Dilution rate as a determinant of mycelial morphology in continuous culture

The morphology of mycelial fungi in liquid culture effects culture rheology and this in turn may affect product yield. It is therefore important to understand how environmental factors influence mycelial morphology and this paper describes the effect of dilution rate on two strains of Fusarium graminearum, the relatively sparsely branched parental strain (A3/5) and a relatively highly branched "colonial" variant (C106). At any given dilution rate, the concentration of mycelial fragments present at steady state of both strains remained approximately constant with time, suggesting that mycelial fragmentation occurred in a regular manner. However, for both strains fragment concentration decreased with increasing dilution rate. The strains had a similar morphology at a dilution rate of 0.07 h(-1). The length of the hyphal growth unit of A3/5 increased with increase in dilution rate, while that of C106 decreased with increase in dilution rate. At all dilution rates, C106 produced up to ten times more macroconidia than A3/5.     

Influence of morphology on the near-infrared spectra of mycelial biomass and its implications in bioprocess monitoring

Bioprocesses that employ mycelial microorganisms are commercially important. The application of optical techniques for the measurement of biomass in such processes is limited by the morphological heterogeneity exhibited by the mycelial microorganism employed. We investigated the influence of morphology on the near-infrared (NIR) spectra of the biomass of Streptomyces fradiae, a filamentous microorganism, by studying the spectra of mycelial suspensions that were manipulated to generate a range of morphological forms. Computerized image analysis was used to characterize the morphological forms. Principal component analysis was used to assess the spectral variations and study correlations to the manipulated mycelial morphology. Although morphology was found to influence the near infrared transmittance spectra of biomass, the influence was less pronounced than in the visible region, the spectral information at longer wavelengths (1600-2350 nm) showing greater stability to morphological variations. Long-wave NIR spectral information is therefore likely to be more useful in estimating biomass in mycelial bioprocesses. Furthermore, the NIR reflectance spectra of dried biomass were found to show correlations to the morphological variations introduced, suggesting that NIR spectra may be useful in obtaining morphology related information.     

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.     

A mathematical model for the growth of mycelial pellet populations

In liquid culture, filamentous organisms often grow in the form of pellets. Growth result in an increase in radius, whereas shear forces result in release of hyphal fragments which act as centers for further pellet growth and development. A previously published model for pellet growth of filamentous microorganisms has been examined and is found to be unstable for certain parameter values. This instability has been identified as being due to inaccuracies in estimating the numbers of fragments which seed the pellet population. A revised model has been formulated, based on similar premises, but adopting a finite element approach. This considers the population of pellets to be distributed in a range of size classes. Growth results in movement to classes of increasing pellet size, while fragments enter the smallest size class, from which they grow to form further pellets. The revised model is stable and predicts changes in the distribution of pellet sizes within a population growing in liquid batch culture. It considers pellet growth and death, with fragmentation providing new centers of growth within the pellet population, and predicts the effects of shear forces on pellet growth and size distribution. Predictions of pellet size distributions are tested using previously published data on the growth of fungal pellets and further predictions are generated which are suitable for experimental testing using cultures of filamentous fungi or actinomycetes. (c) 1995 John Wiley & Sons, Inc.     

Change in medium components and colony morphology due to mycelial growth of ectomycorrhizal fungusTricholoma bakamatsutake

Mycelia ofTricholoma bakamatsutake isolate No. 4 grew at temperatures ranging from 10 to 30°C, and the optimum was around 25°C. In well-buffered media of initial pH 5.0 and 6.0, No. 4 mycelia secreted gluconic acid and lowered medium pH. Mycelial growth then accelerated slightly; and with the exhaustion of glucose, growth and secretion of gluconic acid stopped. In 10 different media of initial pH 4.0–7.0, No. 4 mycelia showed higher gluconic acid secretion with higher initial pH. No. 4 mycelial grew best in pH 5.0 media, in which gluconic acid secretion was low. Mycelia of 29 isolates including No. 4 grew better in the media in which less glucose, total carbon and total nitrogen remained, and almost all isolates secreted gluconic acid. Most of the 29 isolates showed irregular colony shapes with rough mycelial fronts, brown pigmentation and aerial hypha on colony surfaces, and brown pigmentation of media under colonies. Dissimilarities were calculated with coded morphological characters on colonies, and similarity between isolates was found not to correlate with proximity of origin. Chlamydospores were observed on every colony of the 29 isolates. Chlamydospores were present on colonies of No. 4, reaching to 2 mm from the mycelial front, where brown pigmentation had not yet developed, and the numbers of chlamydospores incresed with mycelial aging.     

Heavy metals have different effects on mycelial morphology of Achlya bisexualis as determined by fractal geometry

The morphological response, as measured by changes to mycelial area, radial extension and border fractal dimension, of the oomycete Achlya bisexualis to Cu, Co, Hg, Zn and Cd at concentrations of between 0.05 and 3 mM is described. All of the metals decreased mycelial area and radial extension. Border fractal dimension increased in the presence of Cu, Co and Hg with individual hyphae extending out beyond the mycelial margin. In the presence of 3 mM Hg these hyphae displayed spiral growth. Zn and Cd had no effect on border fractal dimension. We suggest that all of the metals slow growth and that Cu, Co and Hg may also disrupt the relationship between tip growth and branching at the edge of the mycelium.     

Compensatory growth of Phanerochaete velutina mycelial systems grazed by Folsomia candida (Collembola)

Phanerochaete velutina is a major agent of wood decomposition in temperate forests. It grows out of woody resources in search of other resources and is then vulnerable to grazing by invertebrates. The aim of this study was to determine how continuous grazing and grazing for only 2 days by different densities of collembola, Folsomia candida, affect mycelial development (radial extension, hyphal coverage and fractal dimension) of P. velutina growing across non-sterile soil. High density (80 collembola) continuous grazing resulted in different mycelial foraging patterns compared to controls and lower density (20 and 40 collembola) continuous grazing: radial extension rate was reduced from 8.4 mm day(-1) (control) to 6.9 mm day(-1) (80 collembola), hyphal coverage was reduced to 81% of controls and mass fractal dimension increased from 1.68 (control) to 1.72 (80 collembola). There was evidence of over-compensatory growth: when high density grazing ceased the new growth was considerably greater (38%) than in controls. Grazing also resulted in growth stimulation: at low density continuous grazing (20 collembola) hyphal coverage was 15.6% greater than in controls. The ecological implications of compensatory and stimulatory growth in fungal-invertebrate interactions are considered.     

The effect of dissolved carbon dioxide on penicillin production: Mycelial morphology

The effect of carbon dioxide on the morphology of Penicillium chrysogenum was examined. The scanning electron microscopic (SEM) study indicated that the morphology of P. chrysogenum was subject to change when exposed to various dissolved CO₂ concentrations in the medium. At low influent carbon dioxide partial pressures between 0% and 8%, the predominant morphological form of P. chrysogenum was filamentous. At higher influent carbon dioxide partial pressures of 15% and 20%, the appearance of swollen and stunted hyphae predominated, and a significant quantity of spherical or yeast-like cells were observed. It was evident that for production subject to high dissolved CO₂ concentrations the inhibition of cell growth and penicillin production related strongly to the concomitant morphological changes of P. chrysogenum.     

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.     

Different mechanisms of the biochemical adaptation of mycelial fungi to temperature stress: Changes in the lipid composition

A comparative study was conducted concerning the effect of temperature stress on the lipid composition of representatives of the subkingdomsEomycota andNeomycota. Changes in the composition of lipid acyl chains (such as saturation and desaturation, isomerization, and changes in the length of fatty acid carbon chains), in the phospholipid composition, and in the contents of sterols and other neutral lipids were revealed. Hyperthermia resulted in (i) an increase in the phosphatidylcholine level, (ii) a decrease in the phosphatidylethanolamine level, (iii) a rise in the content of reserve lipids (triacylglycerols), and (iv) a decline in the free fatty acid level in the neutral lipids. An inverse pattern occurred under hypothermic conditions. The peculiarities in the patterns of the temperature adaptation-related changes in the lipid bilayer composition are considered in terms of the systematic position of the fungi.     

Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738

AIMS: The objective of the present study was to determine the optimal culture conditions for mycelial biomass and exo-polysaccharide (EPS) by Cordyceps militaris C738 in submerged culture. METHODS AND RESULTS: The optimal temperatures for mycelial biomass and EPS production were 20 degrees C and 25 degrees C, respectively, and corresponding optimal initial pHs were found to be 9 and 6, respectively. The suggested medium composition for EPS production was as follows: 6% (w/v) sucrose, 1% (w/v) polypeptone, and 0.05% (w/v) K2HPO4. The influence of pH on the fermentation broth rheology, morphology and EPS production of C. militaris C738 was carried out in a 5-l stirred-tank fermenter. The morphological properties were comparatively characterized by pellet roughness and compactness by use of image analyser between the culture conditions with and without pH control. The roughness and compactness of the pellets indicated higher values at pH-stat culture (pH 6.0), suggesting that larger and more compact pellets were desirable for polysaccharide production (0.91 g g(-1) cell d(-1). CONCLUSIONS: Under the optimized culture conditions (with pH control at 6), the maximum concentration of biomass and EPS were 12.7 g l(-1) and 7.3 g l(-1), respectively, in a 5-l stirred-tank fermenter. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effect of pH on fungal morphology and rheology presented in this study can be widely applied to other mushroom fermentation processes.     

Choline: Its role in the growth of filamentous fungi and the regulation of mycelial morphology

Abstract Choline is an essential metabolite for the growth of filamentous fungi. It occurs most notably as a component of the major membrane phospholipid, phosphatidyl choline (lecithin), and fulfills a major role in sulphate metabolism in the form of choline- o -sulphate in many species. Choline is usually synthesised endogenously, but exogenous choline can also be taken up, either to compensate for metabolic deficiencies in choline-requiring mutants such as those of Aspergillus nidulans and Neurospora crassa , or as a normal function by species such as Fusarium graminearum which do not require added choline for growth. F. graminearum has a highly specific constitutive uptake system for this purpose. Recent studies have begun to indicate that choline also plays an important role in hyphal and mycelial morphology. Over a wide range of concentrations, choline influences mycelial morphology, apparently influences mycelial morphology, apparently by controlling branch initiation. At high concentrations of added choline, branching is inhibited but specific growth rate is unaffected, leading to the production of rapidly extending, sparsely branched mycelia. Reduction of choline concentration allows a progressive increase in branching. Additionally, in choline-requiring mutants which have a very reduced content of choline, multiple tip-formation and apical branching occurs. Just prior to cessation of growth in choline-starved cultures of A. nidulans choline-requiring mutants, hyphal morphology changes due to a brief phase of unpolarised growth to produce spherical swellings called ballons, at or near hyphal apices. The precise mechanism by which choline affects fungal morphology is not yet known, although in A. nidulans it appears to be at least partially due to the influence of membrane composition on the synthesis of the hyphal wall polymer chitin. Several hypotheses for the possible mode of action of choline in affecting fungal morphology are discussed here.     

Effect of deletion of chitin synthase genes on mycelial morphology and culture viscosity in Aspergillus oryzae

The objective of this study was to quantify the effect of disrupting two chitin synthases, chsB and csmA, on the morphology and rheology during batch cultivation of Aspergillus oryzae. The rheological properties were characterized in batch cultivations at different biomass concentrations (from 3.4-22.5 g kg(-1) biomass) and the power-law model adequately described the rheological properties. In the cultivations there were pellets, clumps, and freely dispersed hyphal elements. The different morphological fractions were quantified using image analysis. The apparent viscosity of the fermentation broth was significantly affected by the biomass concentration, the morphology, and also by pH. The chsB disruption strain had lower consistency index K values for all biomass concentrations investigated, which is a desirable trait for industrial Aspergillus fermentations.     

A mathematical model for the leukocyte filtration process

Leukocyte filters are applied clinically to remove leukocytes from blood. In order to optimize leukocyte filters, a mathematical model to describe the leukocyte filtration process was developed by modification of a general theoretical model for depth filtration. The model presented here can be used to predict the time-dependent leukocyte filtration as a function of cell-cell interaction in the filter, filter efficiency, filter capacity, filter dimensions, and leukocyte concentration in the suspension applied to the filter. The results of different leukocyte filtration experiments previously reported in the literature could be well described by the present model. (c) 1995 John Wiley & Sons, Inc.     

Optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by Cordyceps jiangxiensis JXPJ 0109

AIMS: The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. METHODS AND RESULTS: The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min(-1), seed age 4-8 days, inoculum size 2.5-7.5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26 degrees C and pH 5 and at 28 degrees C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl2 > glycerol > MgSO4 > KH2PO4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH2PO4, 0.2 g MgSO4, and 0.5 g CaCl2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH2PO4, and 0.5 g CaCl2 in 1 l of distilled water. CONCLUSIONS: Under the optimal culture requirements, the maximum exopolysaccharide production reached 3.5 g l(-1) after 10 days of fermentation, while the maximum production of mycelial growth achieved 14.5 g l(-1) after 8 days of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.     

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.