Classification and measurement of fungal pellets by automated image analysis

An automated image analysis method for classifying and measuring pellets of filamentous fungi growing in submerged fermentations has been developed. The method discriminates between pelleted mycelial growth and loose aggregates of dispersed hyphae. Pellets are classified into smooth and hairy types. In both cases, the core of the pellet is identified and its shape and size characterized. For hairy pellets the annular region is also characterized. The method was tested on pellets of Aspergillus niger ATCC 11414 grown in a defined medium in shake flasks. This rapid method makes practical extensive studies on the morphology of pellets in submerged fermentations and the influence of fermentation conditions on that morphology.     

Loss of NAD(P)-reducing power and glutathione disulfide excretion at the start of induction of aerial growth in Neurospora crassa.

When exponentially growing hyphae of Neurospora crassa in aerated liquid cultures are filtered and the resulting mycelial mat is exposed to air, aerial hyphae develop and synchronous conidiation is obtained. The hyphae in direct contact with air adhere to each other within minutes and form aerial hyphae during the following 12 h; the hyphae which are not in direct contact with air do not adhere to each other and do not form aerial hyphae. Previous data indicated that oxidative stress was generated in the adhering hyphae; proteins and specific enzymes were found to be oxidatively modified and degraded. In this work, we report a dramatic fall in the reduced-to-oxidized ratio of NAD and NADP coenzymes during the first 6 min of exposure to air. This drop did not occur in a mycelial mat exposed to a N2-enriched atmosphere. Adding a carbon source to the mycelial mat did not abolish the loss of NAD(P)-reducing power. After the initial fall, the reducing levels of the coenzymes returned to the starting value in about 30 min. A peak of extracellular glutathione disulfide occurred simultaneously with the loss of NAD(P)-reducing power. The reducing power loss and the excretion of glutathione disulfide are thought to be consequences of a hyperoxidant state; the adhesion of hyphae is thought to be a response to the hyperoxidant state.     

Scanning Electron Microscope Studies of Interactions between Agaricus bisporus (Lang) Sing Hyphae and Bacteria in Casing Soil

Relationships between the hyphae of Agaricus bisporus (Lang) Sing and bacteria from the mushroom bed casing layer were examined with a scanning electron microscope. Hyphae growing in the casing layer differed morphologically from compost-grown hyphae. Whereas the compost contained thin single hyphae surrounded by calcium oxalate crystals, the casing layer contained mainly wide hyphae or mycelial strands without crystals. The bacterial population in the hyphal environment consisted of several types, some attached to the hyphae with filamentlike structures. This attachment may be important in stimulation of pinhead initiation.     

Alkane-grown Beauveria bassiana produce mycelial pellets displaying peroxisome proliferation,oxidative stress,and cell surface alterations

The entomopathogenic fungus Beauveria bassiana is able to grow on insect cuticle hydrocarbons, inducing alkane assimilation pathways and concomitantly increasing virulence against insect hosts. In this study, we describe some physiological and molecular processes implicated in growth, nutritional stress response, and cellular alterations found in alkane-grown fungi. The fungal cytology was investigated using light and transmission electron microscopy while the surface topography was examined using atomic force microscopy. Additionally, the expression pattern of several genes associated with oxidative stress, peroxisome biogenesis, and hydrophobicity were analysed by qPCR. We found a novel type of growth in alkane-cultured B. bassiana similar to mycelial pellets described in other alkane-free fungi, which were able to produce viable conidia and to be pathogenic against larvae of the beetles Tenebrio molitor and Tribolium castaneum. Mycelial pellets were formed by hyphae cumulates with high peroxidase activity, exhibiting peroxisome proliferation and an apparent surface thickening. Alkane-grown conidia appeared to be more hydrophobic and cell surfaces displayed different topography than glucose-grown cells. We also found a significant induction in several genes encoding for peroxins, catalases, superoxide dismutases, and hydrophobins. These results show that both morphological and metabolic changes are triggered in mycelial pellets derived from alkane-grown B. bassiana.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

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.     

Isolation and morphological characterization of a mycelial mutant of Candida albicans.

In this paper we describe the isolation of a novel strain of Candida albicans which is a mycelium at ambient temperatures. Mutagenesis of C. albicans ATCC 10261 with N-methyl-N-nitro-N-nitrosoguanidine followed by plating on solid media at 28 degrees C yielded colony morphology variants which were characterized by a raised, rough-surfaced colony of irregular outline in marked contrast to the flat, shiny circular colonies of the parental 10261 strain. One mutant colony, hOG301, was studied in detail. Strain hOG301 was stable and exhibited mycelial morphology over a wide temperature range (5 to 40 degrees C) in several media. The hyphae comprising hOG301 mycelium were examined by light microscopy, scanning electron microscopy, and transmission electron microscopy and showed morphological features described in the literature as being typical of both true hyphae and pseudohyphae. In contrast to 10261, hOG301 was not pathogenic after intraperitoneal injection in mice. This is the first report of a mycelial C. albicans that is stable at ambient temperatures.     

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.     

The role of initial spore adhesion in pellet and biofilm formation in Aspergillus niger

Fungi grow on a great variety of organic and inorganic materials. Colony establishment and growth on solid surfaces require adhesion of spores and hyphae to the substrate, while cell-to-cell interactions among spores and/or hyphae are a prerequisite for the development of three-dimensional mycelial structures such as pellets or biofilms. Surface adherence has been described as a two-step process, comprised of the initial attachment of ungerminated conidia followed by further adhesion of the forming germ tubes and growing hyphae. In the present study, we analyzed the contribution of adhesion of ungerminated spores to pellet and biofilm formation in Aspergillus niger. Mutants deficient in melanin biosynthesis were constructed by the deletion of the alb1 gene, encoding a polyketide synthase essential for pigment biosynthesis. Δalb1 conidia have an altered surface structure and changed physicochemical surface properties. Spore aggregation in liquid culture as well as spore surface attachment differ between the wild type and the mutant in a pH-dependent manner. In liquid culture further pellet formation is unaffected by altered spore-spore interactions, indicating that germ tube and hyphal adherence can compensate for deficiencies in the initial step of spore attachment. In contrast, under conditions promoting adhesion of Δalb1 conidia to polymer surfaces the mutant forms more stable biofilms than the wild type, suggesting that initial spore adhesion supports sessile growth.     

Mycelial pellet formation by Penicillium ochrochloron species due to exposure to pyrene

Five indigenous fungal strains with characteristics of the genus Penicillium capable of degrading and utilizing pyrene, as sole carbon source were isolated from soil of a former gas work site. Two strains were identified as Penicillium ochrochloron. One of the strains was able to degrade a maximum of 75% of 50 mg l⁻¹ pyrene at 22 °C during 28 days of incubation. The presence of pyrene in the medium resulted in an aggregation of hyphae into pellets by the two Penicillium ochrochloron strains. Formation of pellets was observed after 48 h of incubation with difference in size and texture between the two strains. This indicated the individual variation within the same genus of fungi. However, remaining strains did not show this behavior even though they were capable of utilizing pyrene as sole carbon source. The macro- and microscopic morphology of fungal pellets was studied using scanning electron microscopy. It was found that the addition of varying concentration of pyrene ranging from 10 to 50 mg l⁻¹ in the medium influenced shape and structure of the mycelial pellets. A two-fold increase in hyphal branching (with concomitant decrease in the average hyphal growth unit) was observed at a concentration of 10 mg l⁻¹. The relevance of fungal growth and morphology for bioremediation of polycyclic aromatic hydrocarbons (PAHs) contaminated sites are discussed.     

食用外共生菌根真菌兰茂牛肝菌Lanmaoa asiatica纯培养条件下不同发育时期的核相

食用外共生菌根菌是一类可食用并与植物共生的大型真菌,其在纯培养条件下菌丝生长缓慢,不会扭结发育成原基,不能完成生活史。因此关于食用菌根菌纯培养条件下生活史的研究报道极少。本研究的兰茂牛肝菌Lanmaoa asiatica已能在纯培养条件下诱导出原基,这使生活史的研究得以完成。利用扫描电子显微镜、透射电子显微镜及荧光显微镜对兰茂牛肝菌子实体担子、担孢子、纯培养菌丝和原基的核相进行了观察。结果表明,子实体菌丝细胞为双核,担子经过减数分裂形成4个单核担孢子;担孢子萌发时长轴端长出芽状突起伸长为菌丝,原孢子中的细胞核不分裂,直接进入菌丝,形成单核初生菌丝;初生菌丝5d后变为双核菌丝;纯培养菌丝及原基细胞均为双核,其菌丝表面光滑,未观察到锁状联合。     

两种方法形成菌丝球固定2-氯酚降解菌的对比

【目的】系统研究吸附法和同时培养法对所形成混合菌丝球的外观形态、内部结构及其去除2-氯酚效果的影响。【方法】采用吸附法和同时培养法将可降解2-氯酚的光合细菌PSB-1D固定在黄孢原毛平革菌(Phanerochaete chrysosporium)DH-1发酵而成的菌丝球上,形成混合菌丝球。以单一菌丝球为对照,利用光学显微镜、扫描电镜等仪器观察混合菌丝球的外观形态和内部结构,考察2种方法对混合菌丝球成球效果的影响;以无菌培养液为空白对照,考察游离光合细菌、单一菌丝球、2种方法形成混合菌丝球对2-氯酚的降解效能。【结果】在吸附法形成的混合菌丝球上,光合细菌主要集中在过渡区;而同时培养法将光合细菌牢固地包埋在菌丝球内核区,并大量簇状附着生长在菌丝交联的空隙处和每根菌丝上。在接种等量孢子和光合细菌的前提下,同时培养法较吸附法操作时间更短,成球数量更多,形成菌丝球干湿比更大,单位菌丝干重上固定的细菌数量更多。菌丝球降解体系和游离光合细菌对2-氯酚的降解均符合一级动力学特征。同时培养法形成的混合菌丝球降解效果最好,7 d内对初始浓度为50 mg/L的2-氯酚降解率可达89%以上,降解速率常数为0.3286 mg/(L·d),2-氯酚半衰期t1/2为2.8 d。【结论】首次报道黄孢原毛平革菌包埋固定化光合细菌形成混合菌丝球。该研究为生物质固定化材料的实际应用提供理论依据。     

Method To Enhance Growth and Sporulation of Pelletized Biocontrol Fungi

The biocontrol fungi Trichoderma harzianum, used to control soilborne plant pathogens, and Beauveria bassiana, used to control insect pests, were formulated as mycelial biomass in alginate pellets with wheat bran added. After drying for 0, 4, or 16 h, pellets were placed in water or in aqueous solutions of polyethylene glycol (PEG) 8000 for 4 to 24 h and then allowed to continue drying. PEG-treated pellets containing T. harzianum showed significantly greater proliferation of hyphae in soil than untreated pellets or pellets treated with water. Production of conidia of T. harzianum from PEG-treated pellets was lower than production from untreated pellets after 4 days, although rates were equivalent after 7 days. In contrast, production of conidia of B. bassiana was significantly more rapid from PEG-treated pellets than from untreated pellets. Biocontrol of soilborne plant pathogens or insect pests may be enhanced by rapid hyphal growth of T. harzianum in soil or rapid sporulation of B. bassiana on foliage, respectively. Therefore, PEG treatment may improve the efficacy of these biocontrol agents.     

Production of GFP and glucoamylase by recombinant Aspergillus niger: effects of fermentation conditions on fungal morphology and protein secretion

The effect of filamentous fungal morphology on heterologous protein secretion was investigated using the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP], which contained the gene coded for the GLA-GFP (glucoamylase-green fluorescence protein) fusion protein. Three culturing systems were studied to develop different morphological forms of the fungus. Free-cell cultures in conventional stirred-tank bioreactors grew in pellet form with various sizes depending on culturing conditions. Cells immobilized on cotton cloth grew in mycelial form in a rotating fibrous bed (RFB) and a static fibrous bed (SFB) bioreactors. The expression of the fusion protein was growth-associated and dependent on the fungal morphology. Immobilized cells produced 10-fold more GFP and glucoamylase than well-oxygenated free-cell pellets. In free-cell cultures, excretion of the fusion protein occurred mainly from cell autolysis, when oxygen or nutrient were depleted, whereas protein secretion took place from the beginning of the fermentation in immobilized-cell cultures. Also, protein secretion was found to be strongly dependent on morphology. Small pellets of a 1-mm size secreted 82% of GFP produced, whereas 43% of GFP remained intracellular in larger pellets of 5 mm. Complete secretion of GFP was obtained with cells immobilized on the fibrous matrix. The improvement in heterologous protein synthesis and secretion can be attributed to the filamentous mycelial morphology since protein secretion occurred predominantly at the tips of growing hyphae. Secretion of proteases occurred mainly in the stationary phase or when cell autolysis were induced by nutrient depletion and was not dependent on morphology, although immobilizing the cells also reduced protease activity. The RFB bioreactor gave the best fermentation performance because of its ability to control the cell morphology that was amenable to efficient oxygen transfer and protein secretion.     

Appressoria of the nematophagous fungus verticillium suchlasporium

Appressoria of the nematophagous fungus Verticillium suchlasporium infecting eggs of the beetroot cyst nematode Heterodera schachtii were studied by field emission scanning electron microscopy (FESEM). Protoappressoria and mature appressoria were found in both germ tubes and mycelial hyphae, and mucilaginous material was seen where the appressoria contacted the surface of the egg. The role of this material in the penetration of the eggs by the hyphae is discussed.     

Strategies of food substrate colonization by mycelial fungi

This article deals with a new research area related to mycology and the food industry. It is concerned with the strategies used by ascomycete mycelial fungi to utilize and colonize food items. The data of scanning and transmission electron and light microscopy revealed that fungi growing on such food items as cheese and sausage are characterized by the following colonization strategies: (i) spreading growth; (ii) burrowing growth; (iii) growth with the formation of up to 4–5 new branches on the apexes of the old hyphae; (iv) growth with the formation of long strands, and (v) growth with biofilm formation.     

Developmental control of glucosamine and galactosamine levels during conidation in Neurospora crassa.

The glucosamine and galactosamine content of mycelia was measured in cultures of Neurospora crassa grown on the surface of dialysis membranes. The glucosamine content was relatively constant throughout the different regions of the mycelial mat. The galactosamine content, however, was always lower in the growing-front region of the mycelial mat than in the older regions. At most, only low levels of galactosamine were necessary for the formation of hyphae at the growing front of a mycelial mat. Thus, galactosamine-containing polymers cannot be a major shape-determining component of the cell walls of these hyphae in Neurospora. The effect of conidiation on the amino sugar content was determined by using the bd (band) strain of N. crassa. When grown on the surface of dialysis membranes, this strain rhythmically produced regions of conidiating and non-conidiating growth. With this strain, it was concluded that conidiation did not affect the amino sugar levels. Since conidia that contained only very low levels of galactosamine were produced from regions of the mycelial mat that contained much higher levels of this amino sugar, there must be some mechanism of spatial differentiation that prevented the accumulation of galactosamine-containing polymers in conidia.     

Antifungal Activity of Leaf Extracts and Essential Oils of some Medicinal Plants against Didymella bryoniae

The fungitoxicity of crude extracts and essential oils of Achillea millefolium , Cymbopogon citratus , Eucalyptus citriodora and Ageratum conyzoides on the fungus Didymella bryoniae was verified in vitro by means of germination of spores and mycelial growth. In addition, some observations were made using scanning electron microscopy (SEM) to detect possible alterations on the hyphae of Didymella bryoniae . The results revealed that crude extracts of E. citriodora and A. conyzoides were more effective in inhibiting the mycelial growth of D. bryoniae whereas in the germination of spores A. conyzoides and A. millefolium were responsible for most of the inhibition, namely, 52 and 46%, respectively. The essential oils of C. citratus , A. conyzoides and E. citriodora provided 100% inhibition of the mycelial growth and germination of spores of D. bryoniae . SEM observations revealed alterations in the growth pattern of hyphae of D. bryoniae when the essential oil of A. millefolium was present.     

Malic acid accumulation by Aspergillus flavus

Summary Scanning electron microscopy revealed that Aspergillus flavus produced unusual crystals and hair-like processes during its l-malic acid production phase. Crystallinic dendritic aggregates were formed on the hyphae growing as pellets. The size and number of crystal aggregates increased during the fermentation in parallel with l-malic acid accumulation. The crystals (composed of calcium malate as well as small amounts of calcium succinate and calcium fumarate) were removed from the hyphae, after incubation with 6N HCl. On day 5 of the fermentation, about 9% of the total amount of l-malic acid produced was accounted for by the attached crystals. In addition to crystal formation we observed the appearance of hair-like processes during the early phase (2 days) of malic acid production only.     

Growth Characteristics of Polyporales Mushrooms for the Mycelial Mat Formation

Mushroom strains of Polyporales from the genera Coriolus, Trametes, Pycnoporus, Ganoderma, and Formitella were explored in terms of mycelial growth characteristics for the application of mushroom mycelia as alternative sources of materials replacing fossil fuel-based materials. Among the 64 strains of Polyporales, G. lucidum LBS5496GL was selected as the best candidate because it showed fast mycelial growth with high mycelial strength in both the sawdust-based solid medium and the potato dextrose liquid plate medium. Some of the Polyporales in this study have shown good mycelial growth, however, they mostly formed mycelial mat of weak physical strength. The higher physical strength of mycelial mat by G. lucidum LBS5496GL was attributed to its thick hyphae with the diameter of 13 µm as revealed by scanning electron microscopic analysis whereas the hyphae of others exhibited less than 2 µm. Glycerol and skim milk supported the best mycelial growth of LBS5496GL as a carbon and a nitrogen source, respectively.