Copper adsorption by Rhizopus arrhizus,Cladosporium resinae and Penicillium italicum

Summary Copper adsorption by Rhizopus arrhizus, Cladosporium resinae and Penicillium italicum was studied using a copper-selective electrode. Copper adsorption by C. resinae and P. italicum obeyed the Freundlich and Langmuir isotherms for single-layer adsorption whereas R. arrhizus followed the BET isotherm for multi-layer adsorption. Temperature had little effect on adsorption over the range 4–25°C. Mineral acids were effective for desorption of copper from preloaded biomass, the efficiency of desorption increasing with decreasing pH. Other cations were also capable of copper desorption with zinc showing the greatest efficiency and sodium the lowest.     

Biosorption of copper by fungal melanin

Summary Melanin obtained from Aureobasidium pullulans and Cladosporium resinae was an efficient biosorbent for copper. Copper uptake could be expressed using various adsorption isotherms; melanin from A. pullulans obeyed Freundlich and Langmuir isotherms whereas C. resinae melanin followed the BET isotherm indicating a more complex type of adsorption than in A. pullulans. In general, uptake capacities of melanin were greater than for intact biomass and the higher uptake by pigmented rather than albino biomass could be correlated with the presence of melanin. Cu²⁺ was less readily desorbed from melanin by dilute mineral acids than from intact biomass and again, the relative ease of Cu²⁺ desorption from pre-loaded pigmented or albino biomass was correlated with the presence or absence of melanin. Mg²⁺ and Zn²⁺ appeared to be the most effective cations for desorption with Na⁺ and K⁺ the least effective. The addition of melanin to a coppercontaining culture of the albino strain of A. pullulans resulted in some reduction of toxicity.     

Subcellular location of enzymes involved in oxidation of n-alkane by Cladosporium resinae

More than 70% of n-hexadecane-grown cells of Cladosporium resinae ATCC 22711 were converted to spheroplasts when they were treated with chitinase and lytic enzyme from Trichoderma harziamum. The light mitochondrial fraction, containing microbodies, mitochondria and vacuoles, was isolated from spheroplasts. Vacuoles in cells were demonstrated by the inability of acridine orange to stain organelles previously treated with 2.5 μM Bafilomycin A₁, a vacuolar ATPase inhibitor. Microbodies, mitochondria and vacuoles were separated from the light mitochondrial fraction by self-generated density-gradient ultracentrifugation using iodixanol as gradient medium. NADH-dependent n-alkane monooxygenase activity and fatty alcohol oxidase activity were located in the cytoplasm and mitochondrial fractions respectively. Received: 21 September 1998 / Received revision: 21 January 1999 / Accepted: 31 January 1999     

The uptake of n-alkanes from alkane mixtures during growth of the hydrocarbon-utilizing fungus Cladosporium resinae

Summary Cladosporium resinae growing on alkane mixtures removed n-alkanes sequentially in order of increasing molecular weight, each at about the same rate as during growth on it as single alkane. This sequence is not in order of the ability of each alkane to support growth. No alkane-specific extracellular solubilizing agent able to affect the order o metabolism could be detected during the growth of C. resinae on mixed n-alkanes, but supplementing the medium with phospholipids like those produced during growth on specific alkanes increased the rate or removal of the respective alkanes. Kinetic analysis indicated that the uptake of dodecane, hexadecane and octadecane from a mixture could be by a common mechanism, the order being determined, through competition, by the affinity of the system for each alkane.     

Production of biosurfactants by Cladosporium resinae

Summary Cladosporium resinae produces extracellular biosurfactants when growing in a hydrocarbon source such as the jet fuel JP8. This production of biosurfactants was observed by the reduction of the surface tension of the aqueous phase of growing medium, and by the increase in emulsion and foaming properties. A partial purification by collapsed foam gave better physical properties by decreasing surface tension and increasing foaming power and stabilization of emulsions. Surface active substances were purified by reversed phase chromatography. Six compounds representing over 75% of fraction containing surface activity were present. This fraction gave an improvement of all surface properties.     

Fuel-soluble biocides for control ofCladosporium resinae in hydrocarbon fuels

Summary Five fuel-soluble biocides — a benzimidazole fungicide, an organoboron, a pyridinethione and two isothiazolone products — were evaluated for inhibition of a typical hydrocarbon fuel contaminant,Cladosporium resinae, in fuel/water systems. The biocides exhibited marked differences in anti-fungal activity with storage and in the presence of sludge. A methylchloro/methyl-isothiazolone mixture prevented growth of the fungus at a concentration of one part per million and, in contrast to other biocides tested, showed no tendency to be inactivated by storage or the presence of sludge.     

Reductive transformation of benzoate by Nocardia asteroides and Hormoconis resinae

A variety of substituted aromatic acids were reduced to the corresponding alcohols by Nocardia asteroides JCM3016 under aerobic conditions. An isolated mold, Hormoconis resinae F328, could also reductively transform benzoate, the erythro isomer of (1R, 2S)-1-phenyl-1,2-propanediol being yielded as well as benzyl alcohol. C-1 of the diol was found to be derived from the α-carbon of benzoate by ¹³C-NMR analysis. The acyloin condensation between pyruvate and benzaldehyde formed from benzoate is assumed to participate in the diol formation. An ATP-dependent and NADPH-linked benzoate reductase (EC 1.2.1.30) and an NADPH-linked benzaldehyde reductase (EC 1.1.1.91) were demonstrated to participate in the benzoate reduction in both N. asteroides JCM 3016 and H. resinae F328.     

Spheroplast formation and partial purification of microbodies from hydrocarbon-grown cells ofCladosporium resinae

Summary Cells ofCladosporium resinae form greater numbers of microbodies when grown onn-alkanes than when grown on glucose. To facilitate isolation of microbodies, hydrocarbon-grown cells were spheroplasted. Of four spheroplasting agents and five osmotic supports examined, best results were obtained after a 4-h incubation with Novozym 234 plus chitinase and with 0.8 M sorbitol as osmotic support. Equal numbers of spheroplasts were obtained at pH 5.8 and at pH 7.0. Catalase was used as a marker for microbodies and cytochrome-c oxidase as a marker for mitochondria. Urate oxidase, a second marker for microbodies, was not detected in cell extracts. Microbodies were extremely fragile; of eight spheroplast disruption techniques attempted, the best yield of microbodies was obtained using a Teflon homogenizer for 5 min. Microbodies were partially purified by differential and density gradient centrifugation. Best results were obtained with discontinuous Percoll gradients which yielded a fraction enriched in microbodies and one enriched in mitochondria.     

Activity of an isothiazolone biocide against Hormoconis resinae in pure and mixed biofilms

Biofilms containing single or mixed cultures of the fungus Hormoconis resinae and anaerobic sulphate-reducing bacteria (SRB) on stainless steel were incubated with an isothiazolone biocide (Kathon FP) at 28°C for 24 h. H. resinae within the biofilm was enumerated by immunofluorescence microscopy using specific antiserum, and SRB were assayed by culture. Fungal numbers in mixed biofilms were considerably reduced in comparison with those in pure biofilms. The biocide was shown to be effective against H. resinae in pure biofilms at 50 and 100 ppm, but in mixed biofilms only at the higher concentration. This concentration also reduced the sessile SRB numbers by 99%.P.S. Guiamet is with the Sección Biolectroquimica, INIFTA, Suc. 4, C.C. 16, 1900 La Plata, Argentina. C.C Gaylarde is with the Departamento de Solos, Fac. de Agronomia, UFRGS, Av. Bento Gonçalves, 7712, 91540-000 Porto Alegre, RS, Brazil     

Microbial metabolism of alkylbenzene sulphonates Fungal metabolism of 1-phenylundecane-p-sulphonate and 1-phenyldodecane-p-sulphonate

A study was made of the biodegradation of 1-phenylundecane-p-sulphonate and 1-phenyldodecane-p-sulphonate byCladosporium resinae (CMI 88968) which was capable of growth on a number of such alkylbenzene sulphonate homologues as the sole source of carbon and sulphur. The results from both whole-cell and cell-free systems indicated that the alkyl, aryl and sulphonate moieties of detergent homologues were metabolised by the fungus. The alkyl side-chain, after a presumed initial oxidation of the terminal methyl group, was subsequently oxidised by a β-oxidation pathway. Three enzymes of the β-oxidation pathway, i.e. acyl-CoA synthetase, acyl-CoA dehydrogenase and β-hydroxyacyl-CoA dehydrogenase were identified in detergent-grown cell-free extracts of the fungus. The sulphonate moiety was released as sulphate by a desulphonating enzyme. The combined results of continuous sampling programmes monitored by both TLC and sulphate appearance in the growth medium indicated that desulphonation of the aromatic moiety was an early event in the overall biodegradation of synthetic detergent homologues. The presence of 1-phenylvalerate, 1-phenylpropionate, benzoate,p-hydroxybenzoate and 3,4-dihydroxybenzoate in cells after growth on 1-phenylundecane-p-sulphonate was indicated by GLC analysis. Cells grown on 1-phenyldodecane-p-sulphonate were shown to contain 1-phenylhexanoate, 1-phenylbutyrate, phenylacetate,p-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate. The aromatic nuclei remaining after alkyl side-chain biodegradation were further metabolised by an oxidation sequence involving an “ortho-cleavage” pathway. An overall metabolic pathway for the biodegradation of alkylbenzene sulphonates byCladosporium resinae is proposed.     

A novel qPCR protocol for the specific detection and quantification of the fuel-deteriorating fungus Hormoconis resinae

A wide variety of fungi and bacteria are known to contaminate fuels and fuel systems. These microbial contaminants have been linked to fuel system fouling and corrosion. The fungus Hormoconis resinae, a common jet fuel contaminant, is used in this study as a model for developing innovative risk assessment methods. A novel qPCR protocol to detect and quantify H. resinae in, and together with, total fungal contamination of fuel systems is reported. Two primer sets, targeting the markers RPB2 and ITS, were selected for their remarkable specificity and sensitivity. These primers were successfully applied on fungal cultures and diesel samples demonstrating the validity and reliability of the established qPCR protocol. This novel tool allows clarification of the current role of H. resinae in fuel contamination cases, as well as providing a technique to detect fungal outbreaks in fuel systems. This tool can be expanded to other well-known fuel-deteriorating microorganisms.     

A novel method of quantifying root exudation in the presence of soil microflora

A microcosm is described in which root exudation may be estimated in the presence of microorganisms. Ryegrass seedlings are grown in microcosms in which roots were spatially separated from a microbial inoculant by a Millipore membrane. Seedlings grown in the microcosms were labelled with [¹⁴C]-CO₂, and the fate of the label within the plant and rhizosphere was determined. Inoculation of the microcosms with Cladosporium resinae increased net fixation of the [¹⁴C] label compared to plants grown under sterile conditions. Inoculation also increased root exudation. The use of the microcosm was illustrated and its applications discussed.     

Production of β-glucosidase byCladosporium resinae

Summary Cladosporium resinae QM 7998 produced high activities of extracellular and constitutive -glucosidase when grown on a variety of sugars or cellulose. Starch and ribose induced enzyme synthesis several fold.Cladosporium resinae could utilize agricultural waste residues for growth and -glucosidase production. The initial pH of the medium had a marked effect on enzyme prowduction and optimum pH was between 4.0 and 5.0 depending on the assay method. Mixed culturing ofC. resinae with yeasts, viz.Saccharomyces cerevisiae andCandida utilis, increased the -glucosidase production while that with other fungi decreased the enzyme yield. The- glucosidase preparation fromC. resinae significantly increased the saccharification of rice and wheat straw (untreated or delignified) withTrichoderma reesei QM 9414 cellulase preparation.

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Résumé Cladosporium resinae QM 7998 produit des concentrations élevées de -glucosidase tant extracellulaire que constitutive lorsqu'elle croît sur une variété de sucres ou sur la cellulose. On a trouvé que l'amidon et le ribose augmentent de plusieurs fois la quantité d'enzyme synthétisée.Cladosporium resinae peut utiliser des résidus agricoles pour sa croissance et pour la production de -glucosidase. Le pH initial du milieu exerce un effet marqué sur la production d'enzyme et le pH optimum est compris entre 4.0 et 5.0 selon les conditions de l'essai. La croissance mixte deCladosporium resinae avec diverses levures, notammentSaccharomyces cerevisiae etCandida utilis, augmente la production de -glucosidase tandis que celle avec d'autres moisissures diminue le rendement en enzyme. La -glucosidase deCladosporium resinae augmente de manière significative la saccharification des pailles de riz et de froment (non-traitées ou délignifiées) traités par la cellulase deTrichoderma reesei QM 9414.

     

Production of Hormoconis resinae glucoamylase P by a stable industrial strain of Saccharomyces cerevisiae

A stable strain of Saccharomyces cerevisiae secreting glucoamylase (EC 3.2.1.3) with high debranching activity was constructed using recombinant DNA technology. An expression cassette without bacterial sequences, containing Hormoconis resinae glucoamylase P cDNA and the dominant selection marker MEL1 was integrated into the yeast chromosome using ARS1 homology. The glucoamylase expression level of the integrant yeast strain was increased by chemical mutagenesis. The yeast strains secreting glucoamylase were able to grow on soluble starch (5%, w/v) and ferment it to ethanol.Correspondence to: A. Vainio     

Antibiotic production by the cyanobacteriumNostoc muscorum

A broad spectrum antimicrobial antibiotic is produced byNostoc muscorum (Lancashire Polytechnic Culture Collection 23) during the post-exponential phase of growth. The antibiotic inhibits the growth of bacteria, notably multiple-resistantStaphylococcus aureus, and a biocide resistantPseudomonas aeruginosa: fungi such as the biodeteriogens,Cladosporium herbarum andHormoconis resinae and yeasts such asCandida albicans andC. pseudotropicalis. The antibiotic has an apparent molecular weight of 2000–3000 Daltons. Production appears to be dependent upon the limitation of one or more nutrients in the medium. author for correspondence     

Bioactive compound production by thermophilic and thermotolerant cyanobacteria (blue-green algae)

A thermotolerant species of Phormidium produced extracellular anti-microbial material during batch culture. Although this material was inactive when screened against a number of other cyanobacteria, it inhibited the growth of a wide range of Gram-positive and Gram-negative heterotrophic bacteria, Candida albicans and Cladosporium resinae.The authors are with the Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK     

Melanin as a virulence factor of Paracoccidioides brasiliensis and other dimorphic pathogenic fungi: a minireview

Melanin pigments are substances produced by a broad variety of pathogenic microorganisms, including bacteria, fungi, and helminths. Microbes predominantly produce melanin pigment via tyrosinases, laccases, catecholases, and the polyketide synthase pathway. In fungi, melanin is deposited in the cell wall and cytoplasm, and melanin particles (“ghosts”) can be isolated from these fungi that have the same size and shape of the original cells. Melanin has been reported in several human pathogenic dimorphic fungi including Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides posadasii. Melanization appears to contribute to virulence by reducing the susceptibility of melanized fungi to host defense mechanisms and antifungal drugs.     

Characterization of melanin isolated from a dark septate endophyte (DSE), Exophiala pisciphila

Melanin produced by a dark septate endophyte, Exophiala pisciphila, was isolated and purified. The solubility properties, Ultraviolet–visible and Fourier transform infrared spectra of the purified E. pisciphila melanin were similar to those of typical melanin. Inhibition of melanin production was observed when colonies exposed to tricyclazole (1,8-dihydroxynaphthalene (DHN) melanin inhibitor), but not to kojic acid (3,4-dihydroxyphenylalanine melanin inhibitor). Thus the E. pisciphila melanin was a member of DHN melanin family. In addition, the antioxidant activities of E. pisciphila melanin were evaluated in vitro by 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging assay. E. pisciphila melanin exhibited a strong antioxidant activity. Addition of 50–350 mg L⁻¹ Cd(II) to the medium increased the melanin production in E. pisciphila.     

Assignment of biological functions to specific plasmids in a local isolate ofRhizobium cicer

The genetic basis of symbiosis, mucoid structure and melanin biosynthesis in a local isolate ofRhizobium cicer was investigated. The strain was a very effective symbiont and produced substantial amounts of exopolysaccharides and melanin. It harbored three high-molar-mass plasmids of 55, 80 and 130 kb, respectively. Thirty-one melanin production-negative (Mep⁻) derivatives were obtained through plasmid curing. The plasmid patterns of cured derivatives indicated that the loss of 55 and/or 80 kb plasmids was sufficient for obtaining Mep⁻ phenotype. The specific involvement of 55-kb plasmid in melanin production was confirmed upon re-introduction of this plasmid into one of the Mep⁻ derivatives. Further investigation also indicated that the 130-kb plasmid might be necessary for both mucoidal appearance and symbiotic functions.     

Growth Profile and Hydrocarbonoclastic Potential of Microorganisms Isolated from Tarballs in the Bight of Bonny, Nigeria

Summary The growth profile and hydrocarbonoclastic potential of microorganisms isolated from tarballs harvested from Ibeno beach in the Bight of Bonny were examined to determine their role in the degradation of the aquatic pollutants (tarballs). The results of the analysis revealed that the mean heterotrophic bacterial count ranged from 3 (±0.01) × 10³ to 3.18 (±0.2) × 10⁵ c.f.u./g. The mycological count ranged from 1 (±0.3) × 10² to 2 (±0.4) × 10⁴ c.f.u./g while the mean count of biodegraders on tarball mineral salt medium (TMSM) ranged from 1 (±0.3) × 10² to 2 (±0.4) × 10⁴ c.f.u./g. The ability of the microbial isolates to utilize the tarballs as their sole source of carbon and energy was examined and noticed to vary in growth profiles between the isolates. Chromobacterium violaceum, Cladosporium resinae, Bacillus submarinus, Micrococcus varians, Pseudomonas aeruginosa, Candida marina and Saccharomyces estuari were the most efficient utilizers and biodegraders while Corynebacterium glutamicum, Nocardia marina, and Cryptococcus albidus exhibited moderate growth in TMSM. Vibrio parahaemolyticus and Escherichia coli were opportunistic inhabitants, as they could neither grow nor degrade the balls in TMSM. The results imply that the efficient biodegraders like Chromobacterium violaceum could extensively degrade the balls with time.