Intra-particle oxygen diffusion limitation in solid-state fermentation

Oxygen limitation in solid-state fermentation (SSF) has been the topic of modeling studies, but thus far, there has been no experimental elucidation on oxygen-transfer limitation at the particle level. Therefore, intra-particle oxygen transfer was experimentally studied in cultures of Rhizopus oligosporus grown on the surface of solid, nutritionally defined, glucose and starch media. The fungal mat consisted of two layers--an upper layer with sparse aerial hyphae and gas-filled interstitial pores, and a dense bottom layer with liquid-filled pores. During the course of cultivation ethanol was detected in the medium indicating that oxygen was depleted in part of the fungal mat. Direct measurement of the oxygen concentrations in the fungal mat during cultivation, using oxygen microelectrodes, showed no oxygen depletion in the upper aerial layer, but revealed development of steep oxygen concentration gradients in the wet bottom layer. Initially, the fungal mat was fully oxygenated, but after 36.5 hours oxygen was undetectable at 100 microm below the gas-liquid interface. This was consistent with the calculated oxygen penetration depth using a reaction-diffusion model. Comparison of the overall oxygen consumption rate from the gas phase to the oxygen flux at the gas-liquid interface showed that oxygen consumption of the microorganisms occurred mainly in the wet part of the fungal mat. The contribution of the aerial hyphae to overall oxygen consumption was negligible. It can be concluded that optimal oxygen transfer in SSF depends on the available interfacial gas-liquid surface area and the thickness of the wet fungal layer. It is suggested that the moisture content of the matrix affects both parameters and, therefore, plays an important role in optimizing oxygen transfer in SSF cultures.     

Effect of low oxygen concentrations on growth and alpha-amylase production of Aspergillus oryzae in model solid-state fermentation systems

Oxygen transfer in the fungal mat is a major concern in solid-state fermentation (SSF). Oxygen supply into the mycelial layers is hampered by diffusion limitation. For aerobic fungi, like Aspergillus oryzae, this oxygen depletion can be a severely limiting factor for growth and metabolite production. This paper describes the effects of a low oxygen concentration on growth at the levels of individual hyphae, colonies and overcultures, and on alpha-amylase production in overcultures. PDA medium was used to study the effect of a low oxygen concentration on hyphal elongation rate and branching frequency of hyphae, and radial extension rate of colonies of A. oryzae. We found similar saturation constants (K(O2)) of 0.1% (v/v in the gas phase) for oxygen concentration described with Monod kinetics, for branching frequency of hyphae and colony extension rate. When A. oryzae was grown as an over-culture on wheat-flour model substrate at 0.25% (v/v) oxygen concentration, the reduction in growth was more pronounced than as individual hyphae and a colony on PDA medium. Experimental results also showed that the specific alpha-amylase production rate under the condition of 0.25% (v/v) oxygen was reduced. Because the value of K(O2) is relatively low, it is reasonable to simplify the kinetics of growth of A. oryzae to zero-order kinetics in coupled diffusion/reaction models.     

Aspergillus oryzae in solid-state and submerged fermentations. Progress report on a multi-disciplinary project

We report the progress of a multi-disciplinary research project on solid-state fermentation (SSF) of the filamentous fungus Aspergillus oryzae. The molecular and physiological aspects of the fungus in submerged fermentation (SmF) and SSF are compared and we observe a number of differences correlated with the different growth conditions. First, the aerial hyphae which occur only in SSFs are mainly responsible for oxygen uptake. Second, SSF is characterised by gradients in temperature, water activity and nutrient concentration, and inside the hyphae different polyols are accumulating. Third, pelleted growth in SmF and mycelial growth in SSF show different gene expression and protein secretion patterns. With this approach we aim to expand our knowledge of mechanisms of fungal growth on solid substrates and to exploit the biotechnological applications.     

Substrate aggregation due to aerial hyphae during discontinuously mixed solid-state fermentation with Aspergillus oryzae: experiments and modeling

Solid-state fermentation (SSF) is prone to process failure due to channeling caused by evaporative cooling and the formation of an interparticle mycelium network. Mixing is needed to break the mycelium network and to avoid such failure. This study presents the first attempt to quantify and predict the effect of mycelium bonds on particle mixing and vice versa. We developed a novel experimental set-up to measure the tensile strength of hyphal bonds in SSF: Aspergillus oryzae was cultivated between two wheat-dough disks and the tensile strength of the aerial mycelium was measured with a texture analyzer. Tensile strength at different incubation times was related to oxygen consumption, to allow a translation to a rotating drum with A. oryzae cultivated on wheat grain. We performed several discontinuously mixed solid-state fermentations in the drum fermentor and measured the number and size of grain-aggregates remaining after the first mixing action. We integrated data on mycelium tensile strength into a previously developed two-dimensional discrete-particle model that calculates forces acting on individual substrate particles and the resulting radial-particle movements. The discrete-particle model predicted the quantity and size of the aggregates remaining after mixing successfully. The results show that the first mixing event in SSF with A. oryzae is needed to break mycelium to avoid aggregate formation in the grain bed, and not to distribute water added to compensate for evaporation losses, or smooth out temperature gradients.     

Modeling of heat and mass transfer for solid state fermentation process in tray bioreactor

A mathematical model is developed to simulate oxygen consumption, heat generation and cell growth in solid state fermentation (SSF). The fungal growth on the solid substrate particles results in the increase of the cell film thickness around the particles. The model incorporates this increase in the biofilm size which leads to decrease in the porosity of the substrate bed and diffusivity of oxygen in the bed. The model also takes into account the effect of steric hindrance limitations in SSF. The growth of cells around single particle and resulting expansion of biofilm around the particle is analyzed for simplified zero and first order oxygen consumption kinetics. Under conditions of zero order kinetics, the model predicts upper limit on cell density. The model simulations for packed bed of solid particles in tray bioreactor show distinct limitations on growth due to simultaneous heat and mass transport phenomena accompanying solid state fermentation process. The extent of limitation due to heat and/or mass transport phenomena is analyzed during different stages of fermentation. It is expected that the model will lead to better understanding of the transport processes in SSF, and therefore, will assist in optimal design of bioreactors for SSF.     

Aerial mycelia of Aspergillus oryzae accelerate α-amylase production in a model solid-state fermentation system

Aspergillus oryzae is commonly used in solid-state fermentation (SSF) and forms abundant aerial mycelia. Previously, we have shown that aerial mycelia are extremely important for the respiration of this fungus during growth on a wheat-flour model substrate. In this paper, we show that aerial mycelia of this fungus give a strong increase in fungal biomass and α-amylase production. Cultures of A. oryzae on wheat-flour model substrate produced twice the amounts of fungal biomass and α-amylase, when aerial mycelia were formed. Utilization of these findings in commercial solid-state fermenters requires further research; results from packed beds of grain indicate that aerial mycelia are of limited importance there. Probably, substrate pre-treatment and an increase in bed voidage are required.     

Morphological patterns of Aspergillus niger biofilms and pellets related to lignocellulolytic enzyme productivities

AIMS: To study the morphological patterns of Aspergillus niger during biofilm formation on polyester cloth by using cryo-scanning electron microscopy related to lignocellulolytic enzyme productivity. METHODS AND RESULTS: Biofilm and pellet samples obtained from flask cultures were examined at -80 degrees C in a LEO PV scanning electron microscope. Spore adhesion depends on both its rough surface and adhesive substances that form a pad between spore and support. An extracellular matrix surrounding germ tubes and hyphae was also seen. Biofilm mycelia showed an orderly distribution forming surface and inner channels, while pellets showed highly intertwined superficial hyphae and a densely packed deep mycelium. Morphological differences between both types of culture correlated with differences in enzyme volumetric and specific productivities. Biofilm cultures produced higher filter paper cellulase, endoglucanase, beta-glucosidase and xylanase volumetric and specific productivities than submerged cultures. CONCLUSIONS: Fungal biofilms are morphologically efficient systems for enzyme production. Favourable physiological aspects are shared with solid state fermentation, but fungal biofilms present better possibilities for process control and scale-up. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study support the importance of morphology in the productivity of fungal submerged processes, placing biofilms in a preferential category.     

Use of confocal scanning laser microscopy to measure the concentrations of aerial and penetrative hyphae during growth of Rhizopus oligosporus on a solid surface

In order to develop a method for use in investigations of spatial biomass distribution in solid-state fermentation systems, confocal scanning laser microscopy was used to determine the concentrations of aerial and penetrative biomass against height and depth above and below the substrate surface, during growth of Rhizopus oligosporus on potato dextrose agar. Penetrative hyphae had penetrated to a depth of 0.445 cm by 64 h and showed rhizoid morphology, in which the maximum biomass concentration, of 4.45 mg dry wt cm(-3), occurred at a depth of 0.075 cm. For aerial biomass the maximum density of 39.54 mg dry wt (-3) occurred at the substrate surface. For both aerial and penetrative biomass, there were two distinct regions in which the biomass concentration decayed exponentially with distance from the surface. For aerial biomass, the first exponential decay region was up to 0.1 cm height. The second region above the height of 0.1 cm corresponded to that in which sporangiophores dominated. This work lays the foundation for deeper studies into what controls the growth of fungal hyphae above and below the surfaces of solid substrates.     

Regulation of Streptomyces development: reach for the sky!

Streptomyces coelicolor is characterized by a complex life cycle and serves as a model system for bacterial development. After a feeding substrate mycelium has been formed, this filamentous bacterium differentiates by forming aerial hyphae that septate into spores. The bld cascade regulates initiation of aerial growth, whereas the whi genes control spore formation. Recent findings indicate the existence of another regulatory pathway that operates after aerial hyphae have started to grow into the air, which we call the sky pathway. This pathway controls the expression of the chaplin and rodlin genes. These genes encode proteins that assemble into a rodlet layer that provides surface hydrophobicity to aerial hyphae and spores.     

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.     

Significance of bed porosity, bran and specific surface area in solid-state cultivation of Aspergillus oryzae

In this paper, the effects of bed porosity, bran and specific surface area on the oxygen uptake rate and alpha-amylase production during growth of Aspergillus oryzae on wheat grain and wheat-flour substrate are reported. The high oxygen uptake rate found during cultivation of A. oryzae on wheat-flour substrate was not reached on wheat grain. This is mainly due to the bran of the wheat grain. Using wheat-flour substrates, it was shown that extra bed porosity increased the alpha-amylase production and oxygen uptake rates. Furthermore, the peak oxygen uptake rate decreased with increasing surface area-volume ratio of the substrate particles, while the alpha-amylase production and the cumulative oxygen uptake per gram of initial substrate dry matter increased. The present work does not support a direct correlation between aerial mycelia and enzyme production. There is, however, a correlation between the alpha-amylase yield and the cumulative oxygen uptake (not the uptake rate). This implies that aerial mycelia could accelerate alpha-amylase production even if they do not increase the yield.     

Zur Wirkung von Antimycotica auf das Wachstum des Luftmycels bei einigen Pilzen

Zusammenfassung Das Luftmycel von Pilzen reagiert auf Antibiotica im allgemeinen nur schwach. Einige Basidiomyceten schränken aber das Wachstum der Lufthyphen bei geringen Konzentrationen bestimmter Hemmstoffe, vor allem Polyenantibiotica, stärker ein als das des Substratmycels. Die Reaktionsweise des Luftmycels läßt im Gegensatz zur Auffassung mancher Autoren keine Schlüsse auf das Fehlen einer Leitung von Griseofulvin in Pilzhyphen zu. Polyporus versicolor zeigt in seiner Myceldifferenzierung ausgeprägte Unterschiede bei Einwirkung gewisser strukturell verwandter Stoffe.

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Summary The aerial mycelium of fungi is only slightly inhibited by antibiotics in most cases, but in some species of Basidiomycetes little concentrations of some inhibitors, e.g. polyene antibiotics, retard the growth of aerial hyphae more than the growth of substrate mycelium. The reaction of aerial hyphae doesn't permit any conclusion about transport of griseofulvin in fungal hyphae. Polyporus versicolor shows great differences in mycelial differentiation influenced by structurally related compounds.

     

Modelling the contribution of arbuscular mycorrhizal fungi to plant phosphate uptake

In this paper, we investigate the role of arbuscular mycorrhizal fungi in plant phosphorus nutrition. We develop a mathematical model which quantitatively assesses the contribution of external fungal hyphae to plant phosphate uptake.We derive an equation for solute uptake by a growing fungal mycelium which we couple with a model for root uptake. We analyse the model using nondimensionalization and numerical simulations.Simulations predict that removal of phosphate from soil is dominated by hyphal uptake as opposed to root uptake. Model analysis shows that the depletion zones around hyphae overlap within 8 h and that the transfer between fungus and root is a critical step for the behaviour of phosphorus within the mycelial phase. We also show that the volume fraction of mycelium is negligibly small in comparison to other soil phases.This is the first model to quantify the contribution of mycorrhizal fungi to plant phosphate uptake. A full data set for model parametrization and validation is not currently available. Therefore, more complete sets of experimental measurements are necessary to make this model more applicable.     

Expression analysis of the spi gene in the pock-forming plasmid pSA1.1 from Streptomyces azureus and localization of its product during differentiation

The sporulation inhibitory gene spi in the pock-forming conjugative plasmid pSA1.1 of Streptomyces azureus was introduced into cells via a high or low copy number vector to examine the effect of gene dosage on the growth of Streptomyces lividans TK24 as a host. In transformants carrying a high spi copy number, nutrient mycelial growth was inhibited, as was morphological differentiation from substrate mycelium to aerial mycelium on solid media. The degree of inhibition depended on the spi gene dosage, but the presence of pSA1.1 imp genes, which encode negative repressor proteins for spi, relieved the inhibition. Confocal images of Spi tagged with enhanced green fluorescent protein in cells on solid media revealed that spi expression was initiated at the time of elongation of substrate mycelium, that its expression increased dramatically at septation in aerial hyphae, and that the expression was maximal during prespore formation. Expression of spi covered the whole of the hyphae, and the level of expression at the tip of the hyphae during prespore formation was about sixfold greater than during substrate mycelial growth and threefold greater than during aerial mycelial growth. Thus, localized expression of spi at particular times may inhibit sporulation until triggering imp expression to repress its inhibitory effects.     

Microbial production of extra-cellular phytase using polystyrene as inert solid support

Aspergillus ficuum TUB F-1165 and Rhizopus oligosporus TUB F-1166 produced extra-cellular phytase during solid-state fermentation (SSF) using polystyrene as inert support. Maximal enzyme production (10.07 U/g dry substrate (U/gds) for A. ficuum and 4.52 U/gds for R. oligosporus) was observed when SSF was carried out with substrate pH 6.0 and moisture 58.3%, incubation temperature 30 degrees C, inoculum size of 1.3 x 10(7) spores/5 g substrate, for 72 h for A. ficuum and with substrate pH 7.0 and moisture 58.3%, incubation temperature 30 degrees C, inoculum size of 1 x 10(6) spores/5 g substrate for 96 h for R. oligosporus. Results indicated scope for production of phytase using polystyrene as inert support.     

Hyphal death during colony development in Streptomyces antibioticus: morphological evidence for the existence of a process of cell deletion in a multicellular prokaryote

During the life cycle of the streptomycetes, large numbers of hyphae die; the surviving ones undergo cellular differentiation and appear as chains of spores in the mature colony. Here we report that the hyphae of Streptomyces antibioticus die through an orderly process of internal cell dismantling that permits the doomed hyphae to be eliminated with minimum disruption of the colony architecture. Morphological and biochemical approaches revealed progressive disorganization of the nucleoid substructure, followed by degradation of DNA and cytoplasmic constituents with transient maintenance of plasma membrane integrity. Then the hyphae collapsed and appeared empty of cellular contents but retained an apparently intact cell wall. In addition, hyphal death occurred at specific regions and times during colony development. Analysis of DNA degradation carried out by gel electrophoresis and studies on the presence of dying hyphae within the mycelium carried out by electron microscopy revealed two rounds of hyphal death: in the substrate mycelium during emergence of the aerial hyphae, and in the aerial mycelium during formation of the spores. This suggests that hyphal death in S. antibioticus is somehow included in the developmental program of the organism.     

A novel Streptomyces gene, samR, with different effects on differentiation of Streptomyces ansochromogenes and Streptomyces coelicolor

A 1.4-kb DNA fragment from Streptomyces ansochromogenes accelerated mycelium formation of S. ansochromogenes when present on a multicopy plasmid. The DNA fragment contains one complete open reading frame, designated samR, encoding a protein with 213 amino acids that contains a likely DNA-binding helix-turn-helix motif close to its N-terminus. The deduced SamR protein resembles the product of the hppR gene, which is involved in the regulation of catabolism of 3-(3-hydroxyphenyl) propionate in Rhodococcus globerulus. A samR disruption mutant was constructed that presented a bald phenotype and failed to form aerial hyphae and spores. We suggest that samR plays an important role in the emergence of aerial hyphae from substrate mycelium. An almost identical gene of Streptomyces coelicolor was also subjected to gene disruption. Surprisingly, the mutant was able to develop an aerial mycelium, but it remained white and deficient in sporulation instead of forming gray spores.     

Growth and sporulation stoichiometry and kinetics of Coniothyrium minitans on agar media

Coniothyrium minitans was cultivated on agar media with different concentrations of starch, urea, and trace elements. By means of elemental balances, the stoichiometry of growth and sporulation was established. C. minitans produced byproducts on all media, especially in the medium with high urea concentrations, where 30% of the starch was converted into byproducts. Simple empirical models were used to describe the kinetics of growth, sporulation, CO(2) production, and substrate consumption on all media. Total biomass and mycelium could be described reasonably well with the logistic law. Starch, urea, and oxygen consumption and CO(2) production could be described as a function of total biomass by the linear-growth model of Pirt. There were almost no differences between media for the estimates of yield coefficients and maintenance coefficients. Only at high urea concentrations were maintenance coefficients much higher. Similar to substrate consumption and CO(2) production, the kinetics of sporulation could be described as a function of mycelium production with the linear-growth model. It is shown that sporulation of C. minitans is growth-associated. Based on kinetics, the process costs for producing spores are roughly calculated. In addition, it is shown that fermentor costs represent the majority of production costs.     

A surface active protein involved in aerial hyphae formation in the filamentous fungus Schizophillum commune restores the capacity of a bald mutant of the filamentous bacterium Streptomyces coelicolor to erect aerial structures

The filamentous bacterium Streptomyces coelicolor undergoes a complex process of morphological differentiation involving the formation of a dense lawn of aerial hyphae that grow away from the colony surface into the air to form an aerial mycelium. Bald mutants of S. coelicolor, which are blocked in aerial mycelium formation, regain the capacity to erect aerial structures when exposed to a small hydrophobic protein called SapB, whose synthesis is temporally and spatially correlated with morphological differentiation. We now report that SapB is a surfactant that is capable of reducing the surface tension of water from 72 mJ m^?2 to 30 mJ m^?2 at a concentration of 50 μg ml^?1. We also report that SapB, like the surface-active peptide streptofactin produced by the species S. tendae, was capable of restoring the capacity of bald mutants of S. tendae to erect aerial structures. Strikingly, a member (SC3) of the hydrophobin family of fungal proteins involved in the erection of aerial hyphae in the filamentous fungus Schizophyllum commune was also capable of restoring the capacity of S. coelicolor and S. tendae bald mutants to erect aerial structures. SC3 is unrelated in structure to SapB and streptofactin but, like the streptomycetes proteins, the fungal protein is a surface active agent. Scanning electron microscopy revealed that aerial structures produced in response to both the bacterial or the fungal proteins were undifferentiated vegetative hyphae that had grown away from the colony surface but had not commenced the process of spore formation. We conclude that the production of SapB and streptofactin at the start of morphological differentiation contributes to the erection of aerial hyphae by decreasing the surface tension at the colony surface but that subsequent morphogenesis requires additional developmentally regulated events under the control of bald genes.