Hydrocarbon Degradation and Enzyme Activities of Aspergillus oryzae and Mucor irregularis Isolated from Nigerian Crude Oil-Polluted Sites

Abstract

Many free-living saprobic fungi are nature recruited organisms for the degradation of wastes, ranging from lignocellulose biomass to organic/inorganic chemicals, aided by their production of enzymes. In this study, fungal strains were isolated from contaminated crude-oil fields in Nigeria. The dominant fungi were selected from each site and identified as Aspergillus oryzae and Mucor irregularis based on morphological and molecular characterization, with site percentage incidences of 56.67% and 66.70%, respectively. Selected strains response/tolerance to complex hydrocarbon (used engine oil) was studied by growing them on Bushnell Haas (BH) mineral agar supplemented with the hydrocarbon at different concentrations, i.e., 5%, 10%, 15%, and 20%, with a control having dextrose. Hydrocarbon degradation potentials of these fungi were confirmed in BH broth culture filtrates pre-supplemented with 1% engine oil after 15 days of incubation using GC/MS. In addition, the presence of putative enzymes, laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) was confirmed in culture filtrates using appropriate substrates. The analyzed fungi grew in hydrocarbon supplemented medium with no other carbon source and exhibited 39.40% and 45.85% dose inhibition response (DIR) respectively at 20% hydrocarbon concentration. An enzyme activity test revealed that these two fungi produced more Lac than MnP and LiP. It was also observed through the GC/MS analyses that while A. oryzae acted on all hydrocarbon components in the used engine oil, M. irregularis only degraded the long-chain hydrocarbons and BTEX. This study confirms that A. oryzae and M. irregularis have the potential to be exploited in the bio-treatment and removal of hydrocarbons from polluted soils.

Keywords: bio-treatment; dose inhibition response; extracellular enzymes; fungi; hydrocarbon degradation; pollutants; tolerance.

Figure 1

Neighbour-joining phylogenetic tree showing the relationship between the Mucor sequence from strain B-Yorla10 isolated from oil-contaminated soil in this study and selected database relatives. Kimura 2-parameter distances were used. Bootstrap values are based on percentages of 1000 replicates. The tree was rooted with Umbelopsis nana as outgroup.

Figure 2

Neighbour-joining phylogenetic tree showing the relationship between the Aspergillus sequence from strain C-Effurun isolated from oil-contaminated soil in this study and selected database relatives. Kimura 2-parameter distances were used. Bootstrap values were based on percentages of 1000 replicates. Aspergillus peyronelii (EF669715.1) was used as the outgroup.

Figure 3

Plate assay for colorimetric screening of ligninolytic enzymes produced by the isolated fungi. (i) Control—Mucor irregularis strain B-Yorla10 growing on MEA without chemicals (ii) Mucor irregularis strain B-Yorla10 showing reddish brown coloration on reaction with MEA supplemented with CuSO4 and Guaiacol (iii) Mucor irregularis strain B-Yorla10 showing reddish brown coloration on reaction with MEA supplemented with CuSO4 and ABTS (iv) Control—Aspergillus oryzae strain C-Effurun growing on MEA without chemicals (v) Aspergillus oryzae strain C-Effurun showing reddish brown coloration on reaction with MEA supplemented with CuSO4 and Guaiacol (vi) Aspergillus oryzae strain C-Effurun showing reddish brown coloration on reaction with MEA supplemented with CuSO4 and ABTS.

Figure 4

Activities of extracellular enzymes produced by Mucor irregularis B-Yorla10 isolated from a crude oil polluted site in Nigeria.

Figure 5

Activities of extracellular enzymes produced by Aspergillus oryzae C-Effurun isolated from a crude oil polluted site in Nigeria.

Figure 6

Composition of the used engine oil as revealed through GC/MS analysis.

Figure 7

Degradation of hydrocarbon fractions in used engine oil by selected filamentous fungi. Degradation of used engine oil by Mucor irregularis and Aspergillus oryzae. The compounds were grouped in classes of Benzene (yellow), Naphthalene and Indene (dark-yellow), Dimethyl-biphenyl (brown), Naphthalene and substituents (blue), the group of Anthracene, Fluorene and Phenanthrene (dark-blue), Short-chain alkanes (white) and Long chain alkanes (grey).