Morphological engineering of Streptomyces hygroscopicus var. geldanus: regulation of pellet morphology through manipulation of broth viscosity.

Actinomycetes, especially members of the genus Streptomyces, are responsible for producing the majority of known antibiotics. The production of antibiotics by filamentous organisms is often dependent on the morphology and size distribution of the pellet population within the culture. Particle interaction and subsequent pellet formation are primarily dependent on the rate of collision of particles in culture, which is in turn, a function of fluid turbulence. The microbial polysaccharide xanthan gum was used to artificially regulate the apparent viscosity (mu(a)) of S. hygroscopicus fermentation broths with the aim of controlling particle interaction, aggregation and hence pellet formation. An increase in both pellet count and biomass concentration from approximately 2,000 to 8,000 pellets ml(-1) and 0.9-2.1 g l(-1) dry weight of biomass, as well a decrease in the mean pellet volume from 0.014 to 0.004 mm(3) was observed in cultures supplemented with 3 g l(-1) xanthan gum. The addition of xanthan gum significantly alters fluid rheology by increasing the mu(a). Counter-intuitively, an increase in the mu(a) within the experimental range examined resulted in an increase in the rate of gas-liquid mass transfer. This was attributed to the predominantly diffusive nature of oxygen transfer in shake flask cultures.

Development of a robust microtiter plate-based assay method for assessment of bioactivity.

A microtiter plate-based assay was developed for the quantitative monitoring of bioactive compound production in Streptomyces hygroscopicus fermentation samples. The method reported demonstrates the successful application of the theories of disk diffusion based methods of bioactivity assessment, to a microtiter assay for high throughput analysis. The assay method facilitates the generation of the dose-response curve of test organisms (Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae) to a bioactive compound. Using this dose-response curve, the method facilitates definition of three distinct Minimum Inhibitory Concentration (MIC) values for use in the characterisation of the bioactive attributes of a sample. The assay uses established standard procedures to facilitate adaptation of the assay for use with a wider range of test microorganisms. Errors due to the assumption of a linear relationship between turbidity and biomass concentration are also reduced, due to incorporation of a step to convert turbidity to biomass concentration, for use in the calculation of bioactivity.

Morphological quantification of pellets in Streptomyces hygroscopicus var. geldanus fermentation broths using a flatbed scanner.

An image analysis technique has been developed to allow high throughput morphological characterisation of microbial fermentation broths containing spherical pellets greater than 100 microm in diameter. Images of stained Streptomyces hygroscopicus var. geldanus culture samples at three different inoculum levels were captured using a flatbed scanner, at a resolution of 21 microm per pixel (1200 dots per inch) and subsequently analysed leading to the generation of a morphological profile of each sample. The time taken for image capture and analysis of a prepared sample, containing approx. 2000 particles, was 3 min 6 s.

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 brewer's 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.

The effect of culture conditions on the morphology of the dimorphic yeast Kluyveromyces marxianus var. marxianus NRRLy2415: a study incorporating image analysis.

The effect of changing environmental conditions on the morphology of the yeast Kluyveromyces marxianus var. marxianus NRRLy2415 was investigated in batch and continuous culture, using a previously developed computer-aided image analysis protocol [O'Shea and Walsh (1996) Biotechnol Bioeng 51: 679 690]. The morphology of the organism is primarily controlled by the specific growth rate, mu. This finding was contrary to a previous investigation [Walker and O'Neill (1990) J Chem Tech Biotechnol 49: 75-89]. When the organism is cultured in batch with excess oxygen, mu can approach the maximum specific growth rate, microm, and the primary morphology of the culture is yeast-like. However, if the organism is cultured in a chemostat, thereby controlling the growth rate, the morphology reverts to a pseudohyphal form. This response is thought to be an adaptation by the organism to its environment, whereby it assumes a foraging form under adverse environmental conditions. The use of computer-aided image analysis made possible the discrimination of subtle morphological differences between samples and the determination of the relationship between morphology and growth rate.

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, alpha, was found to be related to pressure, DeltaP, through the equation alpha = alpha0(1 + kcDeltaP). The dependence of alpha0/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.

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%.

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.

Models for forecasting residential populations: the geometric programming approach.

PIP: This paper is concerned with the application of geometric programming to a specific land use problem in New South Wales, Australia. Two entropy models are linked to form a predictive model that is used to generate population projections for a planning region for 1986. The results of the projections are then discussed. (summary in FRE, GER)^ieng