ELSEVIER Journal of Biotechnology 46 (1996) 221-234

Cultivation of Aspergillus awamori on wheat bran: Ultrastructural description of growth and localization of xylanase

by immunogold labeling

Stefan Adolph a, Susanne Miller a, Detlef Siedenberg a, Karin J’gger b, Heiner Lehmann b, Karl Schiigerl a* * , Marco Giuseppin ’

a Institute for Technical Chemistry, University of Hannooer,Callinstr. 3, D-30167 Hannouer, Germany b Botanical Institute, Veterinary University of Hannouer, Hannouer, Germany

’ Unilever Research Laboratorium, Vlaardingen, Netherlands

Received 15 August 1995; revised 19 December 1995; accepted 20 December 1995

Abstract

Aspergillus awumori was cultivated in shake flask cultures and in stirred tank reactors on synthetic medium and on complex medium, (with wheat bran substrate), respectively, and samples were taken at different cultivation times. The filamentous mycelia and pellets of the fungus were analyzed using four different techniques: transmission electron microscopy (TEM), electron spectroscopic imaging (ESI), electron energy loss spectroscopy (EELS) and immunocytochemi- cal investigations. The ultrastructure of A. awamori and the colonization and decomposition of the wheat bran by the fungus were determined as a function of the cultivation time. The xylanase concentration in the cytoplasm and cell wall of the fungus was high during the first phase of the cultivation and it gradually decreased due to its secretion. At the same time the xylanase concentration increased on the surface of the solid substrate as well as in the cultivation medium. The variation of the local distribution of the enzyme xylanase during the cultivation was determined as well. It is shown how the fungus penetrates and decomposes the wheat bran. Phosphorous and sulfur inclusions, determined with ES1 and EELS, were found in the vacuoles.

Keywords: Aspergillus awamori; Xylanase; Ultrastrncture; Transmission electron microscopy (TEM); Electron spectroscopic imaging (ESI); Electron energy loss spectroscopy (EELS); Immunogold labeling

1. Introduction ones. Some of the complex medium components are

Several fungal industrial production processes use complex media, consisting of agricultural byproducts (corn steep liquor, peanut flour, sugar beat molasses, pharma medium, etc.>. These media are inexpensive and yield much higher productivity than synthetic

* Corresponding author.

solids. The particular polymer (cellulose, hemicellu- lose, starch, protein), which often forms a suspension in the complex cultivation medium, serves as an inducer for the formation of hydrolyzing enzymes. This is the case for the production of xylanase, which is induced by xylan, an integral component of wheat bran. The aim of the investigations was to monitor changes of the fungal ultrastructures and the

Elsevier Science B.V. SSDI 0168-1656(95)00198-O

222 S. Adolph et al. / Journul of’ Biotechnology 46 (19961 221-234

colonization of wheat bran during the cultivation by means of electron microscopic measurements. Xy- lanase was detected by immunogold labeling, using

the post sectioning-labeling technique.

2. Materials and methods

2. I. Aspergillus awamori cultiration

Cultures of the fungus Aspergillus awamnri

grown on agarose plates (3.8% potato-dextrose-agar) in petri dishes were used to obtain the conidia. The spores were suspended in 0.9% NaCl solution and counted in a Neubauer counting chamber.

400 ml cultivation medium in a 1 1 shake flask was inoculated with the spore suspension (1.8 X lo6 spores per ml final concentration) and cultivated on an orbital shaker at 150 rpm and 30°C for 9.5 h. This preculture was used to inoculate 15 1 complex medium in a 20 1 stirred tank reactor (B. Braun,

Melsungen). The cultivations were performed at 300 rpm, at 25°C and pH 4.5 with 1 vvm aeration rate.

Some cultivations were performed on synthetic medium in 1 1 shake flask, which was inoculated by a spore suspension (10” spores per ml>. These culti- vations were performed on an orbital shaker at 150 rpm and 30°C.

2.2. Preparation of the ,fungus for electron micro-

scopic investigations

Good structural preservation and high immuno- logical reactivity of the sample are difficult to com- bine. Fixation with glutardialdehyde and osmium tetroxide leads usually to a good conservation of the ultrastructure, but is unsuitable for immuno cyto- chemical investigations because the epitopes become covered due to the crosslinking of proteins. In the present investigations four different preparation tech- niques were tested (Table 1). In Preparations 1 to 4 the degree of preservation of the structure decreases, whereas the degree of immunological reactivity in- creases.

For all preparations the fixation was performed immediately after sample collection.

In Preparation 1 the specimens were fixed with 4% glutaraldehyde in 0.025 M PIPES-buffer (final concentrations). After 16 h of fixation at 8°C the samples were rinsed (3 X 10 min) with pure buffer

and stored at 4°C. The post fixation was performed with 1% (final concentration) osmium tetroxide at room temperature for 2 h at pH 7.2. The samples were rinsed three times with distilled water and

dehydrated over night in an desiccator above CaCI, and acetone, then stagewise with 80%, 90%, 95%, 100% and 100% acetone over a molecular sieve for 10 or 20 min, respectively. Resin infiltration was

carried out by replacing half of the acetone by Durcupan resin (Fluka) and shaking in closed glass vials on a rotor with an inclined axis. After 24 h, the vials were opened and the solvent evaporated overnight. The embedding was performed in plastic dishes with fresh embedding medium, which was polymerized at 40°C for 24 h and then for an addi- tional 48 h at 60°C.

Preparation 4 for immunogold labeling was per- formed according to Roth (1982) and Jager and Bergman (1991). The sample was fixed by addition of an equal volume of 6% formaldehyde in phos- phate buffer (pH 7.2) at room temperature for 30 min. After rinsing twice with buffer, the superfluous aldehyde was removed with 50 mM NH,Cl. After 50 min, the sample was rinsed twice again with buffer solution and stored at 4°C.

The dehydration with ethanol was carried out in 13 stepsfor 10 minutes each in: 15% (1 X >, 30% (2 x ), 50% (2 x >, 70% (1 x >, 80% (1 x ), 90% (2 X 1 95% (2 X > and 100% 3 X > ethanol. The specimens were infiltrated with LR-White Resin (a hardgrade, acrylic resin with good immunocytochem ical properties) in 9 steps: with 30% (2 X 10 min), 50% (1 X 10 min), 50% (1 X overnight), 70% (2 X 10 min), 100% (1 X 60 min), 100% (1 X overnight) 100% (1 X 8 h) LR-White.

Afterwards the specimens were embedded in gelatin capsules in fresh LR-White and the resin was polymerized at 50°C (max. 52°C) for 24 h.

The fixations for Preparations 2 and 3 were car- ried out with 2% glutaraldehyde and 1% formalde- hyde in PIPES-buffer (pH 7.2) for 30 min. The sample was divided into two portions. One was processed according to Preparation 1 and the other according to Preparation 4 (Table 1).

2.3. Semithin sectioning and light microscopy

To select areas for ultramicrotomy, semithin sec- tioning was performed with the Pyramitom 11 800

S. Adolph et al. /Journal of Biotechnology 46 (1996) 221-234 223

(LKB) using glass knives. The semithin sections (l-2 pm) were stained with methylene blue and observed with a photomicroscope III (Zeiss) to ob- tain orientation and to select interesting parts, which would be observed by transmission electron mi- croscopy. Occasionally, micrographs were taken, employing the black-white film Agfa Pan 25. To

allow ultrathin sectioning, the resin was brought into

a trapezoid shape with 100-200 pm edge length, the

part of interest being on the top of the trapezium.

2.4. Ultrathin sectioning

The ultrathin sectioning was performed with the Ultracut E (Reichert-Jung) and glass knives. After

treating the sections with chloroform, the thickness

Table 1

Applied preparation techniques

samples from the cultivation

fiXat.iOIl 4% glutaraldehyde

fiXatiOIl 2% glutaraldehyde 1% formaldehyde

rinsing with buffer solution

Preparation 1 Preparation 2 Preparation 3 Preparation 4

224 S. Adolph et ol./Joumal ofBiotechnology 46 (19961221-234

amounted to 50- 100 nm. Diamond knives were used for the preparation of sections containing wheat bran

particles. The sections of Preparations 1 and 2 were transferred to copper grids coated by 0.05% Piolo- form F. The sections of Preparation 3 and 4 were transferred to nickel grids (50 and 100 mesh, respec-

tively) coated with 0.05% Pioloform F.

one sample was taken from a shake flask culture after 25 h of cultivation and treated the same way. All samples were centrifuged and applied to agarose

gel to diffuse against the antiserum. The precipitate was stained with Coomassie Brilliant Blue.

2.6. Staining and transmission electronmicroscopy

The preparation for electron spectroscopy imaging (ESI) and electron energy loss spectroscopy (EELS) were cut with diamond knives (thickness 30-50 nm) and transferred to uncoated copper grids (700 mesh, Plano).

2.5. Immunogold labeling of xylanase and immunod-

ifision test

The fresh ultrathin sections of Preparations 3 and 4 were immuno labeled (with colloidal gold) follow- ing the technique recommended by Roth (I 982) and modified by Jager and Bergman (1991 I. As primary antibody, a polyclonal rabbit anti-xylanase-antibody, directed against Xylanase I of Aspergillus awamori

was used. As secondary antibody. a goat anti-rabbit- antibody labeled with 10 nm colloidal gold particle (GAR GIO, Amersham) was employed. The ultrathin sections on the nickel grids were incubated on a 10 ~1 droplet of the antibody solutions and on a 20 ~1 droplet of the other solutions. The following stock solutions were used: PBS-T (phosphate buffered saline, 0.9% NaCl, 0.1 M Na-phosphate. 0.05% Tween-20); 1% BSA (bovine serum albumin) in PBS-T; 0.1% BSA in PBS-T; primary antibody in 1% BSA/PBS-T diluted: 1: 2000; secondary anti- body in 1% BSA/PBS-T diluted: 1:40. The labeling of the sections was performed in six steps: incuba- tion (30 min 1% BSA/PBS-T), incubation (over- night at 4°C: primary antibody solution); rinsing (2 X 5 min: 0.1 BSA/PBS-T), incubation (30 min: secondary antibody solution); rinsing (2 X 5 min: 0.1% BSA/PBS-T) and rinsing (2 X 5 min: double distilled water). Then, the sections were allowed to dry for 1 h.

The ultrathin sections of the Preparations 1 and 2 (with Durcupan-embedding) were stained with 2% uranylacetate solution for 15 min and for 5 min respectively with 5% lead acetate solution according to Reynolds (1963). The ultrathin sections of Prepa- ration 3 and 4 (with LR-White embedding) were treated with the same solutions, but at shorter incuba- tion periods of 3 min per 30 s(uranyl acetate/lead acetate). After staining, the sections were observed with a transmission electronmicroscope EM IOA (Zeiss) at an acceleration voltage of 80 kV and documented on planefilms (Guilleminot).

2.7. Electron spectroscopic imaging (ESI) and elec-

tron energy Loss spectroscopy (EELS)

Ultrathin sections on 700 mesh copper grids were observed directly with CEM 902 (Zeiss) without staining (Probst and Bauer, 1987; Bauer, 1988). For the ESI-analysis, images were recorded with a SIT 66 DAGE MT1 video camera and processed directly by the microscope’s digital image processing system. The images were transferred to film by a film recorder (Presentation Technologies). The distribu- tion of the elements was measured at a primary magnification of 4400 and an acceleration voltage of 80 kV, and with an aperture of the objective of 90 pm. Phosphorous and sulphur were measured at the L,,,-edge with the two-window-method: the energy windows were 10 eV and were situated for phospho- rous at 122 & 5 eV (background) and 152 + 5 eV (absorptions edge) and for sulphur at 160 * 5 and 200 + 5 eV, respectively. The images showing the distribution of the elements were produced by digital subtraction of the background.

The affinity of the primary antibody to the antigen The EELS analysis was performed with a spec- was controlled by immunodiffusion according to trometer integrated to the microscope and the sys- Ouchterlony (1958). To do this behalf, samples were tem’s program EELS 13. The measurements were taken from the cultivation at 5 different times (see performed at a magnification of 30000 X . The filter results section) and homogenized by freezing, using entrance aperture amounted 100 pm, which means a rotating knife and ultrasonication. Additionally, that the measured diameter was 368 nm. P and S

S. Adolph et al. / Journal of Biotechnology 46 (19%) 221-234 225

were recorded in the energy range 120 eV < 200 eV. The resolution was 1 eV. The background was sub- tracted from the spectrum.

3. Results

Two cultivations of Aspergillus awamori on complex medium in the stirred tank reactor were

monitored by electron microscopy: Runs 1 and 2.

The following measurements were performed: 1. Run 1: Only Preparation 1 at 37.9, 49.8 and 71.2

h.

2. Run 2: Preparations l-4 at 35.9, 51.7, 81.3, 106.9 and 131 h.

In addition, samples were taken from a cultivation on synthetic medium in a shake flask after 25 h of incubation and investigated by Preparations 3 and 4.

Fig. 1. Ultrastructure of Aspergillus awamori. (a) Young hypha with exopolysaccharide layer. Mitochondria of various shapes can be

observed in the cytoplasm. Preparation 3; 35.9 h. (b) Micelle with N = cell nucleus, M = mitcchondria, ribosomes, cytoplasmic membrane

with invaginations (CMI), golgi cisterns (arrow). Preparation 2; 57 h. (c) Cross-section through the center of the pellet, arrow shows:

intrahyphal hyphae; Preparation 1. (d) Cross section of three intrahyphal hyphae, originating from a pellet. Preparation 3; 131 .l h. (e)

Budding of a hypha through the septum, leading to the formation of intrahyphal hyphae. Preparation 1; 71.1 h.

226 S. Adolph rt (11. /Journal of Biotechnology 46 ~IY961221-234

S. Adolph et al. /Journal of Biorechnology 46 (19%) 221-234 227

Fig. 2. Ultrastructure of wheat bran and its decomposition by the fungus. (a) Light microscopy: Cross section through a wheat bran with

pellet; Ep = epidermis, Hy = hypodermis, Qz = cross cells, T = testa, Nut = nucellar tissue, Al = aleurone layer. (b) Light microscopy:

Cross section of wheat bran which surrounds a pellet. Preparation 3; 35.9 h. (c) Light microscopy: Growth of fungus (arrow) between the

inner and outer pericarp. Preparation 3; 57.2 h. (d) Light microscopy: Fungal growth in all wheat cell layers. Preparation 3; 131.1 h (e)

Longitudinal section of the wheat bran after 57.1 h incubation with A. awamori. All of the cell layers are separated from each other. Arrow:

decomposition of the intracellular matter, Al = aleuron layer, Nut = nucellar layer, T = testa, Qz = cross cells. Preparation 3 (f) Dissolution

of the seed coat. iCu/aCu = internal/external cuticle, Pi = pigment (color) layer. Arrows: outer wall of the color layer. (g) Aspergillus awamori grows along a middle lamella in the wheat bran. Preparation 1; 72.1 h. (h) Perforation (arrow) of the wall of a hypodermis cell,

which was completely filled by micelle. Preparation 4, 57.1 h.

The Preparation 1 yielded shrinkage artifacts due to the hypertonic fixing solution used. This was resolved by modification of the buffer solution. Preparation 2 lead to good preservation of the ultra- structure. Preparation 4 caused strong plasmolysis which partly destroyed the ultrastructure. Preparation 3 preserved the ultrastructure and yielded excellent immunocytochemical reactivity.

Preparation 1 and 2 on the one hand showed the

‘normal’ contrast, whereas preparation 3 on the other hand exhibited a kind of negative contrast: The cytoplasm appeared dark, but membranes were stained only lightly.

3.1. Ultrastructure of Aspergillus awamori

The diameters of the hyphae varied between 0.5 and 5 pm. Old hyphae in the center of the pellets

228 S. Adolph et al. /Journal of Biotechnology 46 ( 1996) 22 I-234

b

S. Adolph et al. / Journal of Biotechnology 46 (1996) 221-234 229

had larger diameters, while younger hyphae in the pellet periphery were smaller. The cell wall consisted of several layers and its thickness varied between 150 nm (younger hyphae) and 250 nm (older hy- phae). Young hyphae were often covered by an exopolysaccharide layer (Fig. la). The nuclei had a size of 1 to 1.5 pm; no nucleolus was observed. The mitochondria had irregular (long, elliptical, branched, constricted) shapes, with an average diameter of about 200 nm. The cell membrane exhibited invagi- nations (CM1 cytoplasmic membrane invagination, lomasome) (Fig. lb), which are easily observable in samples of preparation 2. Ribosomes, endoplasmatic reticulum (ER), Golgi-cistemae and electrodense granules (with a diameter of 200 k 50 nm) were observed in the cytoplasm of samples with Prepara- tion 2 (Fig. lb). Sulphur was enriched in these granules. Vacuoles were formed with increasing age, which often had high P- and S-content.

In the center of the pellets, strongly vacuolized or lysed cells were found. At the periphery of the pellets, plasmatic young cells with thin cell walls were observed. In the center of the pellets, intrahy- phal hyphae were found (Fig. lc, d and e).

3.2. Colonization and decomposition of wheat bran

by Aspergillus awamori

Wheat bran is a byproduct of flour production and consists of the pericarp, a seed coat and the outer layers of the endosperm. The outer pericarp consists of the epidermis and hypodermis, the inner per&up of cross cells and tube cells. The testa (seed coat) consists of three layers: the outer cuticle, the colour layer (originating from the inner integument of the immature kernel, and consisting of two layers which become compressed during ripening) and the inner cuticle. Nucellar tissue consists of compressed cells.

The innermost layer of the wheat bran consists of aleurone cells which are rich in fat and proteins (Fig. 2a; Bradbury et al., 1956).

At the beginning of the cultivation, most of tbe hyphae grew on the inner side of the wheat bran, giving rise to pellet formation (Fig. 2b). At this stage, the aleurone layer and the nucellar tissue become especially degraded by the fungus, because the seed coat seemed to form a barrier to fungal growth. In particular, no penetrations of the outer cuticle of the seed coat were observed. The hyphae of the pellets grew over the rim of the wheat bran or through mechanical damage of the seed coat into the per&up.

At first, fungal growth in the pericarp occurs in the inner pericarp (cross cells and tube cells) and in the region between the inner and outer pericarp. This region consists of layers of compressed thin-walled cells (Fig. 2~). Later on, the hyphae penetrated into the cells of the epidermis and hypodermis (Fig. 2d).

By degradation of the middle lamella, fungal hy- phae separate cells and grow into the space between them.

The electron microscopic images confirm these phenomena (Fig. 2e). This micrograph gives a view of wheat bran from the aleurone layer to the cross cells. All of the layers were separated and the inter- space was colonized with hyphae. The testa decom- posed into two layers.

The separation takes place by degradation of the outer wall of the outer (compressed) cell layer which forms the colour layer (Fig. 2f).

Sometimes, the hyphae completely filled the in- terspace along the middle lamella (Fig. 2g). Later on during cultivation, the hyphae colonize cells of the wheat bran by perforation of the cell wall. Such cells were often filled with a branched mycelium (Fig. 2h).

Fig. 3. Detection of phosphorous and sulfur in the hyphae. (a) Electron energy loss spectrum (EELS) of a vacuolar inclusion (see Fig. 3~).

The energy range of phosphorus- and sulfur- absorption had been selected. (b) Same spectrum as in Fig. 3a, after subtraction of background.

Absorption edges of phosphorous and sulphur indicate the location of these elements inside the measured structure. (cl Hyphae (oblique cut)

with two vacuoles and inclusions in them measured by ES1 at different energies. Contrast enhanced ES-image at 240 eV gives the shape of

the section. Background at 122 eV is subtracted from the phosphorous absorption at 152 eV. This gives the phosphorous concentration

distribution in the fungal cell. (d) The same hyphae measured by ES1 at different energies. Contrast enhanced ES1 at 240 eV, background at

160 eV, sulfur absorption at 200 eV. Background image subtracted from the sulfur absorption image gives the sulfur distribution in the

hyphae.. (e) A. awamori grown on synthetic medium. Phosphorous detection in a hypha with ESI. Preparation 3. (f) The same section of A.

awamori. Sulfur detection with ESI. Preparation 3.

230 S. Adolph et al. / Journal of Biotechnology 46 (IYY6I 221-234

3.3. Electron spectroscopic& imaging (ESI) and

electron-energy-loss-spectroscopy (EELS)

Vacuolar inclusions (see Fig. 3c-f) have been subjected to EELS. The crude spectrum is shown in Fig. 3a, whereas in Fig. 3b the background has been subtracted. Two absorption edges, one at 140 eV and another one at 160 eV are visible, representing the absorption caused by phosphorous and sulphur. Fig.

3c shows a longitudinally sectioned hyphae as seen by ESI. The vacuoles exhibit high concentrations of phosphorous inside them. In Fig. 3d, the high sul- phur content in these vacuoles is shown. In Fig. 3e and 3f the net distributions of phosphorous and sulphur in hyphae cultivated on synthetic medium are shown. They show the same features. EELS confirmed these results (not shown).

3.4. tmmunogold labeling

The affinity of the primary antibody was tested by immunodiffusion according to Ouchterlony (1958). Only one ring of precipitate was formed which indi-

cates good affinity (results not shown). Controls were performed by omission of the first

antibody and these yielded no label, whereas im- muno localization of xylanase in Aspergillus

awamori cultivated on synthetic medium, without induction by xylan, gave positive results (not shown).

Since xylanase was also detected in the cultiva- tion medium without induction (Siedenberg, 19961, there seems to be some constitutive enzyme produc- tion.

On account of the labeling results, specific im- muno labelling of the enzyme could be assumed:

Fig. 4a shows a layer of cell wall material of the pericarp which has been colonized by fungal hyphae at only one side. Only on this side, the enzyme

detection by immunogold labeling positive. The de- tected enzyme concentration within the wheat cell wall rapidly decreases. Fig. 4b shows several cross- sectioned fungal cells, growing inside the seed coat. The hyphae separate the seed coat by growing inside the outer layer of the colour layer. This layer was found to be crucial for the degradation of the seed coat, as described above in the section on coloniza- tion. The immunogold assay indicates considerable

enzyme activity near the fungal hyphae and on the surrounding wall material. In particular, filamentous matter was labelled inside the formed cavity. Obvi-

ously, degradation of the seed coat was going on here when fixation occurred. Later on during cultiva- tion, the wheat cell walls become partly fragmented. Fig. 4c shows the particularly high enzyme activity detected on the surface of a fragment of the sec- ondary wall.

In general, enzyme activity was found only on the wheat cell walls in the immediate vicinity of the fungal hyphae. No enzyme activity was detected elsewhere. These results indicate that the immuno- gold assay leads to specific detection of xylanase.

3.5. Variation of the local distribution of xylanase

during the cultivation

Xylanase was detected in three different locations: on the wheat bran, in the cell wall of the hyphae and in the cytoplasm of the fungal cells, including the cell membrane (see Fig. 4e). Fig. 4d serves as an overview, showing a crack in the outer pericarp of the wheat bran. At higher magnification (Fig. 4e), the cytoplasm and cell wall (lighter contrast) of the hyphae exhibit high enzyme concentrations (white

arrows). On the wheat bran fragment, a high local enzyme concentration can be seen (black arrow head,

Fig. 4e).

Fig. 4. Immtmogold labeling of xylanase. (a) Wheat cell walls are labeled by immunogold only at the side of hyphal growth (arrow heads).

Wall matter without contact to ftmgal cells shows no immunolabel (asterisk). Preparation 3. (b) Several hyphae degrading the seed coat of

wheat bran. Immunogold labeling of the substrate (arrows) occurs only nearby the mycelium. Filamentous, dark matter surrounding fungal

cells shows strong label while all other parts of the seed coat remain unlabeled. Preparation 3; 13 1, I h. (c) Strongly immunogold labeled

fragments of partly decomposed wheat cell walls. Preparation 3; 13 1. I h. (d) View of a long section of wheat bran. A dark contrasted hypha

grows through a crack in the epidermis. The arrow indicates the part shown at higher magnification in Fig. 4e. Ep = epidermis.

Hy = hypodermis. Preparation 3; 35.9 h. (e) Enlarged part of Fig. 4d. Immunogold labelled xylanase within the cytoplasm (white arrows),

bound to the fungal cell wall (low contrasted) and on the surface of the substrate (black arrow head). Preparation 3; 35.9 h. (f) Immunogold

labelled xylanase after 57.1 h. Labelling intensity of intracellular xylanase has declined. Only a few gold particles are visible on cytoplasm

(black arrows). The immunolabel of the fungal cell wall remains unchanged, compared with 35.9 h (Fig. 4e) Vesicle (Ve). Preparation 3.

S. Adolph et al./Joumal of Biotechnology 46 (1996) 221-234 231

During the first phase of the cultivation, the de- tected xylanase activity both in the cytoplasm and the cell wall of the hyphae is high (Fig. 4e). With

increasing cultivation time, there is a decline in the concentration of xylanase detected in the cytoplasm, whereas the labeling of fungal cell walls remains

b

232 S. Adolph rt al. /Journal of‘ Biotechnology 46 ~19961221-234

Cultivation time [ h ]

Fig. 5. Process monitoring the concentrations of carbon dioxide in

the off-gas and of xylanase in the cultivation medium filtrate are

shown as a function of the cultivation time.

nearly unchanged (Fig. 4f). Obviously, the enzyme is secreted into the cultivation medium and diluted. However, with increasing time, its concentration be- comes higher as the increase in substrate labeling

indicates.

3.6. Process monitoring of xylanase and phospho-

rous

The concentration of Xylanase I in the cultivation medium as a function of time measured by process monitoring techniques (Siedenberg, 1996) is shown in Fig. 5. Carbon dioxide concentration is also shown in the same figure. Xylanase is detected later by process monitoring than by immuno labelling. Car- bon dioxide in the off-gas served as an indicator of the physiological activity of the microorganism.

4. Discussion

4.1. Fixation of the biological material

For the investigation of any particular biological material, the known techniques must be carefully adapted. In the present investigation, four different preparations were performed simultaneously. On ac- count of the slow diffusion of glutaraldehyde into cytoplasm during Preparation 1, it became homoge- neous and the preservation of ultrastructure was un- satisfactory. In Preparation 2, a combined mixture of glutaraldehyde and formaldehyde was used for the

fixation. Here, formaldehyde produced a quick pre- fixation and glutaraldehyde a slow postfixation, which lead to a good preservation of the ultrastruc- ture. With Preparation 4, good immunogold labelling

of the enzyme was achieved, but the structure of the biological material was partly destroyed by plasmol- ysis. With Preparation 3, a satisfactory immunologi- cal reactivity and good structure preservation were obtained.

4.2. Ultrastructure of Aspergillus awamori

The present ultrastructural investigation on As-

pergillus awamori confirmed the results of Tanaka and Yanagita (1963), McClure et al. ( 1968) and Grove and Bracker (1970) with Aspergillus niger.

Nearly all of the structures and their dimensions in Aspergillus niger agree with those of Aspergillus

awamori, except those for the diameters of the hy- phae inside the wheat bran, which were smaller than those found by Tanaka and Yanagita (1963). Such thin hyphae appeared only inside the wheat bran. This may be due to adaptation of the fungus to the spatial conditions (Fig. 4b). Of interest is the appear- ance of intrahyphal hyphae, often called ‘internal branching’ (Trinci, 1978). Young hyphae grew through the septum of neighboring dead hyphae into those cells (Fig. Id and e). This phenomenon was observed in pellets, but not in fungus grown in wheat bran. With increasing age, both the number and the size of vacuoles increased, thereby leading to the formation of intrahyphal hyphae. Therefore, internal

branching often appeared in aging cultures. Intrahy- phal hyphae can be caused by various effects such as shear or osmotic shock (Belozerskaya and Potapova, 1993). It has been observed in several fungi (Neuro-

spora crassa, Collinge and Trinci, 1974; Belozer- skaya and Potapova, 1993; Aspergillus nidulans,

Fiddy and Trinci, 1976; Coprinus lagopus, Buller, 1958). Miller and Anderson (1961) reported that internal branching of Aspergillus niger occurs under nearly anaerobic conditions. Presumably, the appear- ance of intrahyphal hyphae in the corpus of pellets may be caused by the combination of aging of hyphal cells and the lack of oxygen. On the other hand, no intrahyphal hyphae were found in wheat bran, where oxygen might be expected to be limited as well. A major difference between the hyphae

S. Adolph et al. / Journal of Biotechnology 46 (19%) 221-234 233

inside the bran and those in free pellets is the obvious lack of any influence shear inside of bran. Perhaps, internal branching leads to a selection ad- vantage in free pellets because of protection of hy- phal tips, but is obsolete in bran-protected hyphae.

Plasmarich young cells were becoming gradually thinner at the end of the cultivation.

4.3. Decomposition of wheat bran by Aspergillus

awamori

Aspergillus awamori attacks the wheat bran at locations with the lowest resistance, beginning in the endosperm. When the mycelium reaches the peri- carp, compressed cell layers between the internal and external pericarp seem to be the easiest to dissolve.

The hyphae separate wheat bran cells along the middle lamellae where the intercellular material mainly consists of protopectin. The decomposition of the seed coat occurs along a pectin-rich layer (Brad- bury et al., 1956), the outer wall of the outer cell layer of the colour layer.

The outer cuticle of the seed coat seems to be impenetrable. The pericarp is therefore only accessi- ble from its edges, as long as no mechanical damage of the testa occurs. As a matter of fact, parts of the substrate remain unused over a considerable period. The largest part of the mycelial growth takes place outside the wheat bran. The fungus here uses the enzymatically decomposed and dissolved cell frag- ments.

4.4. Measurements with ESI and EELS

Both these techniques take advantage of the effect of inelastic scattering of beam electrons by elements of the sample, which leads to a characteristic energy loss.

ES1 and EELS measurements indicate that phos- phorous and sulphur are often accumulated in the vacuoles of the fungus as inclusion bodies. Similar granules has been found in other microorganisms as well (Kualaev and Vagabov, 1983). Obviously, they seem to act as storage for these elements.

4.5. Immunogold labeling of xylanase

These investigations indicate that xylanase is

formed in the cytoplasm, transported through the cell membrane and then bound to the cell wall. It is then

released into the medium and adsorbed on the sub- strate surface. Its first appearance in the medium was detected after 36 h cultivation time. This could be due to the start of the autolysis of cells.

The localization of xylanase on the substrate sur- face indicates that it does not penetrate the wheat

cell walls. Thus the substrate decomposition occurs mainly on the substrate surface. The evenly labeled fungal cell wall may be explained with the binding

of the exoenzyme to this cell wall. Reese et al. (1973) found high l3-xylosidase activity in As-

pergillus niger and this was bound to the mycelium and could be detected in the medium after a time lag. According to Biely (19851, the localization of xy- lanolytic enzymes in the cell wall of microorganisms

offers a selection advantage, because of the higher local enzyme concentration. Other exoenzymes, such as cellulase in Trichoderma reesei @prey, 1988) and ligninase in Phanerochaete chrysosporium (Srebot- nik et al., 1988) are also bound to the cell wall, as proved with the immunogold labeling as well.

4.6. Comparison of the results with process monitor- ing (Siedenberg, 1996)

The increase of xylanase concentration in the cytoplasm during the exponential growth phase runs parallel to the strong rise of the CO, production rate. The decrease of xylanase activity in the cytoplasm during the ongoing cultivation is accompanied by its increase in the medium and on the substrate surface. Xylanase is detected later by process monitoring techniques than by immuno labeling. This might be explained by a time lag between enzyme production and its release into the medium.

The decrease in the phosphate concentration in the medium is congruent with the rising intracellular phosphate content and with the enrichment of phos- phorous in the cell vacuoles.

The enzymatic decomposition of the wheat bran in the stirred tank cultures is accompanied by an increase of its decomposition products in the medium which are then gradually consumed by the fungus.

5. Conclusions

Based on these investigations the following con- clusions can be drawn:

234 S. Adolph et al. /Journal qf Biotechno1og.v 46 f I9961 22 I-234

Beginning in the endosperm, the fungus grows inside the bran. At this location, possibly because of

the lower shear, formation of internal pellets is fa- vored (Fig. Zb). The barrier formed by the outer cuticle of the seed coat can only be evaded by the fungus by growing through mechanical damage or via the edge of the bran. In the pericarp, the inner layers show less resistance to fungal colonization

than the outer layers, which are more tightly com- pressed and thicker-walled compared to the inner ones. In particular, the layer between the inner and the outer pericarp can be easily colonized. This leads to earlier utilization of the inner pericarp and a later colonization of the outer pericarp. Two general fea- tures can be distinguished in the decomposition of the substrate: Dissolution of pectin-containing struc- tures and growth into this space, which occurs first, and dissolution of wheat cells by formation of perfo- rating structures and filling the wheat cells with dense mycelium, which happens later. Defects of the seed coat and the dissolution of the pectin-containing structures and penetration into this space seem to accelerate the process of decomposition.

During the early cultivation phase the xylanase

concentration in the cytoplasm and cell wall of the fungus attains high values. During the late cultiva-

tion phase, its intracellular concentration declines whereas it is on the increase in the cultivation medium and on the surface of the substrate.

The formation of phosphorous (and sulphurl gran- ules in the vacuoles is congruent with the increase in intracellular phosphate concentration.

References

Bauer, R. (1988) Electron spectroscopic imaging: an advanced

technique for imaging and analysis in transmission electron

microscopy. Meth. Microbial. 20. I 13- 146.

Belozerskaya, T.A. and Potapova, T.V. (1993) Intrahyphal com-

munication in segmented mycelium. Exp. Mycol. 17, 157- 169.

Biely, P. (1985) Microbial xylanolytic systems, Trends Biotech-

nol. 3(1 l), 286-290.

Bradbury, D.. MacMasters, M.M. and Cull, I.M. (1956) Structure

of the mature wheat kernel. II. Microscopic structure of peri-

carp, seed coat and other coverings of the endosperm and

germ of hard red winter wheat. Cereal Chem. 33, 342-361:

(1956b) III. Microscopic structure of endosperm of hard win-

ter wheat. Cereal Chem. 33, 367-373.

Buller, A.H.R. (1958) Further Observations on the Coprini To-

gether With Some Investigations on Social Organisation and

Sex in Hymenomycetes, Researches in Fungi, Vol. 4. Hafner

Publishing Co., New York.

Fiddy, C. and Trinci, A.P.J. (1976) Mitosis, septation and branch-

ing in the dublication cycle in Aspergillus niduluns. J. Gen.

Microbial. 97, 169-184.

Collinge, A.J. and Trinci, A.P.J. (1974) Hyphal tips of wild-type

and spreading colonial mutants of Neurospora crassa. Arch.

Mikrobiol. 99, 358-368.

Grove, S.N. and Bracker, C.E. (1970) Protoplasmatic organisation

of hyphal tips among fungi: Vesicles and Spitzenkorper. J.

Bacterial. 104(2), 989- 1009.

Jager, K.M. and Bergman, B. (1991) Localization of a multifunc-

tional chaperonin (GroEL protein) in nitrogen fixing An-

abaena PCC 7120. Presence in vegetative cells and hetero-

cysts. Planta 183, 120-125.

Kualaev, IS. and Vagabov, M. (1983) Polyphosphatemetabolisms

in microorganisms. Adv. Microbial. Physiol. 24, 83-158.

McClure, W.K., Park, D. and Robinson, P.M. (1968) Apical

organisation in the somatic hyphae of fungi. J. Gen. Microbial.

50. 177-182.

Miller. C.V. and Anderson, N.A. (1961) Proliferation of

conidophores and intrahyphal hyphae in Aspergillus n&r. Mycologia 57, 433-436.

Probst. W. and Bauer, R. (1987) Technique and biological appli-

cation of the electron spectroscopic imaging (ESI) method and

the electron energy-loss spectroscopy (EELS). Verh. Deutsch.

2001. Ges. 80, 119-128

Ouchterlony, 6. (1958) Diffusion-in-gel methods for immunologi-

cal analysis, In: Kallos, P. (Ed.), Progress in Allergy, Vol. V.

Karger, Basel, pp. I-78.

Reese, E.T., Maguire, A. and Parrish, F.W., (1973) Production of

8.D-xylopyranosidases by fungi. Can. J. Microbial. 19, 1065-

1074.

Reynolds, E.S. (1963) The use of lead citrate at high pH as an

electron-opaque stain in electron microscopy. J. Cell Biol. 17,

‘08-2 12.

Roth. J. (1982) The protein A-gold (pAg) technique-a qualitative

and quantitative approach for antigen localization on thin

sections, In: Bullock, G.R. and Petrusz, P. (Eds.) Techniques

in Immunocytochemistry, Vol. I. Academic Press, London.

pp. 107-133

Siedenberg, D. (1996) Dissertation, University of Hannover, Han-

“over. Germany.

Sprey, B. (1988) Cellular and extracellular localization of endocel- iulase in Trichodemu reesei. FEMS Microbial. Lett. 55.

283-294.

Srebotnik, E., Messner, K. and Foisner, R. (1988) Ultrastructural localization of ligninase of Phanerochaete chyosporium by

immunogold labeling. Current Microbial. 16, 221-227.

Tanaka, K. and Yanagita, T. (1963) Electron microscopy on

ultrathin sections of Aspergillus niger. J. Gen. Appl. Micro-

mol. 9(l), 101-I 18.

Trinci. A.P.J. (1978) The dublication cycle and vegetative devel-

opment in moulds. In: Smith, J.E. and Berry, D.R. (Eds.), The

filamentous fungi, Vol. 3. Edward Arnold, pp. 133-163.