Oszillierende-fed-batch-Technik bei der Cellulasegewinnung

From different fungal genera some mutant strains were selected which in cellulase formation exhibit reduced catabolite-repression related to several soluble carbon sources. The cellulase production by these mutant strains on the basis of soluble carbon sources is possible if a fed-batch-technique is used for feeding the substrate. The optimum procedure is an oscillating-fed-batch-technique (OFB) with feed-back-controlled intermittent addition of the substrate. The substrate concentration in the medium is adjusted in such a manner to realize oscillations between a limitation for mycelium growth and non-limiting conditions under a substrate concentration which is yet below the repressing concentration for cellulase formation. A suitable feed-back-parameter for the control of substrate addition is the specific CO₂-formation of the mycelium. Using OFB the growth in cellulase activity is nearly proportional to the feeded substrate whereas the growth in mycelium is reduced. The productivity of cellulase formation is enhanced in comparison to continuous substrate addition as well as under limiting and non-limiting conditions for growth of mycelium.     

Colony development in Streptomyces carpinensis: a streptomycete with substrate mycelium spores

In this paper we present an ultrastructural study of spore formation in aerial vs. substrate mycelia of Streptomyces carpinensis. Both mycelia initiated spore formation at nearly the same time of colony development but exhibited different patterns of spatial localization of sporulation: spore formation took place throughout the aerial mycelium whereas in the substrate mycelium was confined to a narrow zone at the bottom of the colony. The ultrastructural changes leading to spore formation, however, were quite similar in both mycelia, differing only with respect to the outer components of the sporal wall. Spores formed in the aerial mycelium were covered by a thin sheath whereas the spores formed in the substrate mycelium were covered by an amorphous electron-dense material.     

Changes in ribosomal proteins during colony development in Streptomyces.

The structure and functionality of the ribosomal subunits of the substrate and the aerial mycelium of Streptomyces antibioticus were compared. Using SDS-PAGE and HPLC, several differences between the ribosomal protein pattern from both stages of development were observed, including a clear decrease in the L7/L12 content of the aerial mycelium. The activity of the aerial mycelia ribosomes was also decreased when compared with that of the substrate mycelium. This effect was more pronounced in the 50S subunit. These results suggest that during cell differentiation in Streptomyces important changes occur at the ribosomal level, particularly in the transition from the substrate to the aerial mycelium.     

A model for the formation of ring-shaped structures in colonies of mycelial fungi

Mathematical model of the development of the pattern of colonies is considered. The model represents the systems of differential equations of the first order. It includes non-dimensional parameters characterizing the following features: concentration of substrate, concentration of metabolic products--growth inhibitor, mycelium and spores, radial and specific rate of mycelium growth, rate of substrate consumption and production of metabolic products, coefficients of diffusion of substrate and metabolic products, initial concentration of mycelium and substrate, time of delay of mycelium reaction on metabolic products and spore formation, threshold concentration of metabolic products. The model is adequate to the experiments with cultivation of Penicillium chrysogenum. It was shown that necessary condition for the formation of the circle periodical structures (zoning) in the colonies is an ability for the production of growth inhibitors (antibiotics, etc.). It was proved that formation of colonies of "continuous lawn" type is caused by restrictions on growth because of mycelium satiation or exhaustion of substrate. Such growth scenario is realized in experiments either on reach substrate or on hungry agar. For the appearance of regulating of "zone structure" type limitation on critical level of metabolic product concentration is very important. The number of periodical zone structures and their widths are determined by the above parameters.     

Study of the structure of colonies of active and inactive Actinomyces parvullus variants using luminescence and scanning microscopy]

The colony structure of the active and inactive proactinomycete-like variants of Actinomyces parvullus producing actinomycin D was studied with luminescent and scanning microscopy. Clear differentiation of the colony profile was shown by the structure and functions of the mycelium layers. A zone of active synthesis and accumulation of the antibiotic was observed in the colonies of the active variant in the upper part of the substrate mycelium with reddish-yellow self luminescence in UV light and characteristic close hyphae "cemented" by the intracellular substance. Formations of the granule type were often noted on the hyphae of that layer. The layer of the aerial mycelium was loosely connected with the substrate mycelium and consisted of sporophores and spore chains partially broken into single spores. The colonies of the inactive proactinomycete-like variant had a slightly differentiated profile with a sponge-like structure, no zones of the antibiotic synthesis being found. The presence of the intracellular substance was observed in the upper part of the colony supersubstrate mycelium.     

Effect of polyploidogenic factors on Trichothecium roseum mycelium in the process of trichothecine and fibrinolytic enzyme biosynthesis

The effect of colchicine and boric acid on the substrate mycelium of Trichothecium roseum was studied in the course of trichothecin biosynthesis. Addition of boric aicd (0.01%) and colchicine (0.1%) to the medium for biosynthesis increased the antibiotic activity of the fungus, this being due to the specific effect of polyploidogenous factors on growth of the mycelium and the proportion of nuclei in it. The number of nuclei increased in cells of the substrate mycelium correlating with a higher antibiotic activity.     

New species, Actinomadura fulvescens sp. nov. and Actinomadura turkmeniaca sp. nov. and their antagonistic properties

Two new species of Actinomadura isolated from soil samples of the Turkmen SSR, i.e. Actinomadura fulvescens sp. nov. and Actinomadura turkmeniaca sp. nov. are described. The first species is characterized by formation of short (1-2 turns) spiral spore chains, smooth spores, scanty white aerial mycelium, colourless or yellowish substrate mycelium on synthetic media and brownish-yellow substrate mycelium and soluble pigment of the same colour on organic media. No melanoid pigment is secreted. The type culture is designated as INA 3321. The cultures of A. fulvescens show antibiotic activity. A. turkmeniaca is characterized by formation of short straight or spiral spore chains, smooth spores, scanty white aerial mycelium, substrate mycelium and soluble pigment of pinkish-violet colour, absence of melanoid pigment. The type culture is designated as INA 3344. The strains of this species have low antibiotic activity. The study on the use of carbon sources by the representatives of 7 species (9 strains) of Actinomadura showed that the majority of the cultures (5 species, 7 strains) produced no growth on the Pridham and Gottlieb medium (ISP-9) with various carbon sources, including glucose. Possibly this medium cannot be used as the main medium for investigation of the spectrum of carbohydrate consumption in Actinomadura.     

Actinomycin synthesis in Streptomyces antibioticus. Purification and properties of a 3-hydroxyanthranilate 4-methyltransferase

A methyltransferase, which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate, has been purified to near homogeneity from 30-36-h mycelium of the bacterium Streptomyces antibioticus. The enzyme was obtained in approximately 20% yield with a purification of 130-fold. Polyacrylamide gel electrophoresis under denaturing conditions indicates that the enzyme is composed of a single subunit with Mr of about 36,000. On chromatography in 0.5 M NaCl, the enzyme displays a molecular weight of about 37,000. The specific activity of the enzyme in S. antibioticus mycelium is maximal between 30 and 36 h following inoculation of galactose/glutamic acid medium and, at those times post-inoculation, the specific activity is essentially the same in extracts of mycelium obtained from cultures grown on glucose rather than galactose as the carbon source. The enzyme activity is stimulated by Na2EDTA (in crude extracts) and by 2-mercaptoethanol and the methyltransferase shows a strong preference for HAA as substrate as compared with a number of HAA analogs. Thin layer chromatography of ethyl acetate extracts of large-scale incubation mixtures confirms that the product of the reaction is 4-methyl-3-hydroxyanthranilic acid. The reaction product was also a substrate for phenoxazinone synthase and was incorporated into actinomycin by S. antibioticus mycelium. Kinetic parameters for the methyltransferase reaction was determined.     

New species of Actinomadura isolated from soils in Turkmenia and their antagonistic properties

New species, i.e. Actinomadura polychroma and Actinomadura umbrina are described. The cell wall of the cultures contains meso-diaminopimelic acid galactose, glucose and madurose. The former species is characterized by short spore chains in the form of spirals or pseudosporangia, smooth spore surface, white aerial mycelium and colourless, yellowish-brown or blue-green substrate mycelium. The cultures of this species have no antagonistic activity with respect to various test-microbes. The type culture of A. polychroma is designated as INA 2755. A. umbrina is characterized by formation of short spore chains, which are straight, hooked or spiral, often branching, smooth spore surface, white scanty aerial mycelium and brownish or black-brown substrate mycelium and soluble pigment of the same colour. The strains of this species inhibit the growth of some gram-positive bacteria and have no activity against gram-negative organisms. The type culture of A. umbrina is designed as INA 2309.     

Chitin synthetase from the yeast and mycelial phases of Blastomyces dermatitidis

Chitin synthetase (E.C.2.4.1.16) from mixed membrane fractions of the yeast and mycelial phases of Blastomyces dermatitidis were compared. The behavior of the enzyme from both phases was very similar: N-acetylglucosamine was stimulatory (Km 8.5 mM for yeast and 3.9 mM for mycelium); substrate Michaelis-Menten kinetics were sigmoidal; substrate Km of enzyme from yeast decreased from 3.0 mM at low N-acetylglucosamine (5 mM) levels to 1.4 mM at high (100 mM) levels; substrate Km of enzyme from mycelium was essentially unchanged at 1.4 mM; temperature optimum was 28 ° C; pH optimum was 7–7.5; Mg⁺² optimum was 5–10 mM.The greatest difference was that enzyme from yeast was extracted in a mostly latent form that required trypsin treatment for maximal in vitro activity while enzyme from mycelium was extracted in an active form which was rapidly deactivated by trypsin treatment.     

Protease production by the ontjom fungus,Neurospora sitophila

Summary The production of protease and mycelium byNeurospora sitophila cultured on solid and liquid potato dextrose media was studied. Maximal activity of protease extracted from 4-day-old cultures occurred at pH 6.5 when an unfractionated peanut (groundnut) protein substrate was used. The greatest protease activity and mycelium production occurred during the first day of the 4-day test period. Potato dextrose media containing more than 0.2 M NaCl resulted in decreased protease and mycelium production, while tapioca starch was without affect at concentrations up to 1.4%. Addition of up to 0.3 M sucrose to growth media greatly stimulated protease production and mycelial growth. Maximal proteolytic activity was observed in extracts from mycelium cultured in potato dextrose media adjusted from pH 6.0 to 7.5. Activity was greater when soluble peanut protein was used as a substrate, compared to unfractionated or globulin protein substrates.     

Contribution of aerial hyphae of Aspergillus oryzae to respiration in a model solid-state fermentation system

Oxygen transfer is for two reasons a major concern in scale-up and process control in industrial application of aerobic fungal solid-state fermentation (SSF): 1) heat production is proportional to oxygen uptake and it is well known that heat removal is one of the main problems in scaled-up fermenters, and 2) oxygen supply to the mycelium on the surface of or inside the substrate particles may be hampered by diffusion limitation. This article gives the first experimental evidence that aerial hyphae are important for fungal respiration in SSF. In cultures of A. oryzae on a wheat-flour model substrate, aerial hyphae contributed up to 75% of the oxygen uptake rate by the fungus. This is due to the fact that A. oryzae forms very abundant aerial mycelium and diffusion of oxygen in the gas-filled pores of the aerial hyphae layer is rapid. It means that diffusion limitation in the densely packed mycelium layer that is formed closer to the substrate surface and that has liquid-filled pores is much less important for A. oryzae than was previously reported for R. oligosporus and C. minitans. It also means that the overall oxygen uptake rate for A. oryzae is much higher than the oxygen uptake rate that can be predicted in the densely packed mycelium layer for R. oligosporus and C. minitans. This would imply that cooling problems become more pronounced. Therefore, it is very important to clarify the physiological role of aerial hyphae in SSF.     

Respiratory Activity of the Mycelium of Eremothecium ashbyii

The respiratory activity of the mycelium of Eremothecium ashbyii from submerged cultures was manometrically determined at different stages of its development and the results were statistically analyzed. The experiments were performed in a manner designed to diminish the endogenous respiration without affecting the response to the addition of an exogenous substrate. Lactose was the carbohydrate tested that produced the lowest oxygen consumption. The oxidation of maltose, which was high at 24 hr, decreased by more than 50% at 48 and 55 hr. Glucose and sucrose were actively oxidized by mycelium of three ages. From the intermediates of carbohydrate metabolism, 24-hr mycelium did not produce oxygen consumption with malate, lactate, citrate, fumarate, and α-ketoglutarate. At 48 hr, mycelium did not oxidize either lactate or citrate; 55-hr mycelium showed oxygen consumption with all intermediates tested. Acetate and pyruvate always produced high oxygen consumption. Ethyl alcohol produced high oxygen consumption with mycelium of all tested ages.     

Growth and sporulation behaviour of Penicillium roquefortii in solid substrate fermentation: effect of the hydric parameters of the medium

Both growth and sporulation increase linearly according to the initial water content of the solid substrate when Penicillium roquefortii is cultivated on buckwheat seeds. This indicates that water is the limiting factor for fungal development since neither carbon nor nitrogen sources were exhausted during these experiments. This feature validates the concept of available water for fungal growth, defined as initial water content of substrate minus its residual water content when vegetative growth stops. An efficient methodology, based on drawing a regression line of mycelium dry weight production as a function of the initial water content of substrate is presented; it allows estimation of both available water and water content of mycelium. Results show that growth stops when the residual water in the substrate is close to 0.52 g H₂O/g initial dry matter, corresponding to a water activity (a_w) close to 0.96, and that the initial water content in mycelium is near 76%. Thus, both a_w and water content of the substrate have to be taken into account during the course of solid-state cultivations.Correspondence to: C. Larroche     

The genesis of secondary reproductive structures in streptomycete colonies as revealed by confocal microscopy

Streptomycete colonies were examined by confocal microscopy to elucidate the origin of the secondary structures that form the concentric rings of aerial mycelium. These reproductive structures but no the vegetative ones were found to luminesce upon safranine staining. It is shown that the secondary reproductive structures emerge within the substrate mycelium by lateral branching or apical extension of vegetative hyphae, further developing into numerous secondary branches filled with protospores.     

Spatial distribution of synthetic activity in zone-forming colonies of soil Streptomycetes

The space distribution of synthetic activity in the substrate mycelium gyphs--structure growth units of a quasiorganism of the radiant fungus, was studied by the method of microspectral analysis. A detailed map of the distribution on the alpha-parameter (which reflects the level of synthetic activity) from the cross section of a living streptomyces colony was constructed. The data obtained support with a high reliability the earlier supposition that the maximum of synthetic activity is in the growth zone and the minimum synthetic activity is in the globular regions of formation of air mycelium protogyphs. The use of objectivity characteristics made it possible to thoroughly study the development of globular structures, the precursors and sources of formation of air mycelium concentric zones on colony surface.     

Growth induced translocation effectively directs an amino acid analogue to developing zones in Agaricus bisporus

The vegetative mycelium of Agaricus bisporus supplies developing white button mushrooms with water and nutrients. However, it is not yet known which part of the mycelium contributes to the feeding of the mushrooms and how this depends on growth conditions. Here we used photon counting scintillation imaging to track translocation of the ¹⁴C-radiolabeled metabolically inert amino acid analogue α-aminoisobutyric acid (¹⁴C-AIB). Translocation to the periphery of the mycelium was observed in actively growing vegetative mycelium with a velocity of up to 6.6 mm h⁻¹, which was 30-fold higher than the growth rate. Furthermore, ¹⁴C-AIB translocated to neighboring colonies after fusion by anastomosis depending on the relative growth rate in these colonies. When mushrooms started to develop, translocation of ¹⁴C-AIB was redirected to the fruiting bodies via mycelium and hyphal cords. More abundant mycelial cord formation and a 5-fold higher rate of translocation was observed for cultures growing directionally from inoculum located at one side of the substrate, when compared to non-directional growth (inoculum mixed throughout the substrate). The maximum translocation distance was also greater (≥50 and 22 cm, respectively). In conclusion, ¹⁴C-AIB translocation switches between vegetative growth and towards developing mushrooms, especially via cords and when source–sink relationships change.     

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10⁵ and 10⁷ spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 × 10⁷ to 2 × 10⁷ spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.     

Implication of mycelium-associated laccase from Irpex lacteus in the decolorization of synthetic dyes

The white rot fungus Irpex lacteus is able to decolorize such synthetic dyes as Reactive Orange 16 and Remazol Brilliant Blue R. Here, we demonstrate that this type of dye decolorization is mainly related to a laccase-like enzyme activity associated with fungal mycelium. In its bound form, the enzyme detected showed a pH optimum of 3.0 for the oxidation of ABTS, DMP and guaiacol, and a pH of 7.0 for syringaldazine. The highest enzymatic activity was obtained with ABTS as substrate. Enzyme activity was fully inhibited with 50mM NaN(3). Depending on the chemical structure of dyes, redox mediators had a positive effect on the dye decolorization by fungal mycelium. Enzyme isolated from fungal mycelium was able to decolorize synthetic dyes in vitro.