Black Fungi and Hydrocarbons: An Environmental Survey for Alkylbenzene Assimilation

Abstract

Environmental pollution with alkylbenzene hydrocarbons such as toluene is a recurring phenomenon. Their toxicity and harmful effect on people and the environment drive the search for sustainable removal techniques such as bioremediation, which is based on the microbial metabolism of xenobiotic compounds. Melanized fungi present extremophilic characteristics, which allow their survival in inhospitable habitats such as those contaminated with hydrocarbons. Screening methodologies for testing the microbial assimilation of volatile organic compounds (VOC) are scarce despite their importance for the bioremediation of hydrocarbon associated areas. In this study, 200 strains of melanized fungi were isolated from four different hydrocarbon-related environments by using selective methods, and their biodiversity was assessed by molecular and ecological analyses. Seventeen genera and 27 species from three main orders, namely Chaetothyriales, Cladosporiales, and Pleosporales, were identified. The ecological analysis showed a particular species distribution according to their original substrate. The isolated strains were also screened for their toluene assimilation potential using a simple and inexpensive methodology based on miniaturized incubations under controlled atmospheres. The biomass produced by the 200 strains with toluene as the sole carbon source was compared against positive and negative controls, with glucose and with only mineral medium, respectively. Nineteen strains were selected as the most promising for further investigation on the biodegradation of alkylbenzenes.

Keywords: biodegradation; melanized fungi; toluene.

Figure 1

Schematic representation of the screening test to assess the potential of toluene assimilation by the melanized fungi. Test tubes containing fungal inoculum in a liquid medium were covered with perforated foil to allow gas exchange inside the desiccators. A beaker glass containing pure toluene or toluene diluted in dibutyl phthalate (DBP) were placed inside the desiccators in order to supply toluene as the sole source of carbon and energy via the gas phase at high and low concentrations, respectively.

Figure 2

Relative abundance of strains obtained from the different hydrocarbon-related environments. Strains isolated from the garage soil, Eucalyptus bark, landfarming soil and leaf-cutting ants were obtained using the oil flotation technique (Satow et al., 2008). The ants were also submitted to the agar walk method. Strains recovered from the water samples were obtained by standard serial dilution according to Clesceri et al. (1998).

Figure 3

Two-dimensional (2D) plots of a principal coordinate analysis (PCoA, Bray–Curtis distance) on the relative abundance of melanized strains isolated from different hydrocarbon-associated environments, based on the analysis of their ITS rDNA sequences (Encompassed variance: 32.86% in Axis 1, 20.40% in Axis 2, and 17.72% in Axis 3).