Scanning electron microscopy of immobilizedHumicola lutea during semicontinuous production of acid proteinases

The morphology of the fungusHumicola lutea (strain 120–5), immobilized in polyacrylamide and polyhydroxyethylmethacrylate and used for the semicontinuous production of acid proteinases, was examined by scanning electron microscopy. The fungus developed a dense mycelium below the bead surface as well as in the bead interior after precultivation of entrapped spores. During maximal semicontinuous enzyme biosynthesis, formation of numerous large bulbous cells with a different shape was observed. Lysis of the cells was observed mainly in the centre of the gel beads after 13 successive fermentations with polyacrylamide-immobilized cells or after 21 re-uses of polyhydroxyethylmethacrylate-immobilized mycelia, respectively. Growth and changes in the cellular morphology of immobilizedH. lutea, accompanying biosynthesis of acid proteinases, were comparable in both gel matrices but mycelia immobilized in polyhydroxyethylmethacrylate maintained their productivity twice as long.     

Use of a Novel Sporulation Monitor to Quantify the EVects of Formulation and Storage on Conidiation by Dried Mycelia of the Entomopathogenic Fungus Zoophthora radicans

A novel piece of equipment, the sporulation monitor, is described for the comparison of conidia production from mycelia receiving diVerent formulation and storage treatments. This equipment was used to compare the viability of Zoophthora radicans mycelial samples treated either with 10% maltose solution or with distilled water before drying and storage for 0-12 weeks at 4oC. Freshly dried maltose-treated mycelial mat samples produced significantly more conidia for a significantly longer time than distilled water-treated mat samples of the same age. Very few conidia were produced from mats in either treatment after storage for 4 weeks or longer. There were great diVerences in conidia production from mycelial mat samples produced in diVerent fermenter runs. These results are discussed in relation to the potential for the use of dried mycelia in biological control programmes.     

Morphology of Penicillium chrysogenum strains immobilized in calcium alginate beads and used in penicillin fermentation

Summary The morphology of two strains of Penicillium chrysogenum immobilized in calcium alginate and used in penicillin fermentation was examined. The degree and distribution of mycelial growth inside and on the surface of the beads depended on the strain, the cultivation media and the fermentation time. P. chrysogenum ATCC 12690 developed as a mycelial network inside the beads. The growth tendency of P. chrysogenum S₁ in micropellets was directed to the outer surface of the beads. At the end of the production phase only a trace of mycelia and no micropellets in the center of alginate beads were observed, while the outer surface and the subsurface were completely covered with mycelia.     

Preparation and Regeneration of Mycelial Protoplasts of Alternaria eichhomiae

Preparation and regeneration of mycelial protoplasts from Alternaria eichhorniae were examined. A commercially available muralytic enzyme, Novozym 234, was used for isolation of protoplasts. The mycelial age and the pH of the stabilized buffer affected the formation of protoplasts. The maximum production of protoplasts (3,9 × 10⁸/g fresh weight mycelia) was obtained from 24-h-old mycelia digested with Novozym 234 (20 mg/ml) in a stabilized buffer of pH 6.4 and incubated in the dark at 30°C on a rotary shaker (90 r.p.m.) for 6 h. Morphological characteristics of the protoplasts varied and depended on the age of the mycelia used in protoplast production. Moreover, mycelial age had a highly significant influence (P = 0.0001) on the frequency of protoplast regeneration.     

Controlled mycelial growth for kojic acid production using Ca-alginate-immobilized fungal cells

Summary Conidia of Aspergillus oryzae were immobilized in Ca-alginate beads and then incubated in a nutrient medium to yield an immobilized biocatalyst producing kojic acid. The immobilized cell cultures produced kojic acid linearly during cultivation. Regardless of the size of the immobilized particles, there existed an optimal nitrogen concentration for the maximum production rate of kojic acid, at which smaller bead sizes resulted in a higher production rate. When the growth of mycelia were confined within the bead surface and segregated from each other by gel material, they produced kojic acid with maximal catalytic activity and exhibited the highest conversion yield of glucose. The extent of mycelial segregation was especially higher in cultures of smaller bead particles, and the depth of mycelial growth was 150 to 250 m from the gel bead surface in all cultures of different nitrogen concentrations and bead sizes. Therefore, for the maximum expression of catalytic activities of immobilized mycelial cultures, it was found very critical to optimally control the mycelial distribution in gel beads by the culture conditions affecting mycelial growth.     

Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

Lipase production and Penicillium simplicissimum morphology in solid-state and submerged fermentations

A comparative study of Penicillium simplicissimum morphology and lipase production was performed using solid-state (SSF) and submerged (SmF) fermentation. SSF was carried out on babassu cake as culture medium and SmF on a semi-synthetic medium and a medium based on suspended babassu cake grains. Yield of product on biomass, specific activity and conidia production were 3.3-, 1.3- and 2-fold higher in SSF. In SmF, the type of fungus growth differed according to the medium. Using the semi-synthetic medium, the fungus formed densely interwoven mycelial masses without conidia production, whereas using the babassu-based medium the fungus formed free mycelia and adhered to the surfaces of the grains, producing conidia. The results show that babassu cake induces conidiation in SmF. In SSF, the fungus not only grew on the surface of the grains, producing conidia abundantly, but also effectively colonized and penetrated the babassu particles. The high conidia production and lipase productivity in SSF may be related to the low availability of nutrients or to other stimuli associated with this type of fermentation. Thus, the high production of the thermostable P. simplicissimum lipase, using a non-supplemented, low-cost agro-industrial residue as the culture medium, demonstrates the biotechnological potential of SSF for the production of industrial enzymes.     

A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130±102 and 1200±97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35% of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels.     

Production of glucoamylase using Aspergillus phoenicus immobilized in calcium alginate beads

Summary As a means of better exploiting the growth-dissociated nature of glucoamylase synthesis, a production process in which the growth phase was separated from the enzyme synthesis phase has been developed. Immobilized mycelia arising from a 6-day-old culture of conidia immobilized in calcium alginate beads could be subsequently used repeatedly to produce glucoamylase in a second step using a Dextran T-10 medium. Glucoamylase production was sustained over five sequential batches in a 19-day period and immobilized mycelia remained confined to the subsurface of the beads. Offprint requests to: C. Kuek     

Developmental control of glucosamine and galactosamine levels during conidation in Neurospora crassa.

The glucosamine and galactosamine content of mycelia was measured in cultures of Neurospora crassa grown on the surface of dialysis membranes. The glucosamine content was relatively constant throughout the different regions of the mycelial mat. The galactosamine content, however, was always lower in the growing-front region of the mycelial mat than in the older regions. At most, only low levels of galactosamine were necessary for the formation of hyphae at the growing front of a mycelial mat. Thus, galactosamine-containing polymers cannot be a major shape-determining component of the cell walls of these hyphae in Neurospora. The effect of conidiation on the amino sugar content was determined by using the bd (band) strain of N. crassa. When grown on the surface of dialysis membranes, this strain rhythmically produced regions of conidiating and non-conidiating growth. With this strain, it was concluded that conidiation did not affect the amino sugar levels. Since conidia that contained only very low levels of galactosamine were produced from regions of the mycelial mat that contained much higher levels of this amino sugar, there must be some mechanism of spatial differentiation that prevented the accumulation of galactosamine-containing polymers in conidia.     

Diffusion of lactose in kappa-carrageenan/locust bean gum gel beads with or without entrapped growing lactic acid bacteria

Effective diffusion coefficients (De) of lactose in kappa-carrageenan (2.75% wt/wt)/locust bean gum (0.25% wt/wt) (LBG) gel beads (1.5-2.0-mm diameter)with or without entrapped lactic acid bacteria (LAB) were determined at 40 degrees C. The effects of lactose concentration, bacteria strain (Streptococcus salivarius subsp. thermophilus and Lactobacillus casei subsp. casei) and cell content at various steps of the fermentation process (after immobilization, pre-incubation of the beads and successive fermentations) were measured on De as a first step for process modelling. Results were obtained from transiend concentration changes n well-stirred lactose solutions in which the beads were suspended. A mathematical model of unsteady-state diffusion in a sphere was used, and De was obtained from the best fit of the experimental data. Diffusivity of lactose in cell-tree beads was significantly lower than in pure water mainly because of the obstruction effect of the polymer chains and the hydration region. Furthermore, effective diffusivity and equilibrium partition factor were independent of lactose concentration in the range from 12.5 to 50 g/L. No significant difference was found for De (effective diffusivity) and Kp (partition) coefficients between beads entrapping S. thermophilus (approximately 5 x 10(9) CFU/mL) and cell-free beads. On the other hand higher cell counts obtained with L. casei (close to 1.8 x 10(11) CFU/mL) increased mass transfer resistance resulting in lower effective diffusivities and Kp. Finally, the effects of the type of bacteria and their distribution in the beads on the diffusivity were also discussed.     

Assessment of biofilm formation by Scedosporium apiospermum,S. aurantiacum,S. minutisporum and Lomentospora prolificans

Reported herein is the ability of Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans conidia to adhere, differentiate into hyphae and form biofilms on both polystyrene and lung epithelial cells. To different degrees, all of the fungi adhered to polystyrene after 4 h, with a predominance of those with germinated conidia. Prolonged fungi–polystyrene contact resulted in the formation of a monolayer of intertwined mycelia, which was identified as a typical biofilm structure due to the presence of a viable mycelial biomass, extracellular matrix and enhanced antifungal resistance. Ultrastructural details were revealed by SEM and CLSM, showing the dense compaction of the mycelial biomass and the presence of channels within the organized biofilm. A similar biofilm structure was observed following the co-culture of each fungus with A549 cells, revealing a mycelial trap covering all of the lung epithelial monolayer. Collectively, these results highlight the potential for biofilm formation by these clinically relevant fungal pathogens.     

Immobilization of Trametes versicolor cultures for improving laccase production in bubble column reactor intensified by sonication

The mycelia of Trametes versicolor immobilized in alginate beads provided higher laccase production than that in pelleted form. An efficient ultrasonic treatment enhanced laccase production from the immobilized T. versicolor cultures. The optimized treatment process consisted of exposing 36-h-old bead cultures to 7-min ultrasonic treatments twice with a 12-h interval using a fixed ultrasonic power and frequency (120 W, 40 kHz). Using the intensification strategy with sonication, laccase production increased by more than 2.1-fold greater than the untreated control in both flasks and bubble column reactors. The enhancement of laccase production by ultrasonic treatment is related to the improved mass transfer of nutrients and product between the liquid medium and the gel matrix. These results provide a basis for the large-scale and highly-efficient production of laccase using sonobioreactors.     

Production of acid proteinases byHumicola lutea immobilized in polyacrylamide gel

Summary Fungal spores ofHumicola lutea 120–5 were entrapped in 5% polyacrylamide gel and were cultivated for 44–48 h to form a mycelial network inside the beads. A dense mycelial growth also occurred on the surface of the beads. It was possible to reuse the immobilized mycelium for production of acid proteinases in 12 different batches without loss of mechanical stability. The inoculum size should be controlled prior to its transfer into fresh production medium. Maximal enzyme production exceeding the level of free cell fermentation was registered in the fourth to seventh cycles. According to the size of the inoculum, half of the initial production rate was reached after 7–14 batches.     

Temperature-dependent actinomycin D effect on RNA synthesis during synchronous development in Neurospora crassa

Actinomycin D at a concentration of 5 g/ml of medium inhibited DNA-dependent RNA synthesis by 92% at 35 C, 42% at 30 C, and 28% at 25 C in Neurospora crassa. This concentration also inhibited the development of conidiophores and conidia at 35 C, but not at 30 or 25 C. Mycelia which were induced to synchronous development formed conidiophores in 2.5 hr and conidia in 4.5 hr at 35 C in the absence of drug additives. Addition of actinomycin D to synchronously developing mycelia at zero time and at 0.5-hr intervals thereafter at 35 C indicated that RNA synthesis required for conidiophores occurred before 0.5 hr and for conidia before 2 hr. Addition of cycloheximide at the same times to another synchronous mycelial series at 35 C indicated that protein synthesis required for conidiophores occurred before 2 hr and for conidia before 3.5–4 hr.This work was supported in part by U.S. Public Health Service Training Grant 1 TO1 GM 01968 01.     

Involvement of a conidial endoglucanase and a plasma-membrane-bound beta-glucosidase in the induction of endoglucanase synthesis by cellulose in Trichoderma reesei

The induction of endo-1,4-beta-glucanase synthesis by Trichoderma reesei QM 9414 was investigated in conidia, mycelia and protoplasts. Cellulose induced endoglucanase synthesis only in conidia, but not in glucose-grown mycelia or protoplasts. Cellooligosaccharides and sophorose induced endoglucanase synthesis in mycelia, conidia and protoplasts. Only conidia exhibited detectable basal endoglucanase levels, whereas beta-glucosidase activity was found in conidia, mycelia and protoplasts. The beta-glucosidase was inhibited in vitro by nojirimycin and glucono-delta-lactone. Addition of either of these inhibitors to the induction medium blocked de noro synthesis of endo-1,4-beta-glucanase with cellulose (conidia) or cellooligosaccharides (protoplasts and mycelia) as inducer, whereas induction by sophorose remained unaffected. The results are consistent with the assumption that basal constitutive levels of endoglucanase and beta-glucosidase are involved in the induction of cellulase synthesis by cellulose in T. reesei.     

Characterization of a cobalt-resistant mutant of Neurospora crassa with transport block

A cobalt-resistant strain of Neurospora crassa (cor) was obtained by repeated subculturing of the wild type on cobalt-containing agar medium. N. crassa cor is twentyfold more resistant to cobalt ions compared with the wild type. Resistance was stable on repeated subculturing of cor on cobalt-free media. N. crassa cor is also cross-resistant to nickel (fourfold), but not to zinc or copper. Higher concentrations of iron and magnesium ions are required to reverse growth inhibition due to cobalt toxicity in N. crassa cor, compared with the wild type. Germinating conidia and mycelia of the cor strain accumulated lower levels of cobalt ions compared with the parent N. crassa. The partial transport block for cobalt uptake is shown to be primarily due to decreased surface binding of cobalt to mycelia and cell walls. Efflux of mycelial cobalt was also observed in wild type and cobalt-resistant N. crassa. The characteristics of cor in comparison with wild type N. crassa are discussed in relation to the mechanisms of cobalt resistance.     

Scanning electron microscopic examination of calcium alginate beads immobilizing growing mycelia of Aspergillus phoenicus

Calcium alginate beads inoculated with conidia of Aspergillus phoenicus have been incubated in various culture vessels for 120 h. Scanning electron microscopy revealed that the degree of agitation was a factor in surface stability of the beads. Highly significant was the successful restriction of mycelial growth to the subsurface, a condition required if the full advantages of immobilized fungi are to be realized.     

Factors Affecting the Antigenicity of Trichophyton rubrum

Nitrogen determinations, performed upon the mycelia of Trichophyton rubrum, indicated that both the total nitrogen to mycelial weight ratio and the protein nitrogen to mycelial weight ratio decreased as the age of the mycelia increased. An increase in nitrogen concentration in the medium produced an increase in the total nitrogen to mycelia weight ratio, but did not necessarily increase the protein nitrogen to mycelial weight ratio. The optimal nitrogen source concentration which produced the highest protein nitrogen to mycelium ratio was found to be considerably less than that recommended in most standard Sabouraud medium formulations. Antisera to antigen preparations, grown on low concentrations of Multipeptone, produced more lines in the gel diffusion reaction than did antisera to antigens grown on standard concentrations of Multipeptone. Antisera to antigenic preparations from 2-week-old mycelia exhibited better and sometimes more lines than those of antigens prepared from 1- or 3-week-old mycelia, regardless of the nitrogen concentration in the medium. Dialysis and storage of the antigen produced no change in the quality of the precipitin lines, even though both processes involved considerable loss of Lowry protein. Immunofluorescence studies showed that young mycelia were more antigenic than the old mycelia, since a substantial degree of cell wall fluorescence was exhibited by the young mycelia, especially at the hyphal tips. Older mycelia lacked this fluorescence. An extracellular antigen was also found to be associated with the young mycelia, but cytoplasmic fluorescence was not observed.     

Confining mycelial growth to porous microbeads: a novel technique to alter the morphology of non-newtonian mycelial cultures

In an effort to alter the filamentous morphology of Penicillium chrysogenum cells, a technique was developed to confine the growth of the mycelia to porous celite beads. The pore matrix of these beads was found to be very effective for entrapping mycelial cells and spores. The entrapped spores were used to initiate the fermentations in shake flask cultures. Significant increases in final cell densities were obtained in the confined cell cultures reaching up to 60 g/L cells. This is nearly double the cell concentration attainable in free cell cultures grown in the absence of beads. Cell loadings up to 0.55 g cells per bead were obtained in the confined cell cultures. In the later stages of the fermentations, the specific oxygen uptake rates in the confined cell cultures were found to decrease with respect to free cell cultures.