Morphology of Trichoderma reesei QM 9414 in submerged cultures

The microscopic morphology of Trichoderma reesei QM 9414, growing in submerged culture, was studied by image analysis. The morphology was characterized by the total hyphal length, the total number of tips, the number of actively growing tips, and the length of the main hypha. To describe the growth of a single mycelium a simple model is set-up. The main features of the model are: (1) saturation type kinetics for the tip extension of the individual branches within the mycelium; and (2) random branching with a frequency function, which is proportional to the total hyphal length. The model is used to simulate a population of mycelia, where spore germination is described with a log-normal distribution. From the simulation of the population, the average properties of the mycelia, e.g., the average total hyphal length, are calculated, and by fitting the model to experimental data the model parameters are estimated. Finally, the distribution function with respect to the mycelia properties, that is, number of tips and total hyphal length, is calculated, and it corresponds well with the experimental determination of the distribution function. (c) 1995 John Wiley & Sons Inc.     

A Unique Pattern of Mycelial Elongation of Blakeslea trispora and Its Effect on Morphological Characteristics and β-Carotene Synthesis

The mycelial morphology of Blakeslea trispora was of crucial importance in the production of beta-carotene in submerged cultures of B. trispora. After the spores were inoculated, the time-course variation of mycelial morphology was closely examined under the microscope. With the addition of the non-ionic surfactant (Span 20: Sorbitan monolaurate, E493) to the culture medium, a unique pattern of mycelial elongation was observed: 1) slow formation of germ tubes from spores and 2) appearance of mycelia with very short length, which allowed a well-dispersed growth of B. trispora without significant pellet aggregation. Span 20 appears to act like a paramorphogen. Without Span 20, however, the fungal culture finally formed a big clump of mycelium owing to heavy cross-linking of long mycelia. But the short mycelium maintained in the course of cultivation seemed to be irrelevant to growth inhibition, because the final concentration of dry mycelium was much higher with Span 20 after 3-day cultivation. The 20-fold increase in specific yield of beta-carotene (mg beta-carotene produced per g mycelium) was achieved with this drastic change in the pattern of mycelial elongation. The reason for this result might be more effective mass transfer and/or enhanced sensitivity to environmental oxidative stress in the well-dispersed mycelial cultures of B. trispora.     

Transcriptional analysis of the cyclophilin A gene (cypA) of Streptomyces chrysomallus

In the thermophilic fungus Thermomyces lanuginosus, invertase displays an unusual pattern of development: the induced activity begins to diminish even before any substantial quantity of sucrose has been utilized or an appreciable amount of biomass has been produced. Despite this pattern of invertase activity, neither the growth rate nor the final mycelial yield is affected adversely. T. lanuginosus invertase is a thiol protein and the enzyme is active when specific sulfhydryl group(s) is in the reduced state. Measurements of reduced coenzyme and glutathione pools in sucrose-growth mycelia excluded oxidative stress as the primary reason for the observed decline in invertase activity. Rather, this unusual pattern of invertase is considered to be due to its localization in the hyphal tips. At the early stage of growth, the number of hyphal tips per unit mass of mycelium is maximum, whereas at later times their numbers do not increase in proportion to the biomass. As a result invertase activity shows an apparent inverse relationship with biomass. The enzyme activity disappears when the inducing carbon source is consumed and growth is completed.     

Macroscopic growth of filamentous fungi on solid substrate explained by a microscopic approach.

A quantitative model predicting biomass growth on solid media has been developed. The model takes into account steric interactions between hyphae and tips at the microscopic level (competition for substrate and tip-hypha collisions). These interactions effect a slowing down of the hyphal, population-averaged extension rate and are responsible, at the microscopic level, for the distribution of tip orientations observed at the colony border. At the macroscopic level, a limiting value of the colony radial extension rate is attained. A mathematical model that combines hyphal branching, tip diffusion, and biomass growth was proposed to explain such behavior. Experiments using Gibberella fujikuroi were performed to validate the model; good agreement between experiments and simulations was achieved. Most parameters can be measured by simple image analysis on the peripheral growth zone, and they have clear physical meaning; that is, they correspond to properties of single, leading hyphae. The model can be used to describe two-dimensional (2D) solid media fermentation experiments under varying culture conditions; the model can also be extended to consider growth in three-dimensional (3D), complex geometry substrates.     

A Unique Pattern of Mycelial Elongation of Blakeslea trispora and Its Effect on Morphological Characteristics and β-Carotene Synthesis

The mycelial morphology of Blakeslea trispora was of crucial importance in the production of β-carotene in submerged cultures of B. trispora. After the spores were inoculated, the time-course variation of mycelial morphology was closely examined under the microscope. With the addition of the non-ionic surfactant (Span 20: Sorbitan monolaurate, E493) to the culture medium, a unique pattern of mycelial elongation was observed: 1) slow formation of germ tubes from spores and 2) appearance of mycelia with very short length, which allowed a well-dispersed growth of B. trispora without significant pellet aggregation. Span 20 appears to act like a paramorphogen. Without Span 20, however, the fungal culture finally formed a big clump of mycelium owing to heavy cross-linking of long mycelia. But the short mycelium maintained in the course of cultivation seemed to be irrelevant to growth inhibition, because the final concentration of dry mycelium was much higher with Span 20 after 3-day cultivation. The 20-fold increase in specific yield of β-carotene (mg β-carotene produced per g mycelium) was achieved with this drastic change in the pattern of mycelial elongation. The reason for this result might be more effective mass transfer and/or enhanced sensitivity to environmental oxidative stress in the well-dispersed mycelial cultures of B. trispora.     

Nuclei, septation, branching and growth of Geotrichum candidum.

A study was made of growth, septation and branching in Geotrichum candidum, a mould which forms physiologically complete septa. A correlation was observed between septation and branch initiation; branches were almost invariably formed just behind septa. Primary branches and their parent intercalary compartments initially increased in length at an exponential rate before eventually attaining a constant rate of extension. The whole branching system (which eventually contained seven tips) produced by an intercalary compartment increased in length exponentially until it attained a total length of at least 1-5 mm. The total length and the number of nuclei of undifferentiated mycelia increased exponentially at the same specific growth rate. The results suggest that nuclei divide just before or just after arthrospore formation.     

Mathematical model for apical growth, septation, and branching of mycelial microorganisms

A mathematical model for apical growth, septation, and branching of mycelial microorganisms is presented. The model consists of two parts: the determinstic part of the model is based on fundamental cellular and physical mechanisms; it represents the kinetics for growth of hyphal tips and septation of apical as well as intercalary compartments. In regard to random occurrences of hyphal growth and branching, the stochastic part deals with branching processes, tip growth directions, and outgrowth orientations of branches. The model can explain the morphological development of mycelia up to the formation of pellets. The results, as predicted by the model, correspond very closely to those observed in experiments. In addition, some unmeasured states can be ascertained, such as the distribution functions of hyphal length (biomass) and tips along pellet radii.     

On-line study of fungal morphology during submerged growth in a small flow-through cell

A flow-through cell is designed to measure the growth kinetics of hyphae of Aspergillus oryzae grown submerged in a well controlled environment. The different stages of the growth process are characterized, from the spore to the fully developed hyphal element with up to 60 branches and a total length lt up to 10,000 micrometer. Spore swelling is found to occur without change in the form of the spore (circularity index constant at about 1.06) and the spore volume probably increases exponentially. The germ tube appears after about 4 h. The branching frequency and the rate of germ tube extension is determined. After about 10 h growth at a glucose concentration of 250 mg L-1, 6-7 branches have been set, and both the total hyphal length lt and the number of tips increase exponentially with time. The specific growth rate of the hyphae is 0. 33 h-1 while the average rate of the extension of the growing tips approaches 55 micrometer h-1. The growth kinetics for all the branches on the main hypha have also been found. The main hypha and all the branches grow at a rate which can be modeled by saturation kinetics with respect to the branch length and with nearly equal final tip speeds (160 micrometer h-1). Branches set near the apical tip of the main hypha attain their final tip speed in the shortest time, i.e., the value of the saturation parameter is small. Finally, the influence of substrate (glucose) concentration cs on the values of the morphological parameters has been determined. It is found that saturation type kinetics can be used to describe the influence of cs on the growth. Experiments with recirculation of effluent from the cell back to the inlet strongly suggest that the fungus secretes an inducer for growth and branching.     

Growth of AM fungi on in vitro root organ culture of Sorghum vulgare and Saccharum officinarum

Spores of Gl mosseae and Gig gigantea germinated on minimal medium produced extraradical mycelium. Gl. mosseae infected roots of S. officinarum in in vitro condition were inoculated in M medium with in vitro cultured roots of Sorghum vulgare (test roots). From the infected root of S. officinarum, the mycelium developed and it infected the test roots. The roots developed new mycelia and further the mycelia produced a few hyaline spores. In MS medium combined with soil extract, root exudate, thiamine HCl and inositol combination, spore germination and germ tube growth were higher when compared with other media.     

Measurement and simulation of the morphological development of filamentous microorganisms

Growth of Streptomyces tendae was investigated in submerged culture. Images of several mycelia were analyzed by means of an image-processing system. The studies revealed that tip growth angles and branching outgrowth angles could be regarded as normally distributed. Based on these results, a random model for directional growth of hyphal tips as well as directional growth of branches is proposed. This model shows curved elongation of hyphal tips, so that the morphological development of a mycelium up to the formation of a pellet is predicted, similar to that observed in nature.     

Morphology and growth kinetics of hyphae of differentiated and undifferentiated mycelia of Neurospora crassa.

A comparison was made of the morphology and growth kinetics of hyphae of differentiated and undifferentiated mycelia of Neurospora crassa. Undifferentiated mycelia were formed during exponential growth on solid media or submerged culture. Hyphae at the margin of differentiated mycelia (colonies) differed from undifferentiated mycelia in diameter, extension rate, extension zone length, and intercalary and apical compartment length. The mean hyphal extension rate (E) of an undifferentiated mycelium was a function of the length of the mycelium's hyphal growth unit (G) and the organism's specific growth rate (alpha). Thus, E=Galpha.     

Morphology and physiology of an alpha-amylase producing strain of Aspergillus oryzae during batch cultivations

The microscopic morphology, that is, total hyphal length and total number of tips, has been characterized during batch cultivations of Aspergillus oryzae. The specific growth rate estimated by measuring the total hyphal length (mu(h)) corresponds well with the specific growth rate estimated from dry weight measurements during cultures grown as free hyphal elements. The average tip extension rate can be described with a saturation type kinetics with respect to the average total hyphal length, and the branching frequency is closely related to the total hyphal length. For the applied strain of A. oryzae, pellet formation occurs by coagulation of spores. The agglomeration process is pH dependent and pellets are formed at pH values higher than 5, whereas low pH (<3.5) results in growth as freely dispersed hyphal elements. The maximum specific growth rate has a broad pH optimum between 3 and 7, whereas the alpha-amylase production has a sharper maximum at about pH 6. During batch cultivation with pellets the growth is described well by the cube-root law when pellet fragmentation can be neglected. The kinetic parameter k in the cube-root law is derived from the growth kinetics with no mass transfer limitation, k = mu(h)/3. Based on an oxygen balance, the active growth layer in the pellet is estimated to be 200 to 325 mum and, consequently, up to 50% of the biomass is limited by oxygen for large pellets. Ethanol production (up to 1 g L(-1)) was observed during batch cultivations with pellets, suggesting that ethanol is produced in the oxygen limited part of the biomass. A constitutive, low alpha-amylase production was observed at high glucose concentration. The specific alpha-amylase production was significantly higher for filamentous growth than for pellets and oxygen appears to be necessary for production of alpha-amylase. (c) 1996 John Wiley & Sons, Inc.     

药用蕈菌紫芝液体培养的生长特征分析

使用专门设计的ALR ff蕈菌液体培养反应器 ,研究了有关紫芝液体培养过程中菌丝生长的特征。结果表明 ,液体培养时紫芝菌丝生长对温度有较宽的适应范围 2 5℃～ 3 5℃ ,但生长比速率相差很大。通气量由 0 93vvm提高到 1 6 4vvm时 ,菌丝生长比速率明显地增加 ,最大值为 0 0 4 4 4 (h- 1) ;当通气量进一步提高到 2 5 0vvm时 ,生长比速率反而下降。与低糖浓度比较 ,较高糖浓度 (2 80g 1 0 0mL)可以使生长迟滞期缩短 ;培养后期菌丝缠绕成浓密颗粒 ,较高糖浓度可以促进生长。在整个培养过程中 ,糖浓度较低时葡萄糖转化成菌体的生长效率明显高于较高葡萄糖浓度的效率。碳源限制生长的连续培养研究结果 ,进一步反映了菌丝不同生长活性使菌丝物具有不同形态结构。菌体量、限制生长底物、菌体生成率的三者关系与细菌有明显的差异。并且 ,在 0 0 1 0～ 0 2 2 0 (h- 1)稀释率范围内菌丝生长符合Contois方程     

A model for hyphal growth and branching.

A mathematical model for hyphal growth and branching is described which relates cytological events within hyphae to mycelial growth kinetics. Essentially the model quantifies qualitative theories of hyphal growth in which it is proposed that vesicles containing wall precursors and/or enzymes required for wall synthesis are generated at a constant rate throughout a mycelium and travel to the tips of hyphae where they fuse with the plasma membrane, liberating their contents into the wall and increasing the surface area of the hypha to give elongation. The hypothesis that there is a duplication cycle in hyphae which is equivalent to the cell cycle observed in unicellular micro-organisms is also included in the model. Predictions from the model are compared with experimentally observed growth kinetics of mycelia of Geotrichum candidum and Aspergillus nidulans. The finite difference model which was constructed is capable of predicting changes in hyphal length and in the number and positions of branches and septa on the basis of changes in vesicle and nuclear concentration. Predictions were obtained using the model which were in good agreement with experimentally observed data.     

Regulation of formation of aerial mycelia and spores of Streptomyces viridochromogenes.

Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more casein hydrolysate to the medium stimulated growth while completely repressing the formation of aerial mycelia and spores. This repression was temporary, as evidenced by the fact that transfer of the organisms to media not containing casein hydrolysate resulted in the appearance of aerial mycelia and spores. The effects of individual amino acids were tested. Glycine retarded growth and repressed formation of both aerial mycelia and spores. L-Aspartic acid, L-glutamic acid, and L-histidine stimulated or had little effect on growth and repressed formation of spores but not aerial mycelia. Repression by casein hydrolysate could not be attributed to the carbon/nitrogen ratio or the pH of the medium. Adding 1.25 to 2.5 mM adenine to the medium caused a reversal of the casein hydrolysate repression of aerial mycelium formation but did not reverse repression of sporulation. Dimethyladenine and 8-azaguanine had an effect similar to that of adenine, but a variety of other purine or pyrimidine derivatives had no effect on casein hydrolysate repression. The repression of aerial mycelium and spore formation by casein hydrolysate occurred only in media containing 15 mM or more phosphate. Aerial mycelia and spores were formed in media containing casein hydrolysate and 3 mM or less phosphate.     

Establishment of cultivating strategy for highly aggregated mycelia of Morchella esculenta in a stirred-tank bioreactor

Mycelia of Morchella esculenta were found to aggregate rapidly in a submerged culture, which caused the decrease in dispersed mycelia and the problem of diffusion limitation. The effect of different agitation schemes on the growth of mycelia was investigated in a stirred-tank bioreactor. At the constant speed of 100 or 300?rpm, rapid aggregation caused the biomass concentration to drop to zero in 30?h, which was even worse than achieved under static culture. Intermittent agitation maintained a higher mycelium fragment concentration for 48?h and enhanced the biomass concentration to 4.73?g/L at 120?h. The operation with a polytron connection disrupted effectively mycelium aggregation, thus increasing the specific growth rate, biomass concentration and maximum productivity to 0.0613 1/h, 7.73?g/L and 0.0878?g/L?h at 88?h, respectively. Moreover, logistic equations and genetic algorithm (GA) were used for the simulation of biomass growth and estimation of all kinetic coefficients. The operating strategy developed in this study could be used for the production of highly aggregated mycelia, which could also achieve a high cell-density culture in a stirred tank reactor.     

S-159-06菌株活性产物对植物病原菌的抑菌谱及拮抗机理初探

对S-159-06菌株活性产物的抑菌谱及其拮抗机理作了初步研究,结果表明：S-159-06活性产物具有较广的抑菌谱,对多种病原真菌和细菌具有较好的抑制作用;S-159-06活性产物能强烈抑制病原菌菌丝的生长和孢子的萌发,可引起病原菌菌丝扭曲,菌丝膨大成串珠状,分枝增多,分枝顶端膨胀后细胞壁破裂,原生质外溢,产生溶菌作用;使分生孢子数减少,孢子萌发畸形,萌发率降低;在温室条件下,S-159-06活性产物不仅能阻止番茄灰霉病菌菌丝的入侵,而且在植物体内有内吸作用,对已侵入的菌丝有较好的治疗作用。     

Differences in the species composition of arbuscular mycorrhizal fungi in spore, root and soil communities in a grassland ecosystem

Most studies on the species composition of arbuscular mycorrhizal fungi (AMF) have solely analysed mycorrhizal roots or AM spores collected from soil samples. However, the spore production rate and proportions of AMF mycelium in roots and soils have all been shown to vary substantially in a taxon-specific manner. Therefore, in the study presented here we used a molecular approach to analyse the species composition of AMF in spores, intra-radical and extra-radical mycelium in an intensively farmed meadow in central Germany. By polymerase chain reaction and sequencing of the ITS region members of seven different families and species groups within Glomeromycota were identified. The data revealed remarkable differences in the composition of AMF taxa both between the spores and the mycelia, and between the two types of mycelia. Glomus group Ab was dominant in roots and spores, in accordance with previous research. However, members of this group were rarely detected as extra-radical mycelium, in which Paraglomeraceae were dominant, although we found no evidence for the presence of Paraglomeraceae in roots or spores, even when a specific primer set was used. These results may be interpreted as a further indication that AMF are not necessarily obligate symbionts of plants.     

Structural changes induced by glycine on Streptomyces antibioticus

Abstract Germination and vegetative growth of Streptomyces antibioticus in liquid medium with different concentrations of glycine was examined. Both processes proved to be sensitive to the amino acid, being inhibited by 5 and 2.5% glycine, respectively. At concentrations of 5% or more, lysis of the vegetative mycelium occurred. Subinhibitory concentrations of glycine induced structural changes on germinating spores. These included an increase in the number of germ tubes produced by spore, in relation to the control. Moreover, soon after outgrowth the tubes bifurcate, giving rise to germinated spores with a characteristic aspect, and anomalous formation of cross-walls that appear both within the spores and in the newly formed germinative tubes, at or close to the region of outgrowth. The branching effect of glycine was also observed during vegetative growth of S. antibioticus .     

Fungal fractal morphology of pellet formation in Aspergillus niger

Mycelial fractal values were compared to the conventional fungal morphological parameters: average total mycelial length, average number of tips and average growth unit. The fractal values were between 1.47 to 1.3 for the various submerged culture conditions of Aspergillus niger. The average pellet diameter was 1.4 mm at the fractal value of 1.47. The mycelia with fractal values close to 1 were less branched and slim.