Isolation and characterisation of fungi growing on volatile aromatic hydrocarbons as their sole carbon and energy source

Five fungal strains that are able to grow on toluene were isolated from enrichment cultures. Three different techniques were used: solid state-like batches, air biofilters and liquid cultures. Fungal growth in the latter systems was favoured by combining low pH and low water activity. Soil and groundwater samples from gasoline-polluted environments were used as inocula. The isolates were identified as deuteromycetes belonging to the genera Cladophialophora, Exophiala and Leptodontium and the ascomycete Pseudeurotium zonatum. The previously isolated toluene-degrading fungus Cladosporium sphaerospermum was included in the present study. Results showed that fungi grew on toluene with doubling times of about 2 to 3 days. Some of the strains also grew on ethylbenzene and styrene. The apparent half-saturation constant (Km) for toluene oxidation ranged from 5 to 22 μM. Degradation activity was inhibited by 50t toluene concentrations ranging from 2.4 to 4.7 mM. These kinetic parameters are comparable to analogous data reported for toluene-degrading bacteria. The ability of fungi to grow at low water activities and low pH suggest that they may be used for the purification of gas streams containing aromatic hydrocarbons in air biofilters

Toluene monooxygenase from the fungus Cladosporium sphaerospermum

Assimilation of toluene by Cladosporium sphaerospermum is initially catalyzed by toluene monooxygenase (TOMO). TOMO activity was induced by adding toluene to a glucose-pregrown culture of C. sphaerospermum. The corresponding microsomal enzyme needed NADPH and O2 to oxidize toluene and glycerol, EDTA, DTT, and PMSF for stabilization. TOMO activity was maximal at 35 °C and pH 7.5 and was inhibited by carbon monoxide, Metyrapone, and cytochrome c. TOMO preferred as substrates also other aromatic hydrocarbons with a short aliphatic side chain. Its reduced carbon monoxide difference spectrum showed a maximum at 451 nm. A substrate-induced Type I spectrum was observed on addition of toluene. These results indicated that TOMO is a cytochrome P450. TOMO and its corresponding reductase were eventually purified by a simultaneous purification revealing apparent molecular masses of 58 and 78 kDa, respectively

Toluene monooxygenase from the fungus Cladosporium sphaerospermum

Assimilation of toluene by Cladosporium sphaerospermum is initially catalyzed by toluene monooxygenase (TOMO). TOMO activity was induced by adding toluene to a glucose-pregrown culture of C. sphaerospermum. The corresponding microsomal enzyme needed NADPH and O2 to oxidize toluene and glycerol, EDTA, DTT, and PMSF for stabilization. TOMO activity was maximal at 35 °C and pH 7.5 and was inhibited by carbon monoxide, Metyrapone, and cytochrome c. TOMO preferred as substrates also other aromatic hydrocarbons with a short aliphatic side chain. Its reduced carbon monoxide difference spectrum showed a maximum at 451 nm. A substrate-induced Type I spectrum was observed on addition of toluene. These results indicated that TOMO is a cytochrome P450. TOMO and its corresponding reductase were eventually purified by a simultaneous purification revealing apparent molecular masses of 58 and 78 kDa, respectively

Fungal metabolism of toluene: monitoring of fluorinated analogs by (19)F nuclear magnetic resonance spectroscopy

We used isomeric fluorotoluenes as model substrates to study the catabolism of toluene by five deuteromycete fungi and one ascomycete fungus capable of growth on toluene as the sole carbon and energy source, as well as by two fungi (Cunninghamella echinulata and Aspergillus niger) that cometabolize toluene. Whole cells were incubated with 2-, 3-, and 4-fluorotoluene, and metabolites were characterized by 19F nuclear magnetic resonance. Oxidation of fluorotoluene by C. echinulata was initiated either at the aromatic ring, resulting in fluorinated o-cresol, or at the methyl group to form fluorobenzoate. The initial conversion of the fluorotoluenes by toluene-grown fungi occurred only at the side chain and resulted in fluorinated benzoates. The latter compounds were the substrate for the ring hydroxylation and, depending on the fluorine position, were further metabolized up to catecholic intermediates. From the 19F nuclear magnetic resonance metabolic profiles, we propose that diverse fungi that grow on toluene assimilate toluene by an initial oxidation of the methyl group

Cladophialophora psammophila, a novel species of Chaetothyriales with a potential use in the bioremediation of volatile aromatic hydrocarbons

Cladophialophora is a genus of asexual black yeast-like fungi with one-celled, hydrophobic conidia which is predicted to have teleomorphs in the ascomycete genus Capronia, a member of the order Chaetothyriales. Cladophialophora species are relatively frequently involved in human disease ranging from mild cutaneous lesions to cerebral abscesses. Although the natural niche outside humans is unknown for most opportunistic Cladophialophora species, the fungi concerned are rarely isolated from environmental samples such as dead plant material, rotten wood, or soil. The objective of the present paper is to describe a novel species of Cladophialophora which was isolated from soil polluted with benzene, toluene, ethylbenzene, and xylene (BTEX). It proved to be able to grow with toluene and other related alkylbenzenes as its sole carbon and energy source. This strain is of interest for the biodegradation of toluene and other related xenobiotics under growth limiting conditions, particularly in air biofilters, dry and/or acidic soil. A preliminary genetic analysis using multilocus sequencing typing (MLST) and amplified fragments length polymorphism (AFLP) showed that this fungus was closely related to the pathogenic species Cladophialophora bantiana, sharing a C. bantiana-specific intron in SSU rDNA. However, it was unable to grow at 40 °C and proved to be non-virulent in mice. The clear phylogenetic and ecophysiological delimitation of the species is fundamental to prevent biohazard in engineered bioremediation applications. Keywords AFLP; Air biofilters; Black yeasts; Cladophialophora; In vitro antifungal activity; ITS rDNA; Neurotropism; Pathogenicit