packing and dead-end filter cake compressibility of the polymorphic yeast Kluyveromyces marxianus var. marxianus NRRLy2415

Suspensions of the polymorphic yeast Kluyveromyces marxianus var. marxianus NRRLy2415 were grown in batch and continuous culture, producing broths with a range of mean cell morphologies. The mean cell aspect ratio, Ldm, defined as the mean ratio of total cell length to equivalent cylindrical diameter, was measured for each broth by image analysis and ranged from 1.7 to 51. The porosity of the cell pellet formed in a bench-top centrifuge was measured for each broth and in all cases, the pellet porosity decreased linearly with increasing centrifuge speed. This decrease became more pronounced as Ldm increased. The extrapolated zero-speed pellet porosity, e0, was correlated with Ldm by the expression e0=1−1/(1.24+0.062Ldm). The dead-end specific cake resistance, α, was measured for each broth in the pressure range 30–180 kPa. The cake compressibility constant, kc, defined by the expression α=α0(1+kcΔP) was found to increase with increasing values of Ldm. The increase was most rapid in the region, Ldm<10. Microscopic observations indicate that cell deformation at branching points may contribute to cake compressibility.

CHARACTERISATION OF CAKE COMPRESSIBILITY IN DEAD-END MICROFILTRATION OF MICROBIAL SUSPENSIONS

Abstract Culture broths of Saccharomyces cerevisiae and Kluyveromyces marxianus var marxianus NRRLy2415, suspensions of rehydrated active dry bakers yeast and suspensions or calcium carbonate were filtered in dead-end mode at pressures below 200 kPa. In the case of all the microbial suspensions, the specific cake resistance was found to be a linear function of pressure. Cake compression was found to be reversible or weakly irreversible with respect to changes in pressure, i.e., the specific resistance measured at a given pressure was only weakly dependent on whether the filter cake had previously been exposed to higher pressures. The greatest irreversibility effects were obtained with unwashed active dry yeast suspensions, consistent with the breakdown of cell aggregates in these suspensions. The specific cake resistance of calcium carbonate suspensions was found to be a non-linear function of pressure. The compression of calcium carbonate cakes was irreversible, consistent with the breakdown of the large...

Determination of yeast glycogen content by individual cell spectroscopy using image analysis

A rapid technique has been developed to determine the glycogen content of yeast on an individual cell basis using a combination of image analysis technology and staining of yeast cells with an I(2):KI solution. Changes in mean cellular glycogen content during alcoholic fermentation have been reported using this technique. The glycogen content of stored brewers yeast is heterogeneous compared to freshly propagated yeast which have a more uniform distribution of glycogen. Analysis of the distribution of yeast glycogen during fermentation indicates that a fraction of yeast cells do not dissimilate glycogen. Therefore, conventional analysis of the mean glycogen content of yeast used to inoculate fermentations is of limited use, unless information regarding the proportion of cells which utilize glycogen is known. Analysis of the distribution of glycogen within a yeast population can serve as a useful indicator of yeast quality.

On the Relation between Filtrate Flux and Particle Concentration in Batch Cross flow Micro filtration

Abstract The relation between the filtrate flux and particle concentration in batch cross flow micro filtration is investigated using a model based on classical filtration theory and the Kern–-Seaton theory of surface fouling. The model, which includes the effects of cake compressibility but not of membrane fouling, is solved for both laminar and turbulent tangential flows. It is found that the sole effect of cake compressibility is to reduce the flux without altering the general shape of the flux versus concentration curve. Fluxes which increase with increasing concentration are shown to be a result of enhanced cake removal due to the increased wall shear stress brought about by increased suspension viscosity. A sigmoidal relation between flux and concentration is reproduced by the model only if there is a reduction in the cake removal rate as the tangential flow regime changes from turbulent to laminar.

Determination of the radial distribution of Saccharomyces cerevisiae immobilised in calcium alginate gel beads

The dissolution of alginate gel beads in 20 g sodium citrate /l produces a linear decrease in bead diameter. The rate of dissolution is dependent on the concentration of CaCl2 within the gel beads. This method allows the controlled release of Saccharomyces cerevisiae from alginate gel beads and permits the simple and rapid determination of the radial distribution of cell concentration.