SEARCH

SEARCH BY CITATION

Keywords:

  • dimorphism;
  • life cycle;
  • morphology;
  • Mucor circinelloides;
  • on-line image analysis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References

Aims: The life cycle of the dimorphic fungus Mucor circinelloides was studied in a temperature-controlled flow-through cell, which constitutes an ideal tool when following the development of individual cells, with a view to understanding the growth and differentiation processes occurring in and between the different morphological forms of the organism.

Methods and Results: Mycelial growth and the transformation of hyphae into chains of arthrospores were characterized by image analysis techniques and described quantitatively. The influence of the nature (glucose and xylose) and concentration of the carbon source on specific growth rate and hyphal growth unit length were studied. The organism branched more profusely on xylose than on glucose while the specific growth rates determined were rather similar. Methods were developed to study the yeast-like growth phase of M. circinelloides in the flow-through cell, and combined with fluorescent microscopy which allowed new insights to bud formation. Additionally, numbers and distribution of nuclei in arthrospores, hyphae and yeasts were studied.

Conclusions: The results give essential information on the morphological development of the organism.

Significance and Impact of Study: Development of any industrial process utilizing this organism will be dependent on the information obtained here for effective process optimization.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References

Filamentous fungi are widely applied as production hosts in industry, most notably due to their capacity to secrete large amounts of extracellular proteins. Products range from primary and secondary metabolites to industrial enzymes (Peberdy 1994) and have in recent years also included heterologous proteins of various origins (Punt et al. 2002). The secretion of products is clearly advantageous as the subsequent steps in downstream processing are considerably simplified (Saunders et al. 1989; Peberdy 1994). Fungal morphology is often an important issue for the design of a submerged processes, as entanglement of growing mycelia might adversely affect the broth rheology, resulting in mixing and mass transfer problems and concomitantly reduced productivity (Bocking et al. 1999; McIntyre et al. 2001b). Thus, it is crucial to further the knowledge about growth and morphological development to define efficient methods for optimal fungal fermentations. At present, only few genes are known that directly affect the morphology of filamentous fungi, but alternatively, a lot might be learnt from studying dimorphic fungi. These organisms are capable of isotropic and polarized growth, depending on the environmental conditions, resulting in yeast or hyphal growth, respectively (Gow 1994).

Dimorphism is a trait often seen in medically or agriculturally important pathogens, i.e. Candida albicans or Ustilago maydis, but it occurs in nonpathogenic species, too. Amongst the latter group are many members of the Mucorales (Orlowski 1991). Dimorphic Mucor species are capable of growth as either aseptate filamentous mycelia or in the form of multipolar budding yeasts. Environmental factors determine the morphology the organism will assume. The gaseous atmosphere constitutes a pivotal factor with hyphal growth predominating when conditions are aerobic, whereas strict anaerobiosis is required for the development and maintenance of the yeast form (Bartnicki-Garcia and Nickerson 1962a). Furthermore, the nature of the carbon source is crucial: while the yeast form can only grow on fermentable hexoses (Bartnicki-Garcia and Nickerson 1962b), hyphal growth has been described on a wide range of substrates including complex carbon sources (Orlowski 1991). The exact requirements of each Mucor species to grow in a particular mode may vary. For the Mucor circinelloides strain employed during this study, not only anaerobiosis but also a concentration of 30% CO2 in the sparging gas are necessary to facilitate yeast-like growth (Bartnicki-Garcia and Nickerson 1962a). A lot of research efforts have been put into investigating which conditions favour a particular morphology or what type of compounds might affect the morphological outcome of a cultivation (Orlowski 1991). However, considerably less is known in terms of precise morphological characterization or about growth kinetics. While models about the duplication cycle of Aspergilli or the cell cycle of Saccharomyces cerevisiae are detailed, knowledge about these events is scarce in Mucor.

Furthering the knowledge of Mucor morphology is also important because the organism has recently been suggested as a host for heterologous protein production, exploiting its dimorphic nature (Wolff and Arnau 2002). This approach aims at a phase of yeast growth during the production of biomass while exploiting the secretory capacity of the filamentous form during the phase of protein production.

This work is, therefore, aimed at quantification of the developmental stages M. circinelloides assumes during its life cycle, as depicted in the scheme in Fig. 1. After immobilization of individual spores in a temperature-controlled flow-through cell, germination, growth, and finally the development of hyphae into arthrospores were followed. Filamentous growth was described by quantitative image analysis. The effects of glucose or xylose and the concentration of these carbon sources on key morphological parameters such as hyphal growth unit length and specific growth rate were determined. To study bud formation and obtain kinetic data on M. circinelloides yeasts, methods that promoted yeast-like growth in the flow-through cell were developed. Additionally, samples from submerged batch cultivations were analysed by fluorescence microscopy, the number and distribution of nuclei in the different forms were studied.

image

Figure 1. Life cycle of Mucor circinelloides during submerged growth. After swelling of the sporangiospores growth continues in either a polarized (hyphal) or isotropic (yeast-like) growth depending on the environment. Changing the cultivation conditions triggers a dimorphic shift from one form to the other. After exponential growth septa are produced in the usually aseptate hyphae that differentiate into chains of arthrospores (box). These eventually fragment and again can develop into yeasts or hyphae, governed by the environmental conditions

Download figure to PowerPoint

Microorganism

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References

A M. circinelloides strain obtained from the American Type Culture Collection (ATCC1216B) was used throughout this study.

The spore inocula were derived from rice flasks and prepared as follows: 29 g of rice were autoclaved with 6 ml of Vogel's medium (McIntyre et al. 2002), 0·5 ml vitamin solution (60 mg ml−1 niacin; 60 mg ml−1 thiamine), 3 ml M9 (60 g l−1 Na2HPO4, 30 g l−1 KH2HPO4, 5 g l−1 NaCl, 10 gl−1 NH4Cl) and 7 ml H2O were added by sterile filtration. Spores were harvested with 1% Tween 20 and stored in aliquots of 1 ml at −80°C in 20% glycerol.

Batch cultivations

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References

The bioreactor used in this study was a Braun BIOSTAT® M (B. Braun Biotech. International, Melsungen, Germany) with a working volume of 1·5 l. The culture pH was controlled at 5 by automatic addition of 2 m NaOH. All fermentations were performed with Vogel's medium containing 20 g l−1 glucose at 28°C. Dissolved oxygen in the culture medium was measured using an oxygen probe (Mettler Toledo, Giessen, Germany). The agitation was 300 rev min−1, the stirrer was equipped with three five-blade Rushton turbines. For yeast-like growth, the gas flow was kept constant at 0·3 vvm with a gas mixture of 30% CO2 and 70% N2. Prior to inoculation, the culture vessels were sparged for 90 min to establish anaerobic conditions. For mycelial growth, the fermenter was sparged with air. The airflow rate was gradually increased up to 1 vvm and the stirrer speed up to 700 rev min−1. The inoculum size was 1 × 106 spores per ml medium.

Flow-through cell experiments and image analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References

Spores were immobilized in a temperature controlled flow-through cell mounted on a motorized stage with continuous medium flow, to assess fungal growth and branching in a quantitative manner. The construction and set-up of the flow-through cell has been described in detail previously (Spohr et al. 1998). The cell basically consists of two slides (26 × 76 mm) separated by a single-usage in-house built Parafilm M spacer (thickness 127 μm) clamped into a steel frame equipped with tubes for feed addition and waste withdrawal. To sterilize both the cell and the tubing, 70% ethanol was injected through a filter (0·45 μm), this was followed by addition of distilled water after 20 min to remove the ethanol. One millilitre of 0·1% poly-d-lysine (Sigma) was then filtered into the cell to mediate spore fixation. The cell was inoculated with spores to gain a final number of 50–80 spores. Medium was continuously added and removed from the cell with a flow-rate of 3 ml h−1.

To investigate yeast-like growth, 0·23% (v/v) phenethyl alcohol (PEA) (Terenzi and Storck 1968) was included in the medium, as this facilitated yeast development also under aerobic conditions.

All experiments were carried out at 28°C. Images of up to 40 hyphal elements were obtained at time intervals of 15 min on a Nikon Optiphot 2 microscope equipped with a CCD camera (Bischke CCD-5230P, Image House, Copenhagen, Denmark) connected to the image analysis system (Quantimet 600S; Leica Cambridge Ltd, Cambridge, UK). Automatic image analysis was applied in the detection of hyphal elements and measurements of length or projected area (Spohr et al. 1998), hyphal tips were counted manually. These values were used to calculate the hyphal growth unit length HGUL (μm per tip).

Morphological characterization of yeast-like growth was carried out by applying a PC-based system and image analysis software (Image ProPlus 4·0; Image House). Measurements were made manually on acquired images from the flow-through cell using a circle tool of the software package to mark the projected area of yeast cells.

Values for length and area were directly obtained in μm or μm2, respectively, after calibrating the programme for the different microscope objectives using a Nikon objective micrometer (DFA, Copenhagen, Denmark). The specific growth rate for hyphal elements was obtained from a semi-logarithmic plot of the hyphal length vs time, where the slope of the resulting curve is equivalent to μ.

Fluorescence microscopy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References

Calcofluor white (CFW) (Fluorescent Brightener 28; Sigma) was used to stain deposited chitin in the cell walls and septa. In order to visualize nuclear DNA, 4′, 6-diamidino-2-phenylidole (DAPI) dihydrochloride (Molecular Probes, Leiden, The Netherlands) was applied. 3,3′-Dihexyloxacarbocyanine iodide (DiOC6) (Molecular Probes) was used to stain the mitochondria of live yeast cells. Fifty microlitres of undiluted biomass sample and 400 μl PBS buffer (pH 9·4) were added to an Eppendorf tube coated with tin foil. To this, 10 μl DAPI (2 μg ml−1), 50 μl calcofluor white (0·3 μg ml−1), or 40 μl DiOC6 (0·2 μg ml−1), respectively, were added. The samples were incubated at room temperature for 5 min before fluorescent microscopy. Images were obtained on a Nikon Optiphot 2 microscope using a mercury burner and a filter block with 330–380 nm excitation wavelength and a dichroic mirror with emission >420 nm.

Filamentous growth

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References

Filamentous growth of M. circinelloides is illustrated in the picture panel by the development of an individual hyphal element cultivated at a concentration of 10 g l−1 xylose (Fig. 2). After undergoing a period of isotropic growth during spore swelling, the growth proceeded in a polarized pattern with the emergence of the germ tube and subsequent branch formation. Most hyphal elements possessed several (up to three) germ tubes emerging from the spore. This was more likely to be the case with xylose than with glucose as the carbon source. After an initial lag-phase, the total hyphal length (the length of the skeletonized mycelium after image processing) and the number of hyphal tips increased exponentially and with approx. the same growth rate. The formation of new branches occurred randomly, i.e. no clear pattern in the initiation of branching sites, such as a certain distance between branch points, was discernable. Figure 3 shows the total hyphal length, tip number, and additionally the hyphal growth unit length (HGUL) for a population of 27 hyphal elements grown on 10 g l−1 xylose. The HGUL is defined as the total length of a mycelium divided by its number of tips (Caldwell and Trinci 1973) and constitutes a useful quantity for comparing branching characteristics.

image

Figure 2. Images series of filamentous growth of Mucor circinelloides monitored in the flow-through cell. Bar = 300 μm

Download figure to PowerPoint

image

Figure 3. Average data for hyphal length (bsl00084), tip nr (•), hyphal growth unit length (HGUL) (bsl00001) for 10 g l−1 xylose

Download figure to PowerPoint

Hyphal growth was investigated for two different carbon sources (glucose and xylose) at various concentrations and for a mixture of both sugars (Table 1). Lower HGULs were generally obtained on xylose, i.e. a comparatively larger number of tips per hyphal length were formed than on glucose. Despite differences in the branching frequency, the specific growth rates for the conditions tested were similar, and the highest value was obtained on the mixture of glucose and xylose.

Table 1.  Hyphal growth unit length (HGUL) and specific growth rates obtained from flow-through cell experiments for glucose and xylose at different concentrations
ConcentrationsGlucoseXyloseGlc + Xyl
g l−10·51·0101·01010 + 10
μ h−10·67 ± 0·080·64 ± 0·060·65 ± 0·030·60 ± 0·080·61 ± 0·070·73 ± 0·07
HGUL (μm tip−1)120170140120115100
n221112172716

Formation of arthrospores

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References

Hyphal extension did not continue indefinitely, as illustrated by the example of the single hyphal element in Fig. 4. Once a total hyphal length of almost 8 mm was reached, extension and tip formation abruptly stopped. The mycelium did not become completely inactive but underwent a number of structural changes for the process of arthrospore formation, indicated in the growth curve by the arrowhead. A thickening of the hyphae, particularly in the tip regions became perceptible. These areas appeared denser and their silhouette ‘bumpier’ until septa were laid down and the typical chains of rounded arthrospores formed (Fig. 5).

image

Figure 4. HGUL (bsl00001), tip nr (•), length (bsl00084) for a single hyphal element grown on Vogel's medium containing 10 g l−1 xylose. The arrow marks the onset of arthrospore formation

Download figure to PowerPoint

image

Figure 5. Hyphal tip region undergoing development into chain of arthrospores. Images were obtained at 15·75, 16·9, 17·9, and 18·5 h. Tip extension stops while the filament undergoes several differentiation steps

Download figure to PowerPoint

Yeast-like growth and budding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References

During this study, methods that also allowed investigating yeast growth of M. circinelloides in the flow-through cell were established, which originally had been designed for the study of filamentous fungi (Spohr et al. 1998). The experimental set-up does not allow continuous sparging of the growth medium with CO2 and N2 which is required to obtain yeast growth. Therefore, the medium was sparged with the gas mixture prior to usage, but this resulted in a mixed morphology. Operating the flow-through cell in a batch mode, i.e. sealing both ends after the injection of spores and medium, limited the degree of filament formation, but cells still did not display the same spherical shape as observed during submerged cultivations (results not shown). The most suitable approach was the inclusion of 0·23% phenethyl alcohol (v/v) in the growth medium, which facilitated yeast growth also under aerobic conditions, so fresh medium could be supplied continuously and without any sparging requirements. The yeasts thus obtained (Fig. 6), resembled the spherical forms from submerged cultivations where large mother cells, often covered entirely with buds, were present (Fig. 7a). Multiple buds were also produced by the yeasts in the flow-through cell. Image analysis techniques were employed to quantify yeast growth in the presence of phenethyl alcohol (Fig. 8). The volumes of all buds attached to one mother cell were measured individually and totalled. Thus, the volumetric increase of such a ‘yeast unit’ was determined to be exponential.

image

Figure 6. Yeast-like growth in the flow-through cell in the presence of 0·23% phenethyl alcohol. Bar = 50 μm

Download figure to PowerPoint

image

Figure 7. Mucor circinelloides yeast during submerged growth from a batch cultivation: (a) bright field, (b) DAPI, (c) calcofluor white, (d) DiOC6

Download figure to PowerPoint

image

Figure 8. Volumetric increase (bsl00084) of yeast-like growth during ‘batch’ mode and with 0·23% phenethyl alcohol

Download figure to PowerPoint

No obvious pattern of budding was evident, i.e. there was no strict pattern where a new bud would emerge in relation to an already existing one. Merely a slight spatial preference was detectable, as buds would not emerge directly adjacent to one another as long as free surface area was still available. It was frequently observed that a yeast cell produced two or three buds within a time interval (10 min). These newly formed buds were of the same size and, over time, increased in diameter at approx. the same rate.

Bud detachment was never observed in the flow-through cell. There was no reduction in the number of buds per cell over time, which would correspond to buds being released and subsequently transported away by the constant medium flow.

Budding yeasts from submerged cultures were examined by fluorescent microscopy. Certain areas on the surface of mother cells were stained strongly by calcofluor white (Fig. 7c), similar to bud scars in S. cerevisiae, indicating that at least some of the buds were released. DiOC6 staining revealed that many mother cells were already inactive whilst being covered with highly active buds (Fig. 7d).

Distribution of nuclei in filamentous and yeast forms

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References

Yeasts from submerged batch fermentations were stained with DAPI to investigate the distribution and numbers of nuclei in mother and daughter cells (Fig. 7a,b). All mother cells were multinucleate, often containing several dozens of nuclei whilst most buds possessed between two and 10 nuclei. In an attempt to link cell size with the number of nuclei a cell contained, the radii of M. circinelloides yeasts were measured and their nuclei counted. Up to a number of 35, a linear relationship existed between the nuclei number and the cell size (radius) (Fig. 10). At this point, the cells had almost reached their maximum size. There seemed to be a rather defined limitation to the size of a cell, but the number of its nuclei was controlled less stringently. For example, the number of nuclei contained by cells with a radius of approx. 15 μm ranged between 20 and 60.

image

Figure 10. Number of nuclei (•) per yeast cell during submerged batch cultivations

Download figure to PowerPoint

Nuclei in the filamentous form and in arthrospores were also stained with DAPI (Fig. 9a,b). While the nuclei might have appeared to be regularly spaced in short, young filaments, this was not true for the majority of hyphae studied. Large numbers of nuclei were present at seemingly random locations, and several nuclei could be contained over one diameter of a branch. Arthrospores were multinucleate too, and possessed between five and 12 nuclei.

image

Figure 9. Yeast cells with filaments and developing arthrospores: (a) bright field, (b) DAPI

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References

Mucor circinelloides is able to grow in a filamentous mode on a wealth of carbon sources. In this study, growth on a hexose and a pentose were compared.

Like most other filamentous fungi, with the exception of C. albicans (Gow and Gooday 1984), Mucor mycelia increased in size exponentially (Fig. 3), whereas the individual branches extended linearly. New tips were formed at an exponential rate, and consequently the resultant increase of total hyphal length was exponential. The specific growth rates on both carbon sources were rather similar, however, lower HGULs were determined for growth on Vogel's medium with xylose as the carbon source (Table 1). These findings are interesting for potential industrial applications, as the morphology of an organism constitutes an important process parameter, which can strongly affect the rheology of a fermentation broth. This, subsequently, will affect the mixing characteristics and factors as oxygen or mass transfer, which could have adverse effects on the performance of a cultivation.

In septate fungi like Aspergilli the spacing and distribution of nuclei has been shown to be clearly linked with compartment size (Harris 1997; Müller et al. 2000). Nuclei in M. circinelloides, however, were spaced irregularly throughout the hyphae (Fig. 9b), which might be due to the absence of septa.

Great aberrations from common model organisms were shown upon closer investigation of the yeast form. In S. cerevisiae, strict checkpoints for size and DNA replication exist. Mucor circinelloides might be more ‘primitive’ in this respect and simply produce an abundance of nuclei as this would be a way to ensure that at least one nucleus was passed on to a newly formed bud. Similarly, this might be true for arthrospores where the number of nuclei in compartments of a rather uniform size was not fixed but ranged between five and 12 (Fig. 9b). The finding of considerably different numbers of nuclei for yeast cells of the same size (Fig. 10) might be explained by a poor regulation on DNA replication. Alternatively, this could be suggestive of the fact that DNA synthesis was not linked to synthesis of cytoplasm and thus cell size.

Experiments in the flow-through cell showed that new buds emerged at random locations all over the cell surface (Fig. 5b). Multipolar budding is exhibited rarely, known examples are the dimorphic fungus Paracoccidioides brasiliensis (Hamdan and Ferrari 1995), a strain of U. maydis defective in the gene encoding the regulatory subunit of protein kinase A (Gold et al. 1994), and furthermore occurs in S. cerevisiae strains which exhibit defects in the cell cycle (Zhang et al. 1998). The results gathered on the release of buds are somewhat conflicting. While the presence of chitin patches on the surface of mother cells from submerged cultivations, visualized by calcofluor white (Fig. 7d), indicated the release of buds, this process could never be observed in the flow-through cell which facilitated precise monitoring of individual yeasts and their buds. Additionally, previous analysis on the size of cells bearing buds did not show an increase of smaller cells over time, which would correspond to newly released buds forming daughter cells themselves (Lübbehüsen et al. 2003). Instead, yeasts might continue to bud until they become practically metabolically inactive (Fig. 7c) and merely serve as a platform for the daughter cells. It would also be plausible, that – however loosely attached– buds were more likely to be released during submerged growth in a fermenter as a result of stirring or contact with other cells. In the flow-through cell, although without any agitation and only a very low flow rate, yeasts would remain attached to each other, so that secondary bud formation was observed, i.e. bud formation on existing buds.

Arthrospore formation in Mucor, the process of septation and fragmentation of hyphae into individual new entities, is not very well understood and often referred to as a mere starvation response (Orlowski 1991). Their development is stimulated by specific conditions, including high spore inoculum concentrations and low glucose concentrations (Bartnicki-Garcia and Nickerson 1962a,b; Barrera 1983) but they were also formed in the flow-through cell where only a small number of filaments were cultivated under continuous conditions without any limitations (Fig. 4, Fig. 5). As they constitute the only type of spores occurring during submerged growth, it seems likely that their development is part of the natural life cycle. Given that Mucor is an aseptate organism, how would it propagate asexually? It is commonly accepted that septate filamentous fungi fragment during submerged growth (McIntyre et al. 2001a) and that these fragments branch and develop into new active mycelia. An aseptate organism, on the contrary, would leak its cytoplasm upon fragmentation. Thus, there seems a necessity for a mechanism such as arthrospore formation, if growth is to be directed at an increase in cell number not simply cell size. The process of arthrospore formation by M. circinelloides took place more rapidly than in other fungi. Almost 7 days were required before arthrospore development was completed in Trichophyton mentagrophytes in surface cultures (Bibel et al. 1977). The time scale for Oidiodendron truncatum and Geotrichum candidum was closer to that observed for M. circinelloides (Cole and Kendrick 1969). As for O. truncatum, further hyphal extension ceased with the onset of arthrospore formation for M. circinelloides (Fig. 5).

Mucor circinelloides is a multi-faceted organism, capable of growing on a diverse range of substrates, possessing the ability of multiple modes of growth and having the potential of industrial application for the production of both degradative enzymes and primary metabolites. The growth capabilities of the organism facilitate its attractiveness as an industrial workhorse. However, the poorly understood developmental stages of its life cycle complicate its potential application in large-scale fermentations. As with other filamentously growing organisms, the processes of growth, branching and differentiation should be well understood in order to design optimal fermentation processes. Similarly, it is also necessary to understanding the budding processes in the yeast form of the organism. In studying the life cycle of M. circinelloides, it has been possible to further the available knowledge regarding the growth of particular forms and the transitional processes required in order to complete the life cycle. This information is fundamental to understand the physiology of the organism as well as providing valuable information for those interested in the application of M. circinelloides in industry. Development of efficient bioprocesses will surely be dependent on designing processes with optimal numbers of active (productive) forms regardless of yeast cell or filamentous application.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Microorganism
  6. Cultivation media
  7. Batch cultivations
  8. Flow-through cell experiments and image analysis
  9. Fluorescence microscopy
  10. Results
  11. Filamentous growth
  12. Formation of arthrospores
  13. Yeast-like growth and budding
  14. Distribution of nuclei in filamentous and yeast forms
  15. Discussion
  16. Acknowledgement
  17. References
  • Barrera, C.R. (1983) Formation and ultrastructure of Mucor rouxii arthrospores. Journal of Bacteriology 155, 886895.
  • Bartnicki-Garcia, S. and Nickerson, K. (1962a) Induction of yeastlike development in Mucor by carbon dioxide. Journal of Bacteriology 84, 829841.
  • Bartnicki-Garcia, S. and Nickerson, W.J. (1962b) Nutrition, growth and morphogenesis of Mucor rouxii. Journal of Bacteriology 84, 841858.
  • Bibel, D.J., Crumrine, D.A., Yee, K. and King, R.D. (1977) Development of arthrospores of Trichophyton mentagrophytes. Infection and Immunity 15, 958971.
  • Bocking, S.P., Wiebe, M.G., Robson, G.D., Hansen, K., Christiansen, L.H. and Trinci, A.P.J. (1999) Effects of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity. Biotechnology and Bioengineering 65, 638648.
  • Caldwell, I.Y. and Trinci, A.P.J. (1973) The growth unit of the mould Geotrichum candidum. Archiv für Mikrobiologie 88, 110.
  • Cole, G.T. and Kendrick, W.B. (1969) Conidium ontogeneity in hyphomycetes. The arthrospores of Oidiodendron and Geotrichum, and the endoarthrospores of Sporendonema. Canadian Journal of Botany 47, 17731780.
  • Gold, S., Duncan, G., Barrett, K. and Kronstad, J. (1994) cAMP regulates morphogenesis in the fungal pathogen Ustilago maydis. Genes and Development 8, 28052816.
  • Gow, N.A.R. (1994) The Growing Fungus. pp. 403422. London: Chapman & Hall.
  • Gow, N.A.R. and Gooday, G.W. (1984) A model for the germ tube formation and mycelial growth form of Candida albicans. Sabouraudia 22, 137143.
  • Hamdan, J.S. and Ferrari, T.C.A. (1995) An atypical isolate of Paracoccidioides brasiliensis. Mycoses 38, 481484.
  • Harris, S.D. (1997) The duplication cycle in Aspergillus nidulans. Fungal Genetics and Biology 22, 112.
  • Lübbehüsen, T.L., Nielsen, J. and McIntyre, M. (2003) Morphology and physiology of the dimorphic fungus Mucor circinelloides (syn. racemosus) during anaerobic growth. Mycological Research 107, 223230.
  • McIntyre, M., Dynesen, J. and Nielsen, J. (2001a) Morphological characterisation of Aspergillus nidulans: growth, septation and fragmentation. Microbiology 147, 239246.
  • McIntyre, M., Müller, C., Dynesen, J. and Nielsen, J. (2001b) Metabolic engineering of the morphology of Aspergillus. Advances in Biochemical Engineering/Biotechnology 73, 103128.
  • McIntyre, M., Breum, J., Arnau, J. and Nielsen, J. (2002) Growth physiology and dimorphism of Mucor circinelloides (syn. racemosus) during submerged batch cultivation. Applied Microbiology and Biotechnology 58, 495502.
  • Müller, C., Spohr, A.B. and Nielsen, J. (2000) Role of substrate concentration in mitosis and hyphal extension of Aspergillus. Biotechnology and Bioengineering 67, 390397.
  • Orlowski, M. (1991) Mucor dimorphism. Microbiological Reviews 55, 234258.
  • Peberdy, J. (1994) Protein secretion in filamentous fungi- trying to understand a highly productive black box. Trends in Biotechnology 12, 5057.
  • Punt, P.J., van Biezen, N., Conesa, A., Albers, A., Mangnus, J. and den Hondel, C. (2002) Filamentous fungi as cell factories for heterologous protein production. Trends in Biotechnology 20, 200206.
  • Saunders, G., Picknett, T.M., Tuite, M.F. and Ward, M. (1989) Heterologous gene expression in filamentous fungi. Trends in Biotechnology 7, 283287.
  • Spohr, A.B., Dam-Mikkelsen, C., Carlsen, M., Nielsen, J. and Villadsen, J. (1998) On-line study of fungal morphology during growth in a small flow-through cell. Biotechnology and Bioengineering 58, 541553.
  • Terenzi, H.F. and Storck, R. (1968) Stimulation of fermentative and yeastlike morphogenesis of Mucor rouxii by phenethyl alcohol. Journal of Bacteriology 97, 12481261.
  • Wolff, A.M. and Arnau, J. (2002) Cloning of glyceraldehydes-3-phosphate dehydrogenase-encoding genes in Mucor circinelloides (syn. racemosus) and use of the gpd1 promoter for recombinant protein production. Fungal Genetics and Biology 35, 2129.
  • Zhang, J.W., Parra, K.J., Liu, J. and Kane, P.M. (1998) Characterization of a temperature-sensitive yeast vacuolar ATPase mutant with defects in actin distribution and bud morphology. Journal of Biological Chemistry 29, 18 47018 480.