Quantification of autolysis in Penicillium chrysogenum by semiautomated image analysis

An image analysis method is described for the characterization of empty (autolyzed and inactive) regions within the mycelia of filamentous fungi. It extends a previous method that characterized only regions filled with cytoplasm or vacuoles (i.e., the active biomass). The method is semiautomatic, requiring some manual editing before automated measurements. When the method was used for samples from a batch fermentation of an industrial strain of Penicillium chrysogenum, the empty regions were observed to constitute up to 15% (by projected area) of the biomass during the growth phase. After nutrient exhaustion, however, the proportion of empty regions rose rapidly, eventually representing more than 50% of the biomass by the end of fermentation. The increase in the percentage of empty regions coincided with a decrease in biomass (as measured by dry cell weight) and a fall in penicillin titre. Further morphological analysis revealed that fragmentation of mycelia, particularly clumps, coincided with increases in the levels of empty regions. This new image analysis method gave additional information on hyphal differentiation and a measure of autolysis. It was also a useful indicator of the processes leading to autolysis.     

Computer control of the penicillin fermentation using the filtration probe in conjunction with a structured process model

A structured model for the penicillin fermentation is presented. This model includes three different cell types: (1) hyphae tips, (2) penicillin-producing cells, and (3) degenerated, metabolically inactive cells. Cell degeneration has been described previously as a gradual loss of cytoplasmic material by endogenous metabolism. The rate at which such loss of cytoplasm (and activity) proceeds can be expressed as a linear function of the specific growth rate. At growth rates above some minimum value (0.0115 h(-1)) cell degeneration can be prevented. This model served as the control basis during open-loop as well as closed-loop computer control of the fermentation. Closed-loop control was achieved through feedback information of biomass concentration using a filtration probe and was required when complex nutrients contributed significantly to the overall biomass production.     

菌丝空泡形态比例与青霉素合成的关系

采用图像处理技术量化菌丝空泡区域比例,并研究菌丝空泡与青霉素合成的关系.结果表明:当空泡区域比例达到20％时,青霉素开始大量合成;当空泡比例达到30％～35％时,青霉素合成最为旺盛;当空泡区域比例超过40％时,青霉素效价下降.菌丝空泡的过度膨胀是发酵进入衰退期的重要标志.     

Calibration of quantitative real-time TaqMan PCR by correlation with hyphal biomass and ITS copies in mycelia of Piloderma croceum

DNA-based quantification methods such as real-time TaqMan PCR allow a rapid and highly sensitive species-specific quantification of isolated fungal DNA material, but most quantification systems are only able to measure relative amounts of biomass or biomass changes during different treatments. In this experiment, an already established DNA quantification system for the ectomycorrhizal fungus Piloderma croceum, based on the ITS region of ribosomal DNA, was calibrated to absolute biomass to obtain a direct correlation between mycelial biomass and isolated ITS copies. Thin layers of sterile mycelia were cultured on slides. The mycelial biomass was calculated from measurements of the total hyphal length using image analysis, followed by determination of the mycelial volume, and multiplied by the specific weight of hyphae obtained from literature data. Using the very same mycelium, the number of ITS copies was quantified by TaqMan PCR. The mean value of 1047 (+/- 185) copies per mm hypha results in possible data for a direct conversion: one billion (10 (9)) ITS copies corresponded to 0.79 mg hyphal dry weight. For the ribosomal ITS multi-copy genes, the number of ITS copies could be calculated to approx. 152 (+/- 26) copies per dikaryotic cell. These conversion data now allow determination of the mycelial biomass of Piloderma croceum using real-time TaqMan PCR, a prerequisite for competition experiments with Piloderma croceum.     

Viability, strength, and fragmentation of Saccharopolyspora erythraea in submerged fermentation.

Two fermentations of the commercially important erythromycin-producing filamentous bacterium Saccharopolyspora erythraea were conducted in defined media. One was glucose-limited and the other nitrate-limited. The viability of the hyphae was determined using the fluorescent stain BacLight (Molecular Probes, Eugene, OR). Also, the force required to strain hyphae to breakage was determined using micromanipulation and a sensitive force transducer. In both fermentations, fragmentation coincided with the appearance of regions in the mycelia with permeabilised membranes (considered nonviable). Under glucose-limitation, hyphal breaking force rose to 1,050 +/- 130 nN at the end of the growth phase and fell to an undetectable value as a result of glucose exhaustion. Under nitrate-limitation, hyphal breaking force fell from 900 +/- 160 nN during the growth phase to 550 +/- 40 nN in the stationary phase. In both cases image analysis showed that the dimensions of mycelia were of the same order, suggesting that the major factor influencing fragmentation was the appearance of nonviable regions (assumed to be weak). The location in which nonviable regions first appear within hyphae could not be determined because of their appearance coinciding with fragmentation.     

Use of confocal microscopy to follow the development of penetrative hyphae during growth of Rhizopus oligosporus in an artificial solid-state fermentation system

Two methods were compared for determining the concentration of penetrative biomass during growth of Rhizopus oligosporus on an artificial solid substrate consisting of an inert gel and starch as the sole source of carbon and energy. The first method was based on the use of a hand microtome to make sections of approximately 0.2- to 0.4-mm thickness parallel to the substrate surface and the determination of the glucosamine content in each slice. Use of glucosamine measurements to estimate biomass concentrations was shown to be problematic due to the large variations in glucosamine content with mycelial age. The second method was a novel method based on the use of confocal scanning laser microscopy to estimate the fractional volume occupied by the biomass. Although it is not simple to translate fractional volumes into dry weights of hyphae due to the lack of experimentally determined conversion factors, measurement of the fractional volumes in themselves is useful for characterizing fungal penetration into the substrate. Growth of penetrative biomass in the artificial model substrate showed two forms of growth with an indistinct mass in the region close to the substrate surface and a few hyphae penetrating perpendicularly to the surface in regions further away from the substrate surface. The biomass profiles against depth obtained from the confocal microscopy showed two linear regions on log-linear plots, which are possibly related to different oxygen availability at different depths within the substrate. Confocal microscopy has the potential to be a powerful tool in the investigation of fungal growth mechanisms in solid-state fermentation.     

A new sensor,the “filtration probe,” for quantitative characterization of the penicillin fermentation. I. Mycelial morphology and culture activity

In this article the filtration resistance of fungal (Penicillium) mycelia is shown to be quantitatively related to the morphology of the cells. On this basis, it was possible to determine the intracellular volume by simple filtration measurements. Since the dry weight of the cells can also be measured, a morphological variable-the “hyphae density” (dry weight hyphae/volume hyphae)-can be quantified. This variable typically is measured to have values in the range of 0.35–0.20 g/cm³; the lower values represent the final stages of the fermentation. It can be shown that this loss of cell material during fermentation is limited to cytoplasmic components, and that most (70%) of the components which disappear are proteins. Concurrent with and linearly related to this loss of proteins, the culture activity in terms of both cell maintenance energy (respiration activity) and penicillin synthesis also decreased. The hyphae density measured by filtration is therefore a direct measure of the metabolic potential of the organism.     

红缘拟层孔菌发酵工艺的研究及其升罐培养

为筛选出红缘拟层孔菌发酵的最佳条件,并用于升罐发酵,试验优选出5个基础培养基,再以发酵菌丝体的生物量为指标,确定红缘拟层孔菌发酵的最佳条件。进行升罐发酵,间隔取出少量发酵液进行显微观察并检测葡萄糖含量,描述出菌丝体的生长状态。结果表明:红缘拟层孔菌发酵的最佳条件为马铃薯200 g/L,葡萄糖20 g/L,磷酸二氢钾1 g/L,硫酸镁1.5 g/L,维生素B10.4 g/L。升罐发酵中,菌丝干质量在前60 h处于快速的增长状态,最大为6.890 1 g/L,而60 h之后菌丝的生长变缓慢,到84 h之后,菌丝自溶,菌丝的产量开始下降。菌丝的葡萄糖含量经血糖仪检测在60 h时最高,达到0.296 5 g/L,之后呈现下降趋势。红缘拟层孔菌升罐发酵的最佳发酵时间为56~64 h。     

Fungal growth during rice tapé fermentation

Fungal growth was quantified during Indonesian rice tapé fermentation using an agar-film technique following sample homogenization for 1 min at 25000 rev/min. After 72 h fermentation, mould hyphal length was 0·68 km/g, yeast hyphal length 2·1 km/g and the numbers of mould chlamydospores and single yeast cells were 14 times 10⁵/g and 5·1 times 10⁷/g respectively. The estimated fungal biomass in rice tapé after 72 h was 25 mg/g dry weight with 62% of this being mould hyphae, 24% mould chlamydospores, 13% yeast hyphae and 1% yeast cells.     

菌丝形态分化与头孢菌素C合成的关系

利用显微图像分析法对顶头孢霉菌的菌丝形态进行了定量研究,并统计分析了头孢菌素C发酵过程中的菌丝形态的变化规律,具体对菌丝长度、菌丝宽度和菌丝生长单位进行了定量分析,分析了菌丝形态分化与头孢菌素C合成的关系。研究表明头孢菌素C的合成是在细长菌丝分化成膨大菌丝片段后才启动的,头孢菌素C可能主要是在膨大菌丝分化成节孢子的过程中被合成。     

Macroscopic growth of filamentous fungi on solid substrate explained by a microscopic approach.

A quantitative model predicting biomass growth on solid media has been developed. The model takes into account steric interactions between hyphae and tips at the microscopic level (competition for substrate and tip-hypha collisions). These interactions effect a slowing down of the hyphal, population-averaged extension rate and are responsible, at the microscopic level, for the distribution of tip orientations observed at the colony border. At the macroscopic level, a limiting value of the colony radial extension rate is attained. A mathematical model that combines hyphal branching, tip diffusion, and biomass growth was proposed to explain such behavior. Experiments using Gibberella fujikuroi were performed to validate the model; good agreement between experiments and simulations was achieved. Most parameters can be measured by simple image analysis on the peripheral growth zone, and they have clear physical meaning; that is, they correspond to properties of single, leading hyphae. The model can be used to describe two-dimensional (2D) solid media fermentation experiments under varying culture conditions; the model can also be extended to consider growth in three-dimensional (3D), complex geometry substrates.     

The effects of ropy-1 mutation on cytoplasmic organization and intracellular motility in mature hyphae of Neurospora crassa

We have used light and electron microscopy to document the cytoplasmic effects of the ropy (ro-1) mutation in mature hyphae of Neurospora crassa and to better understand the role(s) of dynein during hyphal tip growth. Based on video-enhanced DIC light microscopy, the mature, growing hyphae of N. crassa wild type could be divided into four regions according to cytoplasmic organization and behavior: the apical region (I) and three subapical regions (II, III, and IV). A well-defined Spitzenk?rper dominated the cytoplasm of region I. In region II, vesicles ( approximately 0.48 micro m diameter) and mitochondria maintained primarily a constant location within the advancing cytoplasm. This region was typically void of nuclei. Vesicles exhibited anterograde and retrograde motility in regions III and IV and followed generally parallel paths along the longitudinal axis of the cell. A small population of mitochondria displayed rapid anterograde and retrograde movements, while most maintained a constant position in the advancing cytoplasm in regions III and IV. Many nuclei occupied the cytoplasm of regions III and IV. In ro-1 hyphae, discrete cytoplasmic regions were not recognized and the motility and/or positioning of vesicles, mitochondria, and nuclei were altered to varying degrees, relative to the wild type cells. Immunofluorescence microscopy revealed that the microtubule cytoskeleton was severely disrupted in ro-1 cells. Transmission electron microscopy of cryofixed cells confirmed that region I of wild-type hyphae contained a Spitzenk?rper composed of an aggregation of small apical vesicles that surrounded entirely or partially a central core composed, in part, of microvesicles embedded in a dense granular to fibrillar matrix. The apex of ro-1 the hypha contained a Spitzenk?rper with reduced numbers of apical vesicles but maintained a defined central core. Clearly, dynein deficiency in the mutant caused profound perturbation in microtubule organization and function and, consequently, organelle dynamics and positioning. These perturbations impact negatively on the organization and stability of the Spitzenk?rper, which, in turn, led to severe reduction in growth rate and altered hyphal morphology.     

Hyphal vocuolation and fragmentation inpenicillium chrysogenum

A link between vacuolation and fragmentation of Penicillium chrysogenum mycelia in stirred tank submerged fermentations is reported. Quantitative information on vocuolation and morphology was obtained by image analysis. In fed-batch fermentations the coincidence of the events of rapid vacuolation and the fall of the mean total and main hyphal lengths suggests that hyphal fragmentation is not necessarily due to "shear" alone. The physiological state of the hyphae, characterized by the proportions of vaccuoles, was found to have a significant influence on the breakage of mycelial hyphae, It was found that the fragmentation was greater when the hyphae became heavily vacuolated following nutrient limitation in the culture, i.e., during the switch from the rapid growth to the production phase. (c) 1994 John Wiley & Sons, Inc.     

Effect of phenylacetic acid feeding on the process of cellular autolysis in submerged batch cultures of Penicillium chrysogenum.

The effects of feeding the 'toxic' penicillin precursor, phenylacetic acid (PAA) at varying rates, upon the process of cellular autolysis, was assessed in batch bioreactor cultures of an industrial strain of Penicillium chrysogenum. Five processes were fed at rates which resulted in extracellular concentrations of PAA ranging from zero (the control) to approximately ten times levels said to be optimal for penicillin biosynthesis. The culture response was assessed chemically and morphologically, using computerised image analysis. High concentrations of PAA reduced biomass and penicillin production, and were associated with increased cellular autolysis. However, the values of classical morphological indices (branch length, main hyphal length and hyphal growth unit) varied little in cultures which showed extensive autolysis and biomass loss. Lower precursor concentrations (0.01 to 1.0 g l-1) had little effect on biomass, penicillin, or upon the levels of autolysis compared with the control process. Therefore, precursor concentration controlled within the optimal range for penicillin production, has little impact upon differentiation or degradation within an industrial culture of P. chrysogenum. By contrast, exploitation of the toxicity of PAA is proposed as a means to bring forward or enhance autolysis, providing a reliable method of 'induction' with which to study the phenomenon in P. chrysogenum.     

Fungal morphology and fragmentation behavior in a fed-batch Aspergillus oryzae fermentation at the production scale

It is well known that high-viscosity fermentation broth can lead to mixing and oxygen mass transfer limitations. The seemingly obvious solution for this problem is to increase agitation intensity. In some processes, this has been shown to damage mycelia, affect morphology, and decrease product expression. However, in other processes increased agitation shows no effect on productivity. While a number of studies discuss morphology and fragmentation at the laboratory and pilot scale, there are relatively few publications available for production-scale fungal fermentations. The goal of this study was to assess morphology and fragmentation behavior in large-scale, fed-batch, fungal fermentations used for the production of protein. To accomplish this, a recombinant strain of Aspergillus oryzae was grown in 80 m(3) fermentors at two different gassed, impeller power-levels (one 50% greater than the other). Impeller power is reported as energy dissipation/circulation function (EDCF) and was found to have average values of 29.3 +/- 1.0 and 22.0 +/- 0.3 kW m(-3) s(-1) at high and low power levels, respectively. In all batches, biomass concentration profiles were similar and specific growth rate was < 0.03 h(-1). Morphological data show hyphal fragmentation occurred by both shaving-off of external clump hyphae and breakage of free hyphae. The fragmentation rate constant (k(frag)), determined using a first-order model, was 5.90 and 5.80 h(-1) for high and low power batches, respectively. At the end of each batch, clumps accounted for only 25% of fungal biomass, most of which existed as small, sparsely branched, free hyphal elements. In all batches, fragmentation was found to dominate fungal growth and branching. We speculate that this behavior was due to slow growth of the culture during this fed-batch process.     

On-line study of growth kinetics of single hyphae of Aspergillus oryzae in a flow-through cell

Using image analysis the growth kinetics of the single hyphae of the filamentous fungus Aspergillus oryzae has been determined on-line in a flow-through cell at different glucose concentrations in the range from 26 mg L-1 to 20 g L-1. The tip extension rate of the individual hyphae can be described with saturation type kinetics with respect to the length of the hyphae. The maximum tip extension rate is constant for all hyphae measured at the same glucose concentration, whereas the saturation constant for the hyphae varies significantly between the hyphae even within the same hyphal element. When apical branching occurs, it is observed that the tip extension rate decreases temporarily. The number of branches formed on a hypha is proportional to the length of the hypha that exceeds a certain minimum length required to support the growth of a new branch. The observed kinetics has been used to simulate the outgrowth of a hyphal element from a single spore using a Monte Carlo simulation technique. The simulations shows that the observed kinetics for the individual hyphae result in an experimentally verified growth pattern with exponential growth in both total hyphal length and number of tips.     

Accurate and rapid viability assessment of Trichoderma harzianum using fluorescence-based digital image analysis

Fluorescence microscopy and image analysis were evaluated in order to assess the viability of Trichoderma harzianum, an economically important filamentous fungus. After the evaluation of the two most commonly used fluorochromes, acridine orange (AO) and fluorescein diacetate (FDA) as metabolic indicator stains, AO gave ambiguous results and therefore FDA was chosen. The lower stability at room temperature and fast fluorescence intensity decay (50% after only 30 s of illumination in UV light) could be overcome by the use of a digital image acquisition system including frame grabber and a video camera. Fresh (live) fungal hyphae emitted bright green fluorescence when stained with this dye (7.5 microg/L), whereas a total absence of fluorescence was observed when using sterilized (dead) fungal cells. Fresh cells were subjected to different lethal and sublethal treatments and the percentage of FDA stained fluorescent hyphae was then measured over the total hyphal area (% of FDA-stained area) by image analysis. At the same time, samples were cultivated in shake flasks in order to correlate this % of FDA-stained area with its growth rate, a functional indicator of viability. The linear correlation (r = 0.979) was: growth rate (g/L x h) = 2.25 x 10(-3) (% of FDA-stained area). This method was used to evaluate the viability of the fungus under two different fermentation conditions in a 10-L bioreactor. Estimated viable biomass during fermentation was strongly influenced by the process conditions. The use of FDA, with computer-aided quantitative image analysis, has made it possible to rapidly and reliably quantify the viability of T. harzianum.     

Transmission of the eect of an antifungal agent within a single hypha

A micro-compartment culture method was devised in which a single hypha of Rhizopus stolonifer growing on an agar section traversed an antifungal non-diffusible barrier to another agar section; thus the local environment of the distal or proximal part of the hypha could be controlled independently. The responses in terms of hyphal extension of the test fungus to local application of amphotericin B in this culture system were estimated by using an automatic analysing system. After hyphae had traversed the barrier, distal application of amphotericin B caused no appreciable effect on the proximal hyphae. In contrast, proximal application of amphotericin B caused inhibition of the extension of distal hyphae. The reversal of polarized cytoplasmic streaming also occured during the inhibition of distal hyphal extension. The extents of inhibition of the distal hyphal extension and the cytoplasmic streaming were dependent upon the hyphal distance between the amphotericin B application site and the hyphal tip. These results show that the effect of an antifungal agent on a hypha depends on the region of the hypha exposed. Cytoplasmic streaming may play key role in the transmission of antifungal effects within a single hypha.     

Oil and air dispersion in a simulated fermentation broth as a function of mycelial morphology

The culture conditions of a multiphase fermentation involving morphologically complex mycelia were simulated in order to investigate the influence of mycelial morphology (Trichoderma harzianum) on castor oil and air dispersion. Measurements of oil drops and air bubbles were obtained using an image analysis system coupled to a mixing tank. Complex interactions of the phases involved could be clearly observed. The Sauter diameter and the size distributions of drops and bubbles were affected by the morphological type of biomass (pellets or dispersed mycelia) added to the system. Larger oil drop sizes were obtained with dispersed mycelia than with pellets, as a result of the high apparent viscosity of the broth, which caused a drop in the power drawn, reducing oil drop break-up. Unexpectedly, bubble sizes observed with dispersed mycelia were smaller than with pellets, a phenomenon which can be explained by the segregation occurring at high biomass concentrations with the dispersed mycelia. Very complex oil drops were produced, containing air bubbles and a high number of structures likely consisting of small water droplets. Bubble location was influenced by biomass morphology. The percentage (in volume) of oil-trapped bubbles increased (from 32 to 80%) as dispersed mycelia concentration increased. A practically constant (32%) percentage of oil-trapped bubbles was observed with pelleted morphology at all biomass concentrations. The results evidenced the high complexity of phases interactions and the importance of mycelial morphology in such processes.