Fungi are recognized for their aptitude to produce a large variety of extra-cellular enzymes . However, most of the fungi studied to date are isolated from forests and other terrestrial environments, while very few studies have focused on the exploration of marine fungal diversity. A large proportion of the diversity of marine-derived fungi would have originated from their terrestrial counterparts, with the appearal of strains able to live in marine harsh environments (high pressure, low temperature, oligotrophic nutrient, high salinity, etc.) [19, 20]. These specific conditions are responsible for the significant differences between the enzymes generated by marine microorganisms and their homologues from terrestrial counterparts . Finally, marine-derived microorganisms have been studied to exploit their potential to generate new natural products and to degrade plant biomass .
In this study, twenty marine derived fungi were isolated from Tunisian marine biotopes and five of them were selected for their oxidative profile on DMP and ABTS. These five strains were identified as ascomycetes belonging to the species Aspergillus nidulans, Stemphylium lucomagnoense and Trichoderma asperellum (three strains belonging to the latter species). Among these marine strains, Aspergillus nidulans, anamorph of Emericella nidulans, is an important model ascomycete for eukaryotic genetics. A few studies are dedicated to marine-derived A. nidulans species, such as two relatively recent works reporting on the production of molecules of interest: melanin precursors with UVB protective properties  and antitumor alkaloids . Another strain identified in this study belongs to the phylum ascomycetes (Dothideomycetes Pleosporales, Pleosporaceae) and more precisely to the Stemphylium genus, that encompasses worldwide distributed saprophytes and plant pathogens affecting a variety of agricultural crops. Molecular analysis branched Stemphylium sp. with both S. vesicarium and Stemphylium lucomagnoense in the phylogenetic tree, but morphological treats confirmed that the isolated species is S. lucomagnoense, anaphorm of Pleospora lucomagnoense. To date, only two studies have been focusing on marine-derived Pleospora. The first deals with the production of antimicrobial compounds  and the second with phylogeny of Pleospora gaudefroyi .
Previously, a number of molecular markers have successfully been used for the taxonomic identification of fungal genera and species, and ITS rDNA region has been often considered as a marker of choice for the fungal kingdom . However, sequencing of the TEF-1α region is considered as a sensitive tool for identification in mycology with superior resolution then ITS, e.g. when studying the genus Trichoderma . In this study, TEF-1α sequence-based phylogeny suggests that the most phylogenetically related species to our three isolates Trichoderma sp 1, 2 and 3 is Trichoderma asperellum, a fungus which is naturally found in soils . Even if Trichoderma species are usually found in terrestrial habitats, some isolates were collected from marine environments, where they live in association to algae  and sponges , in coastal sediments , or as endophyte in mangroves . Among these marine-derived species we found T. asperellum wich was further studied for its production of secondary metabolites, such as sesquiterpenes  and antibacterial peptides .
Different Trichoderma species were extensively studied as sources of cellulases, but also oxidases such as LMCOs . This was the case, for instance, with the terrestrial species Trichoderma reesei , T. harzianum and T. longibrachiatum , as well as for the marine-derived Trichoderma sp. . Moreover, a terrestrial T. asperellum secretome producing oxidases including LMCOs was applied to degrade polycyclic aromatic hydrocarbons in soil . In our study, the secretomes of five fungal isolates showed different amounts of laccase-like activities, in liquid cultures and eventually under saline conditions. The highest laccase-like activity was observed with the strain T. asperellum 1, in cultures with as well as without 1% NaCl. For comparison, while marine-derived A. sclerotiorum produced 9.26 U L− 1 laccase-like activity after 7 day-culture in 3% (w/v) NaCl, for T. asperellum 1 about 190 U L− 1 were obtained. In another study  optimization of laccase-like activity levels from Trichoderma sp. grown in 0.5% NaCl yielded approximately 2000 U L− 1, but activity was assayed using o-tolidine instead of ABTS as a substrate, and as such those results are not directly comparable with ours. The finding of laccase-like activities from fungal cultures grown in NaCl-containing media could be benifical for industrial and biotechnological processes in which saline conditions are high . In our study, we show that high levels of salt-tolerant laccase-like activity could be spot out using synthetic dyes as substrates. These findings pave the way to the discovery of novel biocatalysts for the textile industry, whose effluents contain not only dyes, but also high salt concentrations. Secretome and enzyme characterization will then be the next step of our research.
To maximize the levels of laccase-like activity in T. asperellum 1 cultures, we evaluated the effect of different concentrations of NaCl and known inducers, such as CuSO4 and three carbon sources. These parameters in fact can affect the productivity of various oxidases secreted in the culture medium, due to an inhibition of fungal growth or to effects on enzyme stability and activity, possibly in relationship to protein surface charges and to perturbation of global or local protein folding . In our study, higher levels of laccase-like secreted activity were found when 1% NaCl was added to T. asperellum 1 cultures. Above this concentration, activity gradually decreased with increasing NaCl concentration. The effect of NaCl was also studied for other marine fungi like Cerrena unicolor isolated from mangroves , and was shown to enhance laccase activity in fungal culture supernatants. Similarly, by adding sea salt to T. asperellum 1 cultures, we obtained an increase of the supernantant oxidase activity in time, with a maximum at 75 h, like with NaCl, but no decrease afterwards, unlike with NaCl. In previous studies we demonstrated the activation by sea salt of two LMCOs from the mangrove fungus Pestalotiopsis sp. , while LMCO from Trematosphaeria mangrovei lost 50% of its activity in 1% NaCl . Salt-adapted enzymes are generally characterized by highly negative surface charges that are assumed to contribute to protein stability in extreme osmolytic conditions . Copper has been reported to be a strong laccase inducer in several fungal species , . It has been also reported that the increase in activity is proportional to the amount of copper added . In our study, optimal CuSO4 concentration was 1800 µM for T. asperellum 1 cultures, yielding about 173 U L− 1 laccase-like activity. These results are in agreement with previous ones , showing optimum LMCO activity (32.7 U mL− l) in Pestalotiopsis sp. cultures with 2.0 mM CuSO4, and activity decrease above this concentration. Nakade et al  reported that the best CuSO4 concentration for LMCO production in Polyporus brumalis was 0.25 mM. CuSO4 induction of LMCOs is related to the active site architecture of these enzymes, which contain generally 4 copper atoms per polypeptide. Copper addition to the culture medium was also reported to induce laccase gene transcription . In addition, it has been reported that copper could be toxic as it interacts with nucleic acids, proteins, enzymes and metabolites associated with major cell functions, explaining why CuSO4 concentration should be tested case by case . Several studies have proved that the choice of carbon sources affects the production of ligninolytic enzymes . The purpose of glucose supplementation to lignocellulose for fungal cultures has two reasons. First, it promotes the growth and rapid establishment of the fungus within the solid raw material. Second, the fungus needs an additional, easily metabolizable carbon source to sustain lignin degradation from lignocellulosic substrates . In our study, sucrose is the best substrate for secreted laccase-like activity from T. asperellum 1 cultures (290 U L− 1), as it has previously been showed for Arthrospira maxima .
Industrial dyes usually have a synthetic origin and complex aromatic structures which make them highly resilient and more difficult to biodegrade . Reactive dyes, for exemple, contain chromophoric groups such as azo, anthraquinone and others. Most of these dyes are not toxic by themselves, but after release into aquatic environments they may be converted into potentially carcinogenic amines that have an impact on the ecosystem downstream from the mill . Currently employed physico-chemical methods were showed to have some serious limitations, such as high cost, high salt content utilization, and problems related to disposal of concentrate [57, 58]. In this regard, considerable focus has been placed on developing biological processes, because they are more effective compared to more conventional, physico-chemical methods . The production of LMCOs from marine-derived ascomycetes, zygomycetes and basidiomycetes has been poorly investigated [41, 59]. Similarly, to our knowledge, only one work reports on the application of LMCO-containing secretomes from a marine Trichoderma to degrade synthetic dyes , one describes the production of laccase from marine-derived Aspergillus sclerotiorum  and no work is available on LMCOs derived from Stemphylium species. In this study, the dye decolorization ability of T. asperellum 1 secretome was tested against five different industrial synthetic dyes: Reactive Black 5 (RB5), Remazol Brilliant Blue R (RBBR), Direct Red 75 (DR75), Turquoise Blue (TB) and Acid Orange 51 (AO51). These dyes belong to different dye families: reactive, azo and anthraquinone. It is generally observed that the extent of decolorization is known to depend on the enzyme properties (and as such, the biological source) as well as the chemical properties, structure and size of the dye molecule [2, 60]. Due to its high molecular weight, for example, sulfonated azo dyes are unable to pass through the cell membrane, and therefore degradation of these dyes must occur extracellularly. The role of redox mediators in an azo bond detoxification has also been shown before . For instance, it has been reported that the addition of the mediator HBT to the LMCO-containing secretome of Paraconiothyrium variabile enhanced the decolorization of RB5, RBBR, DR75 and TB .
In a previous study we investigated RBBR decolorization by the culture filtrate of the terrestrial ascomycete Trametes trogii and by the LMCO isolated from it . The purified LMCO decolorized up to 97% of a 100 mg L− 1 dye solution, with only 0.2 U mL− 1 enzyme. In our test conditions, we reached comparable results (60–80% decolorization) with T. asperellum 1 culture supernatant, with or without HBT. In general, different marine strains are able to degrade RBBR to different extents, for exemple Flavodon flavis degraded RBBR to more than 90%  while Cerrena unicolor only to 46% .
Biodegradation of RB5 was investigated using the secretome of the ascomycete Trichoderma atroviride F03 yielding 91.1% decolorization without mediators . Three products of this biodegradation reaction (1, 2, 4-trimethyl benzene, 2, 4-ditert butylphenol and benzoic acid-TMS derivatives) were identified, confirming the validity of enzymatic treatment without generating aromatic amines, which are higly toxic . In comparison, the T. asperellum 1 secretome allowed attaining only 10% of RB5 decoulorization without HBT, and up to 80% in the presence of mediator.
AO51, is a water-soluble anionic azo dye. Typically containing one to three sulfonic groups, it is widely applied to colour wool, silk and polyamide. The nature and level of toxicity of AO51 has not been well established yet , but sulfonated azo dyes (including naphthalene sulfonic acids, naphthols, naphthoic acids, benzidines, etc), and particularly benzidines are in the focus of attention because of their carcinogenicity . AO51 degradation by crude LMCO from Trametes trogii grown in solid cultures on sawdust has been investigated  and above 88% decolorization in the presence of HBT was achieved. Our results show that with T. asperellum 1 culture supernatant, instead, HBT was not essential for achieving AO51 decolorization. To our knowledge, this is the first report of AO51 decolorization without the need of laccase mediators.
To date, only a few studies have dealt with decolorization of the phthallocinine dye TB. Plácido et al. showed that Leptosphaerulina sp. effectively decolorized TB and two real effluents from textile industries . This decolorization was catalyzed by the production of significant quantities of LMCO (650 U L− 1) and manganese peroxidase (100 U L− 1). Leptosphaerulina sp. enzymatic extracts exhibited decolorizing activity when ABTS was added as mediator. Similarly, the secretome of T. asperellum 1 showed maximum TB biodegradation capacity when HBT was added.
Remarkably high levels of DR75 degradation (95–100%) were achieved after 120 h incubation with Penicillium oxalicaum culture supernatant . In that study, high levels of manganese peroxidase activity (659.4 ± 20 U L− 1) were measured in the P. oxalicaum secretome, indicating the involvement of heme peroxidases in the decolorization process. In our study, instead, no peroxidase activity was detected in T. asperellum 1 secretomes, suggesting for the first time, to our knowledge, that LMCO-catalyzed DR75 degradation takes place instead.
Further studies will be necessary to get further insight into the enzymatic mechanisms deployed by marine-derived fungi to cope with their environment. It will be necessary to identify the key enzymes secreted by T. asperellum 1 growing in saline conditions, as well as to produce and characterize them, with a focus on salt-depence and the structure-function relationship underlying enzyme properties. In order to asses the potential of T. asperellum 1 secretomes or enzymes for enzymatic bioremediation of textile effluents, finally, the degradation products of enzymatically treated model dyes and industrial samples should be precisely identified and characterized, and their impact on human health and environment should be determined.