Ethidium bromide stimulated hyper laccase production from bird's nest fungus Cyathus bulleri

AIMS: Effect of ethidium bromide, a DNA intercalating agent, on laccase production from Cyathus bulleri was studied. METHODS AND RESULTS: The bird's nest fungus, Cyathus bulleri was grown on 2% (w/v) malt extract agar (MEA) supplemented with 1.5 microg ml(-1) of the phenanthridine dye ethidium bromide (EtBr) for 7 d and when grown subsequently in malt extract broth (MEB), produced a 4.2-fold increase in laccase production as compared to the untreated fungus. The fungal cultures following a single EtBr treatment, when regrown on MEA devoid of EtBr, produced a sixfold increase in laccase in MEB. However, on subsequent culturing on MEA in the absence of EtBr, only a 2.5-fold increase in laccase production could be maintained. In another attempt, the initial EtBr-treated cultures, when subjected to a second EtBr treatment (1.5 microg ml(-1)) on MEA for 7 d, produced a 1.4-fold increase in laccase production in MEB. CONCLUSIONS: The white-rot fungus Cyathus bulleri, when treated with EtBr at a concentration of 1.5 microg ml(-1) and regrown on MEA devoid of EtBr, produced a sixfold increase in laccase production in MEB. SIGNIFICANCE AND THE IMPACT OF THE STUDY: The variable form of C. bulleri capable of hyper laccase production can improve the economic feasibility of environmentally benign processes involving use of fungal laccases in cosmetics (including hair dyes), food and beverages, clinical diagnostics, pulp and paper industry, industrial effluent treatment, animal biotechnology and biotransformations.     

Evaluation of plant extracts as an antagonist to mycelial growth of Mycosphaerella fijiensis morelet

Three plant extracts (rice husk, wood and bamboo) at different concentration were evaluated in vitro as an antagonist to mycelial growth of Mycosphaerella fijienesis on different culture media using spread plate and mycelia dry weight method. The plant extracts had significant effects on the mycelial growth of Mycosphaerella fijiensis. Rice husk extract at concentration of 1, 1.5, 2.5 and 5% completely inhibited the mycelia growth of M. fijiensis in malt extract broth (MEB) and at 2.5 and 5% on malt extract agar (MEA). Wood extract at concentration of 1 and 1.5% inhibited the mycelial growth of M. fijiensis and completely at concentration of 2.5 and 5% on MEA. Although complete inhibition was only observed at 5% concentration on MEA for bamboo extract, the evaluated plant extracts could be recommended for the control of M. fijiensis on a large-scale farming.     

An alternative, banana peel-based medium used to investigate the catalytic properties of peroxidase from a fungus, Inonotus sp SP2, recently isolated in southern Chile

Recycling industrial wastes is one of the major goals of bioengineering research. Agricultural wastes are often rich in natural sources of organic and inorganic compounds. The present study investigates the use of banana peel waste as a non-conventional alternative to nitrogen-enriched glucose media for a white rot fungus (WRF), Inonotus sp. SP2, recently isolated in southern Chile. WRF are known to produce biodegrading enzymes, such as peroxidases, that can have industrial and biotechnological applications. To that end, the metabolic characteristics and catalytic properties of peroxidases produced by Inonotus sp. SP2 were compared between glucose and banana peel-based growth mediums. The results establish that this strain of WRF produces high concentrations of a Mn⁺²-dependent peroxidase, with greater enzymatic activity in extracellular fluid and crude enzyme extracted from fungus grown in banana peel and glucose media, respectively. H₂O₂ has an inhibiting effect that is greater for enzymes produced in glucose media, and greater biomass can be obtained in banana-peel based media. This demonstrates that banana peel is a suitable and more cost-effective alternative to conventional glucose-based media for the production of biodegradative enzymes, such as peroxidase. Unlike other strains of WRF, the metabolic characteristics of Inonotus sp. SP2 demonstrate that it enters secondary metabolism with the production oxidative enzymes after both carbon and nitrogen sources are depleted. This suggests that with further investigation, this strain of WRF may be useful in industrial applications that require the biodegradation of nitrogen and carbon-based wastes and recalcitrant compounds.     

Comparison of Biomass Dry Weights and Radial Growth Rates of Fungal Colonies on Media Solidified with Different Gelling Compounds

Penicillium commune, Aureobasidium pullulans, and Paecilomyces farinosus were grown on two different media solidified with agar, Pluronic F-127, Carrageenan X-4910, or Carrageenan X-4910 overlaid with cellophane. Growth on Carrageenan X-4910 was generally the same as that on agar, as was the visual appearance of the colonies, e.g., the pigmentation. The Carrageenan X-4910 gels had a melting point, depending on the medium, of 41 to 46(deg)C, and the dry weights of the colonies were readily determined at 60(deg)C. To determine the dry weights of the colonies grown on agar plates, the gels were boiled for 10 min to melt the agar. Comparison of these two procedures showed that the boiling procedure resulted in a 22% reduction of the biomass dry weight. Cellophane membranes did not affect the radial growth rate profoundly. The biomass density was almost halved for P. commune and P. farinosus grown with membranes, whereas the presence of the membrane did not affect the biomass density of A. pullulans. The biomass densities of the colonies grown on Pluronic F-127 were significantly reduced, while in most cases, the radial growth rates of colonies grown on Pluronic F-127 were significantly higher than those obtained on agar or Carrageenan X-4910. Furthermore, the morphology of the leading hyphae was altered, and the hyphal growth unit length was more than twice that obtained on agar and Carrageenan X-4910. Carrageenan X-4910 is a valuable gelling compound for the study of the growth of fungi, as the biomass dry weight is readily determined and growth is similar to that obtained on agar gels.     

An extracellular matrix glues together the aerial-grown hyphae of Aspergillus fumigatus

Pulmonary infections due to Aspergillus fumigatus result from the development of a colony of tightly associated hyphae in contact with the air, either in the alveoli (invasive aspergillosis) or in an existing cavity (aspergilloma). The fungal ball observed in vivo resembles an aerial colony obtained in agar medium in vitro more than a mycelial mass obtained in liquid shaken conditions that have been classically used to date to study A. fumigatus physiology. For this reason, we embarked on an analysis of the characteristics of A. fumigatus colonies grown in aerial static conditions. (i) Under static aerial conditions, mycelial growth is greater than in shaken, submerged conditions. (ii) The colony surface of A. fumigatus revealed the presence of an extracellular hydrophobic matrix that acts as a cohesive linkage bonding hyphae into a contiguous sheath. (iii) The extracellular matrix is composed of galactomannan, alpha1,3 glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. (iv) A. fumigatus colonies were more resistant to polyenes than shake, submerged mycelium. This is the first analysis of the three dimensional structure of a mycelial colony. Knowledge of this multicellular organization will impact our future understanding of the pathobiology of aerial mold pathogens.     

Gari as culture medium for moulds

No significant differences (P<0.05 occurred in the frequency of isolation from soil of four common moulds – Rhizopus stolonifer, Monilia sitophila, Aspergillus niger and Penicillium sp., on malt extract agar (MEA) and gelled gari (carbohydrate gel). Growth of the moulds on slide microcultures of potato dextrose agar (PDA) and gari showed typical diagnostic features of the organisms, which were often clearer on gari. It is concluded that gari, which is much cheaper and far more readily available in Nigeria than MEA or PDA, is an effective alternative to the two standard mycological media.     

In vitro evidence of mycoparasitism of the ectomycorrhizal fungus <Emphasis Type="Italic">Laccaria laccata</Emphasis> against <Emphasis Type="Italic">Mucor hiemalis</Emphasis> in the rhizosphere of <Emphasis Type="Italic">Pinus sylvestris</Emphasis>

Interactions between the ectomycorrhizal fungus Laccaria laccata and the soil fungus Mucor hiemalis f. hiemalis in co-culture, and in the rhizosphere of in vitro-grown Pinus sylvestris seedlings were investigated by light- and scanning electron-microscopy. In co-culture, mycelial growth away from the L. laccata colony reduced the number of aerial hyphae at the contact zone and increased the density and compactness of the mycelium-characterized gross morphology of the saprobic fungus. Although the growth of M. hiemalis was suppressed, no penetration of M. hiemalis hyphae after the colony was entered by L. laccata was observed. Instead, dense coiling of L. laccata hyphae around sporangiophores, overpowering them and causing them to disappear, was quite common. On nonmycorrhizal roots, sporangiospores germinated heavily and formed long hyphae for 2 days post inoculation, whereas their germination was totally inhibited on mycorrhizal roots. At 3 days after inoculation, only sporangia were seen with mycelial mats firmly attached to the roots by the mantle hyphae, whereas some remnants of sporangiophores, ruptured sporangial walls and degraded hyphae of M. hiemalis were overgrown by the mantle hyphae. During the next 3 days, the mantle-hyphae-invading sporangia formed short, thin branches that grew directly towards individual spores, tapering off upon contact.     

Characterization of the Sclerotinia sclerotiorum cell wall proteome

We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)‐anchored cell wall proteins and 30 non‐GPI‐anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes.     

Comparative study of the interactions between bisphenol-A and its endocrine disrupting analogues with bovine serum albumin using multi-spectroscopic and molecular docking studies

Interaction studies of bisphenol analogues; biphenol-A (BPA), bisphenol-B (BPB), and bisphenol-F (BPF) with bovine serum albumin (BSA) were performed using multi-spectroscopic and molecular docking studies at the protein level. The mechanism of binding of bisphenols with BSA was dynamic in nature. SDS refolding experiments demonstrated no stabilization of BSA structure denatured by BPB, however, BSA denatured by BPA and BPF was found to get stabilized. Also, CD spectra and molecular docking studies revealed that BPB bound more strongly and induced more conformational changes in BSA in comparison to BPA. Hence, this study throws light on the replacement of BPA by its analogues and whether the replacement is associated with a possible risk, raising a doubt that perhaps BPB is not a good substitute of BPA.     

Effect of sealing and Tween 80 on the antifungal susceptibility testing of essential oils

Concentrations of essential oils showing high volatility decreased substantially in broth and agar media when incubated under open conditions. The decrease in the half life was from 0.7 to 38 hr in broth medium at 27 C. When evaporation was prevented by sealing, MIC values against Aspergillus fumigatus, Candida albicans and Trichophyton mentagrophytes by broth or agar dilution assay were lowered two to eight-fold, as compared with those obtained under open conditions. Addition of Tween 80 caused a rise of the MICs against A. fumigatus by two to four-fold in broth dilution assay, but little affected the MICs in agar dilution assay.     

Conventional laboratory agar media provide an iron-limited environment for bacterial growth

Abstract The outer membrane proteins of Escherichia coli and Pseudomonas aeruginosa grown in a number of conventional laboratory media were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) High-molecular-weight proteins similar to those produced by these strains in an iron-limited chemically defined medium were detected in cells grown on the surface of various agar media. In contrast, these proteins were not produced or were only poorly expressed by the corresponding broth cultures or by cells grown an agar supplemented with iron. A catecholic substance could be detected in DST agar extracts subsequent to bacterial growth which was produced to a lesser extent in IST agar and in broth cultures.     

Keratinolytic potential of a novel Bacillus sp. P45 isolated from the Amazon basin fish Piaractus mesopotamicus

Bacillus sp. P45, isolated from the intestine of the Amazon basin fish Piaractus mesopotamicus, showed proteolytic activity when grown on skimmed milk and feather meal agar plates. The keratinolytic potential of this strain was evaluated on whole feather broth and human hair broth. Bacillus sp. P45 degraded almost 90% of chicken feathers after 72 h of submerged cultivation on whole feather broth, and the production of extracellular proteases was observed. The formation of thiol groups was also detected during growth, indicating the contribution of sulphitolysis to the efficient hydrolysis of feather keratin. Nevertheless, Bacillus sp. P45 was unable to degrade hair keratin, possibly due to the conformational diversity of this substrate in comparison to feather keratin. Additionally, preliminary results demonstrated that this strain might be utilized in the degradation of recalcitrant collagen-containing wastes. The keratinolytic character of Bacillus sp. P45 might be utilized in environmental-friendly processes such as bioconversion of waste feathers, representing an alternative way of waste management that could lead to the production of value-added products such as microbial biomass, protein hydrolysates and proteolytic enzymes.     

Real-time detection of common microbial volatile organic compounds from medically important fungi by Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS)

We describe a new method, Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS) for the rapid and sensitive real-time detection and quantification of volatile organic compounds from medically important fungi, grown on a range of laboratory media. SIFT-MS utilises the chemical ionisation reactions of mass-selected ions to characterise volatile organic compounds (VOCs) that are produced as metabolites from fungi. This technique has the distinct advantage over others in that it readily detects low molecular weight, reactive volatiles, and allows for real-time, quantitative monitoring. The fungi examined in this study were Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Mucor racemosus, Fusarium solani, and Cryptococcus neoformans grown on or in malt extract agar, Columbia agar, Sabouraud's dextrose agar, blood agar, and brain-heart infusion broth. Common metabolites (ethanol, methanol, acetone, acetaldehyde, methanethiol, and crotonaldehyde) were detected and quantified. We found the fingerprint of volatiles, in terms of presence and quantity of volatiles to be strongly dependent on the culture medium, both in terms of variety and quantity of volatiles produced, but may form the basis for species specific identification of medically important fungi.     

A NOTE ON THE USE OF ROGOSA AND MODIFIED ROGOSA MEDIA FOR THE ENUMERATION AND CULTURE OF LACTOBACILLI

SUMMARY: The modified Rogosa media (acetate broth and acetate agar) developed by Mabbitt & Zielinska (1956) have been compared with tomato juice media for the growth of representative Lactobacillus species.
Most of the species tested grew markedly better in acetate broth than in tomato dextrose broth, but some cultures did not grow well in the acetate broth although they grew normally in tomato dextrose broth. The acetate agar was satisfactory for counting those lactobacilli cultured at 30° but was inferior to tomato dextrose agar for counting those grown at 37°.     

Cell-wall structure and composition of Penicillium janczewskii as affected by inulin

Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.     

Effective and Low-Cost Saccharification of Pineapple Peel by <Emphasis Type="Italic">Trichoderma viride</Emphasis> Crude Extract with Enhanced β-Glucosidase Activity

Efficient, low-cost enzymatic hydrolysis of lignocellulosic biomass is essential for cost-effective production of bioethanol. The aim of this study was to establish a fungal fermentation-based strategy for the economic enzymatic conversion of pineapple peel into fermentable sugars. Trichoderma viride was grown on passion fruit peel in order to improve its β-glucosidase production, and a crude extract was then used to hydrolyze pineapple peel. The effects of medium pH, cultivation time, and passion fruit peel concentration on β-glucosidase production were evaluated using a central composite rotational design (CCRD) combined with response surface methodology (RSM). Optimal β-glucosidase activity of 2.40 U mL^?1 was found after 6.5 days of cultivation in medium at pH 6.0, containing 2.0 % passion fruit peel. Saccharification of pineapple peel was also optimized by RSM and CCRD with respect to pH, temperature, β-glucosidase concentration, and reaction time and proceeded optimally at pH 4.0, 55 °C, with a β-glucosidase loading of 31.25 U g^?1 dry feedstock and 75 h of reaction. Under these conditions, T. viride crude extract hydrolyzed pineapple peel with a glucose yield of 65.3 %. This study therefore presents passion fruit peel as an attractive raw material for the production of β-glucosidases. In addition, it describes an improved, effective, and low-cost enzymatic method for the production of fermentable sugars from pineapple peel, an abundant and inexpensive agro-industrial waste.     

Growth and Polysaccharide Production by Methylocystis parvus OBBP on Methanol

Methylocystis parvus OBBP, an obligate methylotroph originally isolated as a methane-utilizing bacterium, was cultivated on methanol as a sole source of carbon. After adaptation to high methanol levels, this organism grew on methanol with a maximum specific growth rate of 0.65 h. The pH optimum for growth was between 7 and 9, and the temperature optimum was between 30 and 37 degrees C. Methanol concentrations higher than 5% (by weight) were toxic. Formaldehyde, at a concentration greater than 1 mM, inhibited growth. Formate was neither a substrate nor an inhibitor. An extracellular viscous heteropolysaccharide was produced during growth. The maximum production of the total biomass was 14.5 g (dry weight) per liter of broth. The dried biomass contained 22% (wt/wt) crude protein and 62% (wt/wt) polysaccharide. The main components of the polysaccharide were d-glucose (82%) and l-rhamnose (14%).     

Notes on the Biology of Trypanosoma ambystomae Lehmann 1954. I. The Cultivation of T. ambystomae

SUMMARY. Trypanosoma ambystomae has been cultivated in NNN agar for a period of 45 days. The flagellates can be subcultured in NNN agar and in dried beef-blood nutrient agar; growth in the latter medium is not as prolific as in NNN agar. No growth was obtained by attempted subcultures in dried beef-blood glucose broth.     

GROWTH RESPONSE OF SALMONELLA SPP. TO CYCLOHEXIMIDE AMENDMENT IN MEDIA

The present study was designed to evaluate cycloheximide as a potential media amendment to prevent fungal overgrowth on selective media for salmonellae enumeration. The objectives were to determine the effect of cycloheximide on Salmonella spp growth rates and to determine the effect of cycloheximide addition on Salmonella enumeration in selective media. The bacteria tested included two strains of Salmonella typhimurium (NO/NA and LT2) and one strain of Salmonella arizonae. All strains were grown in tryptic soy broth containing cycloheximide to determine the effect of cycloheximide on bacterial specific growth rates. The growth rate of all strains grown in tryptic soy broth were not significantly influenced by addition of cycloheximide at concentrations up to 1,000 mg/L. Growth rates of S. typhimurium NO/NA in minimal media were significantly decreased by addition of cycloheximide aerobically (300 mg/L) and anaerobically (600 mg/L). However, S. typhimurium NO/NA populations on brilliant green agar, MacConkey agar, and from selenite cysteine broth and tetrathionate broth were not affected by cycloheximide additions at concentrations up to 1,000 mg/L. Cycloheximide has potential as a fungistat additive for salmonellae selective media.     

Differentiation and anaerobiosis in standing liquid cultures of Streptomyces coelicolor

Streptomyces coelicolor differentiates on solid agar media by forming aerial hyphae that septate into spores. We here show that differentiation also occurs in standing liquid minimal media. After a period of submerged growth, hyphae migrate to the air interface, where they become fixed by a rigid reflecting film. Colonies that result from these hyphae form sporulating aerial hyphae. In addition, submerged hyphae in the liquid minimal medium may attach to the surface. Liquid standing cultures easily become anoxic only 1 to 2 mm below the surface. Yet, biomass increases, implying the existence of metabolic pathways supporting anaerobic growth.