Improved assessment of aggregate size in Taxus plant cell suspension cultures using laser diffraction

In suspended culture, most relevant for biotechnological application, plant cells form aggregates. This phenomenon is of importance as it is related to productivity, leads to local heterogeneities, and might be a reason for the considerable shear sensitivity of these cultures. The valid measurement of plant cell aggregates, however, is not trivial, due to a rather large size distribution and measurement artifacts implied by the measuring method. In this study, laser diffraction was used as a novel method for characterization of Taxus chinensis cells, a major source for the antitumor agent paclitaxel. Aggregate size measured in shaking flask cultivations over 10 days revealed an increase during the growth phase of a batch cycle and a decrease during the stationary phase. During growth, the increase in bio dry weight was proportional to aggregate size. Laser diffraction was found superior to microscopy and image analysis, which had a tendency to underestimate aggregate size up to 20%. This novel approach provides a practicable, rapid, robust, and reproducible way to analyze a 100‐fold more samples in considerably less time than image analysis and is therefore of especial value for quality control in industrial plant cell cultivation.     

A mathematical model for the growth of mycelial pellet populations

In liquid culture, filamentous organisms often grow in the form of pellets. Growth result in an increase in radius, whereas shear forces result in release of hyphal fragments which act as centers for further pellet growth and development. A previously published model for pellet growth of filamentous microorganisms has been examined and is found to be unstable for certain parameter values. This instability has been identified as being due to inaccuracies in estimating the numbers of fragments which seed the pellet population. A revised model has been formulated, based on similar premises, but adopting a finite element approach. This considers the population of pellets to be distributed in a range of size classes. Growth results in movement to classes of increasing pellet size, while fragments enter the smallest size class, from which they grow to form further pellets. The revised model is stable and predicts changes in the distribution of pellet sizes within a population growing in liquid batch culture. It considers pellet growth and death, with fragmentation providing new centers of growth within the pellet population, and predicts the effects of shear forces on pellet growth and size distribution. Predictions of pellet size distributions are tested using previously published data on the growth of fungal pellets and further predictions are generated which are suitable for experimental testing using cultures of filamentous fungi or actinomycetes. (c) 1995 John Wiley & Sons, Inc.     

Narrow-angle forward light scattering from individual algal cells: implications for size and shape discrimination in flow cytometry

Single-cell forward light scattering patterns have been examinedfor four algal species (one pennate diatom, two green flagellatesand one filamentous cyanobacterium), mounted statically in afocused laser beam. In all cases, the distribution of lightintensity at narrow angles (within the first scattering lobe)is well described by diffraction theory. Narrow-angle forwardscattering measurements can therefore be used in principle todeduce the size and approximate shape of algal cells. The feasibilityof using this technique in flow cytometry has been tested usingan instrument which orientates elongated cells uniformly inthe flow stream, and uses fibre optics to make azimuthally resolvedforward scatter measurements at sub-degree polar angles. Withthis instrument it is possible to discriminate between specieswith similar volume and fluorescence characteristics using forwardlight scattering as a shape-sensitive parameter.     

Classification and measurement of fungal pellets by automated image analysis

An automated image analysis method for classifying and measuring pellets of filamentous fungi growing in submerged fermentations has been developed. The method discriminates between pelleted mycelial growth and loose aggregates of dispersed hyphae. Pellets are classified into smooth and hairy types. In both cases, the core of the pellet is identified and its shape and size characterized. For hairy pellets the annular region is also characterized. The method was tested on pellets of Aspergillus niger ATCC 11414 grown in a defined medium in shake flasks. This rapid method makes practical extensive studies on the morphology of pellets in submerged fermentations and the influence of fermentation conditions on that morphology.     

Characterization of pellet morphology during submerged growth of Streptomyces tendae by image analysis

Many filamentous bacteria and fungi tend to form pellets, or mixtures of dispersed mycelium and pellets in liquid fermentation broths. In some cases, a specific kind of morphology is required for optimum product yield. When quantitative analysis and characterization of the pellet morphology are needed, an image processing system can be used. It allows a fast and reproducible analysis of the frequency distribution of pellet size, mean pellet size, contents of pellets, or their shape. The use of such a system allows for an on-line analysis. For a demonstration of the method, results of two fermentations of Streptomyces tendae are shown.     

Image analysis of pellet size for a control system in industrial feed production

When producing aquaculture fish feed pellets, the size of the output product is of immense importance. As the production method cannot produce pellets of constant and uniform size using constant machine settings, there is a demand for size control. Fish fed with feed pellets of improper size are prone to not grow as expected, which is undesirable to the aquaculture industry. In this paper an image analysis method is proposed for automatic size-monitoring of pellets. This is called granulometry and the method used here is based on the mathematical morphological opening operation. In the proposed method, no image object segmentation is needed. The results show that it is possible to extract a general size distribution from an image of piled disordered pellets representing both length and diameter of the pellets in combination as an area.     

Measurement of bubble and pellet size distributions: past and current image analysis technology

Measurements of bubble and pellet size distributions are useful for biochemical process optimizations. The accuracy, representation, and simplicity of these measurements improve when the measurement is performed on-line and in situ rather than off-line using a sample. Historical and currently available measurement systems for photographic methods are summarized for bubble and pellet (morphology) measurement applications. Applications to cells, mycelia, and pellets measurements have driven key technological developments that have been applied for bubble measurements. Measurement trade-offs exist to maximize accuracy, extend range, and attain reasonable cycle times. Mathematical characterization of distributions using standard statistical techniques is straightforward, facilitating data presentation and analysis. For the specific application of bubble size distributions, selected bioreactor operating parameters and physicochemical conditions alter distributions. Empirical relationships have been established in some cases where sufficient data have been collected. In addition, parameters and conditions with substantial effects on bubble size distributions were identified and their relative effects quantified. This information was used to guide required accuracy and precision targets for bubble size distribution measurements from newly developed novel on-line and in situ bubble measurement devices.     

Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application.

Measurement of receptor distributions on cell surfaces is one important aspect of understanding the mechanism whereby receptors function. In recent years, scanning fluorescence correlation spectroscopy has emerged as an excellent tool for making quantitative measurements of cluster sizes and densities. However, the measurements are slow and usually require fixed preparations. Moreover, while the precision is good, the accuracy is limited by the relatively small amount of information in each measurement, such that many are required. Here we present a novel extension of the scanning correlation spectroscopy that solves a number of the present problems. The new technique, which we call image correlation spectroscopy, is based on quantitative analysis of confocal scanning laser microscopy images. Since these can be generated in a matter of a second or so, the measurements become more rapid. The image is collected over a large cell area so that more sampling is done, improving the accuracy. The sacrifice is a lower resolution in the sampling, which leads to a lower precision. This compromise of precision in favor of speed and accuracy still provides an enormous advantage for image correlation spectroscopy over scanning correlation spectroscopy. The present work demonstrates the underlying theory, showing how the principles can be applied to measurements on standard fluorescent beads and changes in distribution of receptors for platelet-derived growth factor on human foreskin fibroblasts.     

Effect of mycelial morphology on ergothioneine production during liquid fermentation of Lentinula edodes

The morphological type of a microorganism generally influences its metabolite production. In the present study, we investigated the effects of the mycelial morphology of shiitake (Lentinula edodes) on the production of 2-mercaptohistidine trimethylbetaine (ergothioneine, ESH) during liquid fermentation. Analyses of the distribution of ESH in mycelial cells of different morphological types revealed that the ESH content of pellets obtained from the liquid fermentation media was much greater than the content in the free mycelia and clumps. The concentration of ESH in pellets on day 15 of liquid fermentation reached 0.79 mg/g dry weight (DW), which is approximately three times the concentration found in mycelia clumps (0.28 mg/g DW) and free mycelia (0.31 mg/g DW). Macroscopic image analysis of the development and morphological changes of the pellets during a liquid fermentation period of up to 25 days indicated that pellet growth showed a highly positive correlation with the increase in ESH concentration (r ² = 0.9851). A reduced agitation rate of 50 rpm for the culture medium was suitable for pellet formation and size enlargement. The results obtained in this work would be helpful in guiding the intentional manipulation of the distribution and enrichment of ESH in L. edodes through changes in liquid fermentation conditions.     

A computer-aided measuring system for the characterization of yeast populations combining 2D-image analysis, electronic particle counter, and flow cytometry

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.     

A model for pellet size distributions in submerged mycelial cultures

Fungal liquid cultures differ from those of bacteria in that clumps, called pellets are formed. Diffusional limitations constrain growth to the surface of such clumps. Previous models for pellet growth have neglected the effect of clump size distributions on growth rates. The model derived by Edelstein (1981) can be used to approach this question, and to demonstrate that fragmentation can accelerate the overall biomass growth. Experimental observations reported in this paper are in agreement with one version of this model incorporating loss of part of the pellet population due to mechanical damage or washout.     

A method for determining ploidy distributions in liver tissue by stereological analysis of nuclear size calibrated by flow cytometric DNA analysis

A method is presented for determining ploidy distributions in mouse liver from image analysis with stereological estimations of nuclear size in tissue sections. Nuclear profile distributions obtained from profile measurements were subjected to a mathematical unfolding procedure in order to obtain the nuclear size distributions. Based on the assumption that nuclear size increases monotonically with nuclear DNA content, flow cytometric DNA analysis of suspensions of liver cell nuclei was used to calibrate the method, thus yielding the mean nuclear size of each ploidy class, i.e., diploid, tetraploid, and octaploid nuclei. After the size interval for each of the ploidy classes was determined, the method allowed determination of ploidy distributions in mouse liver by stereological image analysis alone. The method was established from combined stereological and flow cytometric measurements on liver tissue representing two different stages of liver regeneration after two-thirds partial hepatectomy, and it was tested against an independent set of data representing a marked increase in the portion of S-phase cells.     

Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope.

Fluorescence photobleaching methods have been widely used to study diffusion processes in the plasma membrane of single living cells and other membrane systems. Here we describe the application of a new photobleaching technique, scanning microphotolysis. Employing a recently developed extension module to a commercial confocal microscope, an intensive laser beam was switched on and off during scanning according to a user definable image mask. Thereby the location, geometry, and number of photolysed spots could be chosen arbitrarily, their size ranging from tens of micrometers down to the diffraction limit. Therewith we bleached circular areas on the surface of single living 3T3 cells labeled with the fluorescent lipid analog NBD-HPC. Subsequently, the fluorescence recovery process was observed using the attenuated laser beam for excitation. This yielded image stacks representing snapshots of the spatial distribution of fluorescent molecules. From these we computed the radial distribution functions of the photobleached dye molecules. The variance of these distributions is linearly related to the diffusion constant, time, and the mobile fraction of the diffusing species. Furthermore, we compared directly the theoretically expected and measured distribution functions, and could thus determine the diffusion coefficient from each single image. The results of these two new evaluation methods (D = 0.3 +/- 0.1 micron 2/s) agreed well with the outcome of conventional fluorescence recovery measurements. We show that by scanning microphotolysis information on dynamical processes such as diffusion of lipids or proteins can be acquired at the superior spatial resolution of a confocal laser scanning microscope.     

Statistical quantification of detachment rates and size distributions of cell clumps from wild-type (PAO1) and cell signaling mutant (JP1) Pseudomonas aeruginosa biofilms

The detachment of cells from bacterial biofilms is an important, yet poorly understood and largely unquantified phenomenon. Detached cell clumps from medical devices may form microemboli and lead to metastasis, especially if they are resistant to host defenses and antibiotics. In manufacturing plants detached clumps entering a process stream decrease product quality. Two strains of Pseudomonas aeruginosa, a wild type (PAO1) and a cell signaling mutant (JP1), were studied to (i) quantify and model detachment patterns and (ii) determine the influence of cell signaling on detachment. We collected effluent from a biofilm flowthrough reactor and determined the size distribution for cell detachment events by microscopic examination and image analysis. The two strains were similar in terms of both biofilm structure and detachment patterns. Most of the detachment events were single-cell events; however, multiple-cell detachment events contributed a large fraction of the total detached cells. The rates at which events containing multiple cells detached from the biofilm were estimated by fitting a statistical model to the size distribution data. For events consisting of at least 1,000 cells, the estimated rates were 4.5 events mm(-2) min(-1) for PAO1 and 4.3 events mm(-2) min(-1) for JP1. These rates may be significant when they are scaled up to the total area of a real biofilm-contaminated medical device surface and to the hours or days of patient exposure.     

An X-ray microtomography-based method for detailed analysis of the three-dimensional morphology of fungal pellets

Filamentous fungi are widely used in the production of biotechnological compounds. Since their morphology is strongly linked to productivity, it is a key parameter in industrial biotechnology. However, identifying the morphological properties of filamentous fungi is challenging. Owing to a lack of appropriate methods, the detailed three-dimensional morphology of filamentous pellets remains unexplored. In the present study, we used state-of-the-art X-ray microtomography (µCT) to develop a new method for detailed characterization of fungal pellets. µCT measurements were performed using freeze-dried pellets obtained from submerged cultivations. Three-dimensional images were generated and analyzed to locate and quantify hyphal material, tips, and branches. As a result, morphological properties including hyphal length, tip number, branch number, hyphal growth unit, porosity, and hyphal average diameter were ascertained. To validate the potential of the new method, two fungal pellets were studied—one from Aspergillus niger and the other from Penicillium chrysogenum. We show here that µCT analysis is a promising tool to study the three-dimensional structure of pellet-forming filamentous microorganisms in utmost detail. The knowledge gained can be used to understand and thus optimize pellet structures by means of appropriate process or genetic control in biotechnological applications.     

Particle morphology characterization and manipulation in biomass slurries and the effect on rheological properties and enzymatic conversion

An improved understanding of how particle size distribution relates to enzymatic hydrolysis performance and rheological properties could enable enhanced biochemical conversion of lignocellulosic feedstocks. Particle size distribution can change as a result of either physical or chemical manipulation of a biomass sample. In this study, we employed image processing techniques to measure slurry particle size distribution and validated the results by showing that they are comparable to those from laser diffraction and sieving. Particle size and chemical changes of biomass slurries were manipulated independently and the resulting yield stress and enzymatic digestibility of slurries with different size distributions were measured. Interestingly, reducing particle size by mechanical means from about 1 mm to 100 μm did not reduce the yield stress of the slurries over a broad range of concentrations or increase the digestibility of the biomass over the range of size reduction studied here. This is in stark contrast to the increase in digestibility and decrease in yield stress when particle size is reduced by dilute-acid pretreatment over similar size ranges.     

Statistical Quantification of Detachment Rates and Size Distributions of Cell Clumps from Wild-Type (PAO1) and Cell Signaling Mutant (JP1) Pseudomonas aeruginosa Biofilms

The detachment of cells from bacterial biofilms is an important, yet poorly understood and largely unquantified phenomenon. Detached cell clumps from medical devices may form microemboli and lead to metastasis, especially if they are resistant to host defenses and antibiotics. In manufacturing plants detached clumps entering a process stream decrease product quality. Two strains of Pseudomonas aeruginosa, a wild type (PAO1) and a cell signaling mutant (JP1), were studied to (i) quantify and model detachment patterns and (ii) determine the influence of cell signaling on detachment. We collected effluent from a biofilm flowthrough reactor and determined the size distribution for cell detachment events by microscopic examination and image analysis. The two strains were similar in terms of both biofilm structure and detachment patterns. Most of the detachment events were single-cell events; however, multiple-cell detachment events contributed a large fraction of the total detached cells. The rates at which events containing multiple cells detached from the biofilm were estimated by fitting a statistical model to the size distribution data. For events consisting of at least 1,000 cells, the estimated rates were 4.5 events mm⁻² min⁻¹ for PAO1 and 4.3 events mm⁻² min⁻¹ for JP1. These rates may be significant when they are scaled up to the total area of a real biofilm-contaminated medical device surface and to the hours or days of patient exposure.     

The influence of morphology on geldanamycin production in submerged fermentations of <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis> var. <Emphasis Type="Italic">geldanus</Emphasis>

The diverse morphology of the filamentous organism Streptomyces hygroscopicus var. geldanus was characterised by image analysis under various environmental conditions. In the presence of surfactant compounds, a significant decrease in the mean pellet diameter was observed. Cell aggregation was also influenced by spore inoculum level, with high concentrations reducing pellet size. In addition, the dispersion of pellets was found to increase with the inclusion of glass beads to submerged shake-flask cultures. In all cases, production of the secondary metabolite geldanamycin was determined to be dependent on the morphological profile of the organism, with a concomitant increase of 88% in geldanamycin yield observed as the mean pellet diameter was reduced by 70%. Thus, to maximise the yield of geldanamycin, it is necessary to limit pellet formation in Streptomyces hygroscopicus var. geldanus to an appropriate size.