Enhancing gas-liquid mass transfer rates in non-newtonian fermentations by confining mycelial growth to microbeads in a bubble column

The performance of a penicillin fermentation was assessed in a laboratory-scale bubble column fermentor, with mycelial growth confined to the pore matrix of celite beads. Final cell densities of 29 g/L and penicillin titres of 5.5 g/L were obtained in the confined cell cultures. In comparison, cultures of free mycelial cells grown in the absence of beads experienced dissolved oxygen limitations in the bubble column, giving only 17 g/L final cell concentrations with equally low penicillin titres of 2 g/L. The better performance of the confined cell cultures was attributed to enhanced gas liquid mass transfer rates, with mass transfer coefficients (k(L)a) two to three times higher than those determined in the free cell cultures. Furthermore, the confined cell cultures showed more efficient utilization of power input for mass transfer, providing up to 50% reduction in energy requirements for aeration.     

Transformation of the ryegrass endophyte Neotyphodium lolii can alter its in planta mycelial morphology

The fungus Neotyphodium lolii grows in the intercellular spaces of perennial ryegrass as a mutualistic endosymbiont. One of the benefits it conveys to the plant is the production of alkaloids toxic to herbivores. We wanted to determine in planta expression patterns of the N. lolii 3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA reductase) gene, believed to be involved in the synthesis of two of these alkaloid toxins, lolitrem B and ergovaline. We transformed the N. lolii strain Lp19 with plasmids, in which DNA fragments upstream of the open reading frame of the N. lolii HMG CoA reductase gene controlled expression of the GUS (gusA; Escherichia coli β-glucuronidase) reporter gene. In exponentially growing cultures, the GUS gene was not expressed if the length of upstream sequence was less than 400 bp, and >1100 bp were required for maximum expression. When reintroduced into ryegrass plants, transformants often showed highly increased hyphal branching compared to the wild-type parent strain, although in culture their growth kinetics and morphology were indistinguishable from that of the wild-type. Deterioration of hyphae and the hypha–plant interface occurred and in one transformant reduced tillering (formation of new plants, referred to in agronomy as tillers) and death of infected plants. We found no evidence that these abnormalities were caused by interference of the construct with the function of the native gene, as judged by analysis of the site of integration of the promoter-GUS cassette, expression of the native gene and lolitrem B and ergovaline levels in infected plants. However, there was some correlation between GUS expression and the degree of hyphal branching, suggesting that high levels of β-glucuronidase may disturb the symbiotic interaction. Levels of another alkaloid, peramine, were also not significantly affected by transformation. In previous studies increased in planta branching of the endophyte has been shown to be associated with a severe reduction of alkaloid production. Our results show that a plant–endophyte association in which increased branching occurs is still able to produce alkaloids.     

Unstructured model for growth of mycelial pellets in submerged cultures

An unstructured model is presented to describe growth of mycelial pellets in submerged cultures. This model integrates growth kinetics at the scale of the hyphae with the physical mechanisms of mass-transfer processes at the scale of the pellets and the fermentor. The main elements of the model are biomass, substrate, and oxygen balances for the liquid phase and the pellets. The possible occurrence of oxygen limitation in the pellets is introduced in analogy with catalyst theories by means of an effectiveness factor. To simulate the growth of pellets the model is transferred into a computer program. The model is tested by means of fermentation experiments in a bubble column. Results of the growth experiments compare favorably with the outcome of computer simulations.     

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.     

Distribution functions of variables characterizing the mycelial morphology of Streptomyces hygroscopicus grown in glucose-limited chemostat cultures

The distribution of variables characterizing the morphology of the mycelium of Streptomyces hygroscopicus grown in glucose-limited chemostat cultures at different specific growth rates were investigated statistically. The values of the hyphal growth unit (L/N) and the values of the distance from the apex to the first branch (Lp) are normally distributed, but the values of the distance between neighbouring branches are logarithmically normal distributed. The distribution functions are discussed from the biological point of view.     

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.     

The composition of Camembert cheese-ripening cultures modulates both mycelial growth and appearance

The fungal microbiota of bloomy-rind cheeses, such as Camembert, forms a complex ecosystem that has not been well studied, and its monitoring during the ripening period remains a challenge. One limitation of enumerating yeasts and molds on traditional agar media is that hyphae are multicellular structures, and colonies on a petri dish rarely develop from single cells. In addition, fungi tend to rapidly invade agar surfaces, covering small yeast colonies and resulting in an underestimation of their number. In this study, we developed a real-time quantitative PCR (qPCR) method using TaqMan probes to quantify a mixed fungal community containing the most common dairy yeasts and molds: Penicillium camemberti, Geotrichum candidum, Debaryomyces hansenii, and Kluyveromyces lactis on soft-cheese model curds (SCMC). The qPCR method was optimized and validated on pure cultures and used to evaluate the growth dynamics of a ripening culture containing P. camemberti, G. candidum, and K. lactis on the surface of the SCMC during a 31-day ripening period. The results showed that P. camemberti and G. candidum quickly dominated the ecosystem, while K. lactis remained less abundant. When added to this ecosystem, D. hansenii completely inhibited the growth of K. lactis in addition to reducing the growth of the other fungi. This result was confirmed by the decrease in the mycelium biomass on SCMC. This study compares culture-dependent and qPCR methods to successfully quantify complex fungal microbiota on a model curd simulating Camembert-type cheese.     

Scaling from mycelial growth to infection dynamics: a reaction diffusion approach

Numerous models have been proposed for the dynamics of fungal growth, and also for the dynamics of infection. Few models, however, have combined the mechanistic interpretation of mycelial growth with epidemiological models for the transmission of infection. Many of the mechanistic models seek to include considerable biological detail, which necessarily leads to a proliferation of state variables and parameters. Including such models within an epidemiological framework makes interpretation of underpinning processes difficult. A simple reaction diffusion model for the growth and spread of fungal mycelium is introduced and analysed, scaling from the small-scale parameters for mycelial dynamics to the large-scale properties of the colony. By coupling the output to a parsimonious epidemiological model for the dynamics of primary infection, we analyse the sensitivity of the probability of successful infection of a host to the colony dynamics associated with local bulking-up, extension, growth and nutrient consumption by the mycelium. In particular we identify optimal trade-offs in bulking-up versus dispersal in controlling infection dynamics.     

Effects of cultivating conditions on the mycelial growth of Ganoderma lucidum in submerged flask cultures

In this paper the effects of environmental conditions on the mycelial growth of Ganoderma lucidum were investigated in shake flask cultures. The optimal temperature and pH were found to be around 30–35?°C and 4, respectively, in a glucose-ammonium chloride medium. The maximum mycelial concentration reached to around 350?mg/100?ml. The formation of mycelial pellets and their ultra structure was demonstrated to be greatly affected by cultivating conditions. Increasing surface aeration would be beneficial for mycelial growth. However, too high rotating speed in shake cultures had a detrimental effect on the formation of mycelial pellets and the optimum was found to be 100?rpm.     

The effect of heat shock on the growth and mycelial morphology of Streptomyces chrysomallus]

The effect of various conditions of heat shock (1 hour at 35, 38, 40, 42, 45 and 50 degrees C) on the growth and morphological features of Streptomyces chrysomallus, an organism producing actinomycin, was studied. A definite regularity in the mycelium morphological changes at high temperatures was observed. After the shock at 35 and 38 degrees C the biomass volume and morphological features of the streptomycete did not markedly differ from those in the control. The shock at 40 degrees C induced the growth inhibition with decreasing the biomass volume by 50 per cent and appearance of submerged spores. When the shock conditions were more rigid (42, 45 and 50 degrees C) the mycelium growth lacked. It is of interest that the temperature of 42 degrees C induced abundant formation of the spores. With further increasing of the temperature to 45 and 50 degrees C the spore formation was not so abundant. The changes in the growth and development of the streptomycete are discussed in relation to the molecular mechanism of the cell protection from temperature shock.     

Effects of agitation intensity on mycelial morphology and protein production in chemostat cultures of recombinant Aspergillus oryzae

The effects of agitation on fragmentation of a recombinant strain of Aspergillus oryzae and its consequential effects on protein production have been investigated. Constant mass, 5.3-L chemostat cultures at a dilution rate of 0.05 h-1 and a dissolved oxygen level of 75% air saturation, have been conducted at 550, 700, and 1000 rpm. These agitation speeds were chosen to cover a range of specific power inputs (2.2 to 12 kW m-3) from realistic industrial levels to much higher values. The use of a constant mass chemostat linked to a gas blender allowed variation of agitation speed and hence gas hold-up without affecting the dilution rate or the concentration of dissolved oxygen. The morphology of both the freely dispersed mycelia and clumps was characterized using image analysis. Statistical analysis showed that it was possible to obtain steady states with respect to morphology. The mean projected area at each steady state under growing conditions correlated well with the 'energy dissipation/circulation" function, [P/(kD3tc)], where P is the power input, D the impeller diameter, tc the mean circulation time, and k is a geometric constant for a given impeller. Rapid transients of morphological parameters in response to a speed change from 1000 to 550 rpm probably resulted from aggregation. Protein production (alpha-amylase and amyloglucosidase) was found to be independent of agitation speed in the range 550 to 1000 rpm (P/V = 2.2 and 12.6 kW m-3, respectively), although significant changes in mycelial morphology could be measured for similar changes in agitation conditions. This suggests that mycelial morphology does not directly affect protein production (at a constant dilution rate and, therefore, specific growth rate). An understanding of how agitation affects mycelial morphology and productivity would be valuable in optimizing the design and operation of large-scale fungal fermentations for the production of recombinant proteins. Copyright 1999 John Wiley & Sons, Inc.     

Species interactions alter evolutionary responses to a novel environment

Studies of evolutionary responses to novel environments typically consider single species or perhaps pairs of interacting species. However, all organisms co-occur with many other species, resulting in evolutionary dynamics that might not match those predicted using single species approaches. Recent theories predict that species interactions in diverse systems can influence how component species evolve in response to environmental change. In turn, evolution might have consequences for ecosystem functioning. We used experimental communities of five bacterial species to show that species interactions have a major impact on adaptation to a novel environment in the laboratory. Species in communities diverged in their use of resources compared with the same species in monocultures and evolved to use waste products generated by other species. This generally led to a trade-off between adaptation to the abiotic and biotic components of the environment, such that species evolving in communities had lower growth rates when assayed in the absence of other species. Based on growth assays and on nuclear magnetic resonance (NMR) spectroscopy of resource use, all species evolved more in communities than they did in monocultures. The evolutionary changes had significant repercussions for the functioning of these experimental ecosystems: communities reassembled from isolates that had evolved in polyculture were more productive than those reassembled from isolates that had evolved in monoculture. Our results show that the way in which species adapt to new environments depends critically on the biotic environment of co-occurring species. Moreover, predicting how functioning of complex ecosystems will respond to an environmental change requires knowing how species interactions will evolve.     

A fractal model for the characterization of mycelial morphology

A new technique based on a fractal model has been developed for the quantification of the macroscopic morophology of mycelia. The morphological structuring is treated as a fractal object, and the fractal dimension, determined by an ultrasonic scattering procedure developed for the purpose, serves as a quantitative morphological index. Experimental observations reported earlier and simulations of mycelial growth, carried out using a probabilistic-geometric growth model developed for the purpose, both validate the applicability of the fractal model. In experiments with three different species, the fractal dimensions of pelletous structures were found to be in the range 1.45-2.0 and those of filamentous structures were in the range 1.9-2.7, with values around 2.0 representing mixed morphologies. Fractal dimensions calculated from simulated mycelia are in rough agreement with these ranges. The fractal dimension is also found to be relatively insensitive to the biomass concentration, as seen by dilution of the original broths. The relation between morphology and filtration properties of the broths has also been studied. The fractal dimension shows a strong correlation with the index of cake compressibility and with the Kozeny constant, two filtration parameters that are known to be morphology dependent. This technique could thus be used to develop correlations between the morphology, represented by the fractal dimension, and important morphology-dependent process variables. (c) 1993 John Wiley & Sons, Inc.     

Elimination of mycoplasmas from cell cultures by a novel soft agar technique

Summary Mycoplasmal infection of cell cultures remains a significant threat to diagnostic and research procedures. In certain defined situations, curing of mycoplasmal infected cultures is a reasonable exercise. Four methods of curing were compared: treatment with BM-cycline, 5 bromouracil, use of specific antisera and treatment of infected cells suspended in soft agar with antibiotics. Antisera treatments were of low efficiency of curing: 50%. None of nine infected cell lines treated with 5-bromouracil were consistently cured of mycoplasmas. The use of BM-cycline was effective for some, but not all lines and required long periods of treatment, 12–21 days. 35 naturally or deliberately infected cultures were treated in soft agar a total of 119 times. This procedure which consisted of suspending infected cultures in soft agar containing appropriate antibiotics resulted in successful mycoplasmal elimination 118/119 times. This soft agar technique took 1–3 days. In separate studies, it was shown that certainMycoplasma fermentans strains were resisted to this and other curing methods. This may be due to their intracellular location. Such strains may be more amenable to antibiotics that penetrate mammalian cells. It is concluded that the soft agar technique is a rapid, efficient and reliable method to eliminate cell culture mycoplasmas. These studies were supported in part by grant 15748 from the National Institute of Allergy and Infectious Diseases and the W. W. Smith Charitable Trust.     

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.     

Lanostane triterpenoids from mycelial cultures of the basidiomycete Ganoderma weberianum

Eight undescribed lanostane triterpenoids (1–8) were isolated from cultures of the basidiomycete Ganoderma weberianum TBRC-BCC 60642. The structural types of lanostane constituents were different between mycelial cultures and fruiting bodies. Lanostanes 2, 3 and 6 showed moderate antimalarial activity against Plasmodium falciparum K1 (IC₅₀ 10–15 μM).     

Effect of fatty acids on the mycelial growth and polysaccharide formation by Ganoderma lucidum in shake flask cultures

Fatty acids were added into the media to investigate their effects on the mycelial growth and polysaccharide formation by Ganoderma lucidum. The experiments were carried out in freely suspended cultures or immobilized cultures using shake flasks. The results indicate that the extent of stimulation or inhibition were associated with the types and levels of fatty acids. Oleic acid at the level of 0.15 g/100 ml led to a significant increase in cell concentration from 0.20 to 0.46 g/100 ml in a suspended culture and palmitic acid was of great advantage to polysaccharide production. In contrast, linoleic acid (0.1 g/100 ml) drastically suppressed both mycelial growth and polysaccharide formation. In immobilized cultures with fatty acids, the stimulation of mycelial growth remained the same level, but the enhancement of polysaccharide production became less. In addition, the growth of G. lucidum in the pattern of immobilization might be beneficial to the production of mycelia and polysaccharide.     

FKBP133: a novel mouse FK506-binding protein homolog alters growth cone morphology

FK506-binding proteins are the peptidyl prolyl cis-trans isomerases that are involved in various intracellular events. We characterized a novel mouse FK506-binding protein homolog, FKBP133/KIAA0674, in the developing nervous system. FKBP133 contains a domain similar to Wiskott-Aldrich syndrome protein homology region 1 (WH1) and a domain homologous to FK506-binding protein motif. FKBP133 was predominantly expressed in cerebral cortex, hippocampus, and peripheral ganglia at embryonic day 18.5. FKBP133 protein was distributed in the axonal shafts and was partially co-localized with F-actin in the growth cones of dorsal root ganglion neurons (DRG). The number of filopodia was increased in the DRG neurons overexpressing FKBP133. In contrast, the overexpression of a mutant deleted the WH1 domain reduced the growth cone size and the number of filopodia. Furthermore, the neurons overexpressing FKBP133 became significantly resistant to Semaphorin-3A induced collapse response. These results suggest that FKBP133 modulates growth cone behavior with the WH1 domain.     

Positive reinforcement training as a technique to alter nonhuman primate behavior: quantitative assessments of effectiveness

Many suggest that operant conditioning techniques can be applied successfully to improve the behavioral management of nonhuman primates in research settings. However, relatively little empirical data exist to support this claim. This article is a review of several studies that discussed applied positive reinforcement training techniques (PRT) on breeding/research colonies of rhesus macaques (Macaca mulatta) and chimpanzees (Pan troglodytes) at The University of Texas M. D. Anderson Cancer Center and measured their effectiveness. Empirical analyses quantified the amount of time required to train rhesus monkeys to come up, station, target, and stay. Additionally, a study found that time spent affiliating by female rhesus was changed as a function of training low affiliators to affiliate more and high affiliators to affiliate less. Another study successfully trained chimpanzees to feed without fighting and to come inside on command. PRT is an important behavioral management tool that can improve the care and welfare of primates in captivity. Published empirical findings are essential for managers to assess objectively the utility of positive reinforcement training techniques in enhancing captive management and research procedures.