Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications

Abstract

Background: Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost-effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization.

Results: Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum, Stemphylium lucomagnoense and Aspergillus nidulans. Among these five isolates, one T. asperellum strain (T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator.

Conclusions: The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO₄ as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.

Keywords: Dyes; Laccase; Laccase-like activity; Marine-derived fungi; Trichoderma asperellum.

Fig. 1

Phylogenetic reconstruction for the strain Stemphylium sp., based on ITS analysis [17] using the neighbor-joining algorithm (NJ) method and 1000 replicate bootstraps. ITS sequences were deposited in the NCBI under accession number MK691703. Culture plate of Stemphylium sp. and conidiophores and conidia (scale bar = 10 μm)

Fig. 2

Phylogenetic reconstruction for the strain Aspergillus sp, based on ITS analysis using the neighbor-joining algorithm (NJ) method and 1000 replicate bootstraps, ITS sequences were deposited in the NCBI under accession number MK691704

Fig. 3

Phylogenetic reconstruction for the strains Trichoderma sp. 1, 2 and 3 based on elongation factor 1-alpha (EF1a) analysis using the neighbor-joining algorithm (NJ) method and 1000 replicate bootstraps (MK966034, MK966035 and MK966036 are the accession numbers of Trichoderma 1, 2 and 3 respectively)

Fig. 4

a Laccase activity of Trichoderma asperellum 1 (●), Trichoderma asperellum 2 (■), Trichoderma asperellum 3 (▲), Stemphylium lucomagnoense (♦) and Aspergillus nidulans (○) during 5 days of culture with ABTS as the substrate at pH 5.5 without (a) or with (b) 1% NaCl. Each data point (mean +/− standard deviation) is the result of triplicate experiments

Fig. 5

a Effect of different concentrations of NaCl (0% (●), 1% (■), 2% (▲), 3% (♦), 4% (□) and 5% (○)) on Trichoderma asperellum 1 laccase-like activity. b Effect of 1% of sea salt on T. asperellum 1 laccase-like activity. Each data point (mean +/− standard deviation) is the result of triplicate experiments

Fig. 6

Effect of different concentrations of CuSO₄ (0 mM (●), 0.8 mM (■), 1 mM (▲), 1.8 mM (♦) and 2 mM (ӿ)) on Trichoderma asperellum 1 laccase-like activity

Fig. 7

Decolorization of the five reactive dyes (50 mg L^− 1 each), namely industry Reactive Black 5 (RB5) (a), Remazol Brilliant Blue R (RBBR) (b), RR75 (c), Blue Turquoise (d) and Acid Orange (E) in 48 h (% of decolorization in the presence of 1-hydroxybenzotriazole (HBT) (●), % of decolorization in the presence of enzyme (■) and % of decolorization in the presence of enzyme and HBT (▲)). The disappearance of the color by Trichoderma asperellum 1 culture supernatant was monitored at specific wavelengths (585, 597, 520, 438 and 606 nm) with time (1, 2, 3, 4, 5, 24 and 48 h). Each data point (mean +/− standard deviation) is the result of duplicate experiments