Isolation of marine-derived filamentous fungi and their potential application for bioremediation process

Abstract

We evaluated the bioremediation potential of petroleum-derived compounds using fungal strains isolated from marine samples collected on the coast of the states of Paraná, Brazil. About 75 isolated filamentous fungi were subjected to assays including decolorization of the synthetic dye Remazol Brilliant Blue R (RBBR), tolerance to diesel oil, production of bioemulsifying and degradation of pyrene. Nine isolates could decolorize RBBR between 3.4% and 88.16%. Ten were able to tolerate diesel oil and/or pyrene. One isolate was able to produce compounds with emulsifying properties. Three strains, Trichoderma sp. FM14 (Penicillium spp. FM02 and FM16, and FM14) were able to degrade pyrene between 33.0 and 42.4%, after 8 days. The results of the present work encourage future studies to optimize enzymatic conditions using isolates with biotechnological potential in bioremediation studies of marine environments contaminated with industrial pollutants including hydrocarbons derived from petroleum such as diesel oil and PAHs and synthetic dyes.

Introduction

With the rise in anthropogenic activity on the planet in recent decades, due to the increasing intensity of human practices, including agricultural activities, rapid industrialization, and the need to generate cheap forms of energy, environmental pollution is constantly increasing. The intensity of human activities has led to an increase in the undue release of various toxic and recalcitrant organic products into the marine and terrestrial environment, such as plastics, petroleum derivatives, pesticides, industrial dyes, and pharmaceuticals [1, 2].

Petroleum hydrocarbons, mainly composed of alkanes, olefins, and aromatics, are considered one of the organic pollutants that most affect marine ecosystems. They are easily deposited in seabed sediments, persisting for long periods of time, causing serious impacts on aquatic communities [2]. Polycyclic aromatic hydrocarbons (PAHs), especially those with more than 4 aromatic rings in the molecules and their metabolites, have adverse effects on both humans and the environment. Due to their recalcitrant, bioaccumulative, persistence, toxicity, mutagenicity, and carcinogenicity, PAHs have caused significant environmental concern. The PAHs naphthalene, anthracene, phenanthrene, pyrene, and benzo [a] pyrene are considered one of the largest groups of environmental pollutants [1–4]. The increase in industrialization means that in the textile industry, there is an increase in the use of different types of synthetic dyes, compounds that can be released indiscriminately into water bodies to reach the marine environment. Remazol Brilliant Blue R (RBBR) dye, one of the most important dyes in the textile industry, is a compound derived from anthraquinone, a class of toxic and recalcitrant organopollutants [5, 6].

The marine ecosystem has been the focus of biodiversity studies and bioprospecting of enzymes and secondary metabolites from microbial communities responsible for producing a wide range of compounds that can be used in biotechnological processes [3, 7, 8]. Marine microbial communities are important ecological components in these environments, which play roles in biogeochemical processes. Due to their genetic and biochemical diversity, fungi of marine origin are considered promising sources of compounds of biotechnological interest [9]. These microorganisms are adapted to extreme temperatures, acidity, pressure, and salt concentration, so they can be sources of new enzymes with interesting biotechnological characteristics due to these environmental stresses [2]. One of the applications for the marine fungal enzymes market is the use of these biomolecules for cleaning compounds derived from the textile, fuel, cellulose, and paper industries [8].

Microbial degradation can biotransform xenobiotic compounds into less complex metabolites, which can be mineralized into water and carbon dioxide, using intra and or extracellular enzymes [2, 10]. Strains of filamentous fungi isolated from substrates of marine origin with the ability to degrade and/or metabolize petroleum derivatives and textile dyes are reported in previous studies [11–13]. Marine-derived filamentous fungi capable of biologically transforming these compounds have been isolated from various substrates in the marine environment, including seawater, sediments, marine organisms, and estuaries [2, 3, 14].

Due to the great human activity of inappropriate disposal of these compounds listed here, there is great concern about treating marine environments impacted with polluting xenobiotic compounds. Therefore, the application of a sustainable and eco-friendly in comparison with physical and chemical approach, the bioremediation technologies can be a promising strategy [15]. The use of species of filamentous fungi from marine ecosystems in the marine environment itself becomes more easily carried out due to the microbial environment in this saline environment. In this sense, the present work evaluated the ability of filamentous fungi isolated from different marine substrates to tolerate petroleum-derived compounds, decolor synthetic dyes, as well as degrade the hydrocarbon pyrene, investigated by HPLC–FLD analysis, aiming for future bioremediation studies in marine environments.

Materials and methods

Sample collection and fungal isolation

The samples were collected in the cities of Bombinhas on the coast of the State of Santa Catarina, Brazil, in the years 2015 (27º07’54"S 48º31’40"W) and Matinhos on the coast of the State of Paraná, Brazil, in the years 2017 (25º49’03"S 48º31’54"W). Samples were collected using sterile plastic tubes. Samples of coral and sea urchin (2015 collection) and algae and barnacle (2017 collection) were collected. The samples were first sterilized with 70% ethanol and then washed twice with sterilized seawater. The samples were crushed in a Polytron homogenizer and processed in two ways: (i) by the serial dilution method (10^− 1, 10^− 2, 10^− 3, and 10^− 4), with aliquots of 50 µL of each sample dilution used for inoculation in culture media; (ii) direct plating: 1 cm³ pieces of each macroorganisms were placed onto Petri dishes [7]. The culture medium used for the isolation of filamentous fungi was Potato Dextrose Agar– (PDA) (10 g L^− 1 of glucose, 15 g L^− 1 of agar in 1000 mL of potato infusion, added 500 mg L^− 1 of chloramphenicol). The plates were kept in 28 °C for 30 days for filamentous growth. Fungal colonies were purified and preserved at − 80 ºC (20% glycerol) [16]. All culture media used in the present study were prepared using 3% NaCl.

Screening for ability to decolorization RBBR on liquid medium

Assays to analyze the ability of the fungal strains to decolorize RBBR dye in liquid culture medium was performed according to Bernal [16], modified. Three discs of fungal mycelium (0.5 cm in diameter) of the edge of the colony, grown in Petri dishes for 5 days at 28 °C, were transferred to the Erlenmeyer flasks containing Potato Dextrose Broth (PDB) (glucose 10 g L^− 1, in 1,000 mL of potato infusion) culture medium with RBBR added at concentrations of 500 mg L^− 1 and 1000 mg L^− 1. The flasks were incubated in a shaker at 150 rpm for 12 days at 28 °C. One-milliliter aliquots were collected at 3, 5, and 7 days of incubation, centrifuged at 12,000 rpm for 2 min and the supernatant was diluted 10 times with distillate water. From these dilutions, the reduction in absorbance compared to time zero, in a spectrophotometer at a wavelength of 580 nm was observed. Erlenmeyer flasks containing medium altered with RBBR, cell-free, and inoculum medium without RBBR were used as control. All tests were performed in triplicate. The efficiency of the decolorization was expressed by the formula: Decolorization % = (A_{λ, initial} - A_{λ, final})/ A_{λ initial} x 100; where A _{λ initial} = initial absorbance and A_{λ final} = final absorbance.

Tolerance to petroleum derivative

Diesel oil and pyrene

All isolates were subjected to diesel oil and pyrene assay as described by Elshafie [17], with modifications, and Carlos [18], respectively. For diesel oil tolerance, two discs of mycelium (0.5 cm) were inoculated in 125 mL flasks containing 50 mL of specific minimal medium (MM) (KCl 250 mg L^− 1: MgSO₄ 0.5 g L^− 1; NaH₂PO₄ 1 g L^− 1; NH₄NO₃ 1 g L^− 1; pH 7.0). One milliliter of diesel oil was added separately. The flasks were inoculated at 28 °C for 12 days at 150 rpm in triplicate. Erlenmeyer containing MM and fungal inoculum were used as controls. For pyrene tolerance, isolates were grown in 50% composition of PDB media. After 24 h of cell growth, 1 mg L^− 1 of pyrene was added to the culture medium. The samples were incubated at 28 °C, 150 rpm 12 days. The negative control consisted of culturing the isolates without the addition of pyrene. Experiments were performed in triplicate.

Dry weight analysis was carried out to verify microbial growth in tolerance tests to diesel oil and pyrene, according to Carlos [18]. The isolates had their weight determined after vacuum filtration in a dry and previously weighed 0.22 μm filter, followed by subsequent drying at 70 °C. The difference in weight of filter papers with and without cells was calculated using the formula: dry weight = mass of dry paper (with cells)– mass of dry paper (without cells).

Biosurfactant production

Microbial culture and emulsification index (EI24)

The culture medium was adapted based on the study of Kiran [19]. Two discs of mycelium (0.5 com diameter) were transferred to a 125 mL Erlenmeyer containing 50 mL of modified Sabouraud Dextrose medium (peptone 10 g L^− 1, dextrose 40 g L^− 1), with the addition of 1% glucose and yeast extract, 3% NaCl, 0.1 mM of FeSO₄ and 0.5 g L^− 1 of olive oil, pH 7.0 and incubated for 120 h at 20 ºC at 150 rpm.

The emulsification activity was carried out according to Bernal [16]. The isolates were cultured in modified Sabouraud Dextrose Agar medium, and filtered through filter paper to generate cell-free supernatant, followed by centrifugation at 8000 rpm at 4 ºC for 20 min. Two millilitiers of kerosene was added to the cell-free supernatant (CFS) and vortexed for 2 min. Emulsification stability was measured after 24 h and the emulsification index (EI24) was calculated by dividing the measured height of the emulsion layer by the total height of the liquid layer (Height of emulsion/total height of solution) x 100. Tests were carried out in triplicate. Sodium Dodecyl Sulfate (SDS) was used as a positive control and uninoculated culture medium was used as a negative control.

Pyrene degradation analysis

Microbial culture

Each isolate was cultured in 3% malt extract agar for 7 days at 28 ºC. Three fungal culture discs (0.5 cm diameter) taken from the edge of a pure colony, were transferred to 125 mL Erlenmeyer flasks containing 50 mL of minimal medium (MM) (KCl 250 mg L^− 1: MgSO₄ 0.5 g L^− 1; NaH₂PO₄ 1 g L^− 1; NH₄NO₃ 1 g L^− 1; pH 7.0), plus 5% dextrose (w/v). After 48 h of growth, 2 mg L^− 1 of pyrene dissolved in dimethyl sulfoxide was added [3]. The samples were incubated for 8 days at 28 ºC and 150 rpm. The experiments were performed in triplicate.

Sample preparation, extraction, and cleaning

The fermented broths were subjected to the lyophilization process followed by the QuEChERS (an acronym for Quick, Easy, Cheap, Effective, Rugged, and Safe) sample preparation method. The samples were frozen in an ultra-freezer at -80 ºC for 24 h and subsequently taken to the freeze-dryer (SL-404, SOLAB) for 72 h at -30 ˚C and 430,000 μm Hg. The QuEChERS method for sample extraction and cleaning was adapted from Slámová [20]. The lyophilized samples were resuspended in 10 mL of acetonitrile and 5 mL of ultrapure water, and homogenized with ultraturrax (IKA, T18 Ultra Turrax^®) at 9,000 rpm for 5 min. Precipitation was carried out with 4 g of MgSO₄ anhydrous and 1 g of NaCl, vortexing for 1 min and centrifugation at 4000 rpm for 15 min. During cleaning, 2 mL of the organic phase was transferred to 15 mL falcon tubes containing 100 mg of C18, 50 mg of PSA (primary secondary amime) and 300 mg of MgSO₄, vortexing for 1 min and centrifuging at 4000 rpm for 15 min. The 1 mL of supernatant was filtered with syringe filters (0.22 μm hydrophobic PTFE) and transferred to 1.5 mL vials for further analysis.

HPLC–FLD analysis

A high-performance liquid chromatography (HPLC) system (Dionex UltiMate 3000 series, Thermo Fisher Scientific) was used, coupled to a fluorescence detector (FLD) and equipped with a quaternary pump (LGP-3400SD) and an autoinjector (20 µL) [21, 22]. The mobile phase was composed of 85% acetonitrile and 15% ultrapure water, pumped through the column at a constant flow rate of 1 mL min^-1. Separation was performed under isocratic conditions using an ACE 5 C18 column (lot number V13-7473) (250 mm × 4.6 mm; 5 μm particle size; 110 Å particle porosity), maintained at 30° W. The fluorescence detector (FLD) recorded the spectra (λex: 260 nm, λem: 430 nm) for pyrene identification.

Molecular identification

The molecular identification of the isolates was carried out by the company GoGenetic. The extraction of genomic DNA was carried out in accordance with the protocols used in the company. Amplifications were performed with primers for the ITS rDNA region. The amplicons were sequenced on an ABI 3500 automatic sequencer. The sequences obtained were compared with sequences deposited in the Genbank databases (www.ncbi.nem.nih.gov) using BLASTn sequence alignment. Sequences were aligned using the BioEdit program version 7.7.1, and evolutionary phylogenetic analyzes were performed using MEGA 11 software [23] with Kimura’s evolutionary distance substitution model [24]. Phylogenetic trees were constructed using the neighbor-joining (NJ) algorithm [25] using bootstraps calculated from 1,000 replicates. GenBank accession numbers: FM02 (PQ388185); FM14 (PQ388186) and FM16 (PQ388187).

Statistical analyses

Statistical analysis was performed according to Ottoni [26], by using variance analysis (ANOVA) and Tukey test (at 5% significance) in software PAST version 2.17c.

Results and discussion

Isolation of marine-derived fungi

A total of 64 filamentous fungi were isolated from several samples recovered from marine subtracts including coral, different algae species, sea urchin and barnacles. The substrates with the highest number of isolates were the 3 different types of seaweed (n = 30), followed by coral (n = 14), sea urchin (n = 12) and barnacles (n = 8).

Marine fungi are classified as obligate (growing and sporulating exclusively in a marine or estuarine habitat) or facultative (of freshwater or terrestrial origin that are capable of growing, and possibly sporulating, in marine environments). However, the term “fungi of marine origin” has been used to classify these organisms because most fungi recovered from samples of marine origin are not proven to be classified as obligatory or facultative marine [8, 27].

Marine-derived fungi have been documented in association with marine organisms including corals, algae, sea urchins and barnacles. Marine fungi belong to Ascomycetes, Basidiomycetes and Deuteromycetes Phylum and play important roles in coral reefs which can facilitate the dissemination, diversification, and ecological adaptation of marine fungi in various tropical coral reefs [28]. Many fungal representatives from different taxonomic groups have already been recovered from coral samples and explored biotechnologically, including for processes of treating environmental pollutants, including synthetic dyes and PAHs [3, 29, 30].

Like those associated with corals, marine-derived fungi associated with macroalgae are also considered an ecologically important group with great biotechnological potential [31]. The search for metabolites produced by fungal isolates is one of the main drivers in the search for fungal microbial strains recovered from marine samples, including algae, corals, sea urchin and other marine organisms [32, 33]. In the search for information regarding marine-derived fungi associated with the barnacle, we observed that the presence of fungal microbial cells in these organisms is also associated with the obtaining of marine substrates that had been introduced anthropically, such as aged wood-based materials, submerged in water with periods of exposure to air, which could have led to the presence of other decomposing marine organisms, such as barnacles [34]. In these studies, wood samples were collected to obtain lignicolous marine fungi, to produce enzymes with biotechnological applications [35]. In this sense, with the immense biotechnology potential that fungi derived from marine environments have been presenting in synthesizing bioactive metabolites, the present study focused on the search for secondary metabolites and/or enzymes potentially used in biotechnological processes for bioremediation of environmental pollutants.

RBBR dye decolorization ability

All isolates were subjected to selection for those capable to decolorizing the RBBR dye in a liquid medium. After analyzing the isolates, nine showed an ability to decolorize RBBR between 3.4% and 88.2%. Five, three, and one strains were isolated from the substrates, sea urchin, coral, and seaweed, respectively (Fig. 1 Table 1).

Fig. 1.

Analysis of RBBR dye decolorization by marine-derived fungi

Table 1.

Biotechnological potential of marine-derived fungi

Strain	Marine-substrates	Biotechnological potential						Molecular ID
		% DecolorizationRBBR	Tolerance to diesel oil (mg.mL− 1)	Tolerance to pyrene (mg.mL− 1)	% Pyrene degradation		% IE24
					4 days	8 days
FM02	Sea urchin	43,33	0,1573	0,0638	33.76	33.06	n.d	Penicillium sp.
FM07	Sea urchin	9,14	0,0842	0,0331	n.p		n.d	n.p
FM08	Sea urchin	5,12	n.d	n.d	n.p		n.d	n.p
FM09	Sea urchin	n.d	0,0435	0,032	n.p		n.d	n.p
FM10	Sea urchin	n.d	0,0422	0,0339	n.p		n.d	n.p
FM12	coral	87,28	n.d	n.d	n.p		n.d	n.p
FM13	coral	88,16	n.d	n.d	n.p		n.d	n.p
FM14	coral	n.d	0,0467	0,0371	29.38	34.48	17.5	Trichoderma sp.
FM16	coral	42,77	0,0679	0,0351	35.18	42.45	n.d	Penicillium sp.
FM17	coral	n.d	0,1185	0,02695	n.p		n.d	n.p
FM22	Sea urchin	30,99	0,03985	0,03475	n.p		n.d	n.p
FM26	coral	n.d	0,1103	0,0012	n.p		n.d	n.p
FM29	Sea urchin	3,36	n.d	n.d	n.p		n.d	n.p
FM42	seaweed	7,90	0,06505	0,05	n.p		n.d	n.p

n.d = not detected; n.p = not performed; FM = fungal marine

Decolorization of synthetic dyes, including RBBR, by fungi derived from marine environments has been documented in bioprospecting and bioremediation work on these dyes [8, 30, 36]. Works in the literature have been shown the use of sea urchins in the bioprospecting for compounds of pharmaceutical interest produced by invertebrates themselves or by microbial cells that inhabit these organisms [37, 38]. Other studies have shown the use of marine-derived fungi in the decolorization of the synthetic dye RBBR by marine fungi isolated from sponges and corals [30, 39]. According to Ben Ali [36], a strain of Trichoderma asperellum recovered from a polluted water sample in the Tunisian sea, was able to decolorize RBBR by almost 80% after 48 h. The authors used this collection site to isolate fungi capable of metabolizing aromatic compounds. However, it was not possible to observe in the literature studies that used marine-derived fungi isolated from seaweed to decolorization of synthetic dyes thus demonstrating the importance of the present study in treatments of textile dyes in marine environments.

Tolerance to petroleum derivative

All marine-derivate fungi were subjected to selection for tolerance to petroleum derivative including diesel oil and pyrene. Ten isolates showed an ability to tolerate diesel oil and/or pyrene as a unique carbon source ranging from 0.04 to 0.16 and 0.0012 to 0.06 mg mL^− 1 of dry mass for diesel oil and pyrene, respectively (Fig. 2 Table 1). As described in the RBBR assay, for tolerance to petroleum derivative, marine-derivate fungi that showed tolerance potential were associated to sea urchin (n = 5), coral (n = four), and seaweed (n = 01) (Table 1).

Fig. 2.

Analysis of tolerance to petroleum-derived compounds by marine-derived fungi

Filamentous fungi associated with marine substrates have been reported in the literature to grow and/or tolerate the presence of polycyclic aromatic hydrocarbons and diesel oil [40–42]. PAHs contaminate marine environments through various anthropogenic actions, being toxic compounds that threaten the environment due to their xenobiotic characteristics [40].

An aquatic model organism used as a bioindicator to verify the potentially harmful effects that environmental pollutants, including petroleum derivatives, can cause in living organisms, sea urchins has been used in studies monitoring the impact of these compounds in the oceans, because it accumulates more pollutants than other organisms during the same period [43]. In this way, we can say that sea urchins can be considered reservoirs of microbial cells potentially applicable in bioremediation studies of compounds derived from petroleum, a characteristic that justifies the potential of the five marine isolates recovered from sea urchin samples in the present study.

Concerning marine fungi associated with coral reefs, it can co-exist with these organisms as endoliths, endobionts, saprotrophs, and pathogens. Fungi that are unique as endoliths (endemic) in corals appear to play a role in the coral reef system. Corals can provide an environment rich in organic compounds for a diverse microbial community composed of viruses, bacteria, archaea, cyanobacteria, fungi, and endolithic algae thus forming a mutualistic ecosystem [44].

The marine macroalgae or seaweed are eukaryotic and photosynthetic organisms, which are distributed worldwide on the coasts and oceans. In the ocean, seaweed and microorganisms form an inseparable relationship [45]. Microbes, including archaea, bacteria, fungi, microalgae, protozoa, and viruses, play an indispensable role in the growth, development, and maturity of marine algae, thus forming a holobiont. This interaction has already been related to bioremediation processes involving the degradation of organic pollutants, including black oil and some PAHs [46].

In this way, the use of microorganisms derived from marine environments including organisms considered a rich source of microbial diversity such as sea urchins, coral reefs, and seaweeds, that tolerate the salinity conditions and pressure exerted by this environment can be used as bioremediation agents for compounds derived from petroleum, including diesel oil and PHAs, more efficiently if microorganisms from other biomes and not the marine one.

Biosurfactant production

In the present study, Trichoderma sp. FM14 strain could produce an IE₂₄ of 17.5% (Table 1). The production of biosurfactants by marine-derived fungi has already been reported in the literature [47, 48]. Trichoderma harzianum MUT 290, isolated from the Mediterranean Sea with a history of oil spills, produced proteins identified as keratoplatanins with biosurfactant properties. Furthermore, this protein presented an IE₂₄ of up to 83% [49].

Pyrene degradation

The degradation of HPA pyrene was observed over an interval of 8 days, with a variation in degradation between 29.4% and 42.4%. Penicillium sp., FM16 was the best pyrene degrader, reaching 42.4% after eight days of cultivation (Fig. 3).

Fig. 3.

Analysis of pyrene degradation by marine-derived fungi

Work in the literature has already demonstrated the degradation of PAHs by marine-derived fungi, including the genera Penicillium and Trichoderma [3, 50–52]. These results demonstrated that in just eight days Penicillium sp. FM16 degraded almost half of all HPA in the cultivation environment. If the cultivation time had been longer, we could have seen an even greater percentage of degradation. Even more impressive results were observed with the marine-derived fungal strain Aspergillus sclerotiorum CBMAI 849, reaching 84.9% pyrene biodegradation after 4 days of growth in liquid medium [3]. In the present study, it is possible to state that the Penicillium spp. FM16 and FM02 as well as Trichoderma sp. FM14, presented excellent results, both for tolerance to petroleum-derived compounds and in the degradation of HPA pyrene. However, it was not possible to find less toxic intermediate metabolites arising from pyrene degradation, such as pyrenylsulfate [3]. Other states have also observed degradation of PAHs, including pyrenes, by marine-derived fungal strains, some of them achieving almost 100% and 95% of pyrene degradation [50, 53]. Polycyclic aromatic hydrocarbons can be degraded both by intracellulares, such as monooxygenase, as well as by extracellular enzymes, known as ligninolytic enzymes, including manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac) [3, 6, 54].

Phylogenetic analyzes showed that filamentous fungi recovered from the marine environment, which presented the best results when applied as PAH bioremediation agents, were related to the species Trichoderma sp. (FM14) (Fig. 4a) and two strains of the genus Penicillium spp. (FM02 and FM16) (Fig. 4b). By sequencing the ITS rDNA, FM02 showing 100% sequence similarity with Penicillium sp. CBS:126,084 (MH864074), Penicillium infrabuccalum DAOMC 250,537 (NR_172032), Penicillium simplicissimum (KR296862), 99.8% Penicillium pedernalense type material (NR_146250), and 98.7% with, Penicillium panissanguineum CBS 140,989 (NR_158825), Penicillium mariae-crucis CBS:271.83 (MH861585). Likewise, FM14 showing 99.8% sequence similarity with Trichoderma koningiopsis CGMCC40722 (OR646746), 99.8% with Trichoderma sulphureum (MT529835), and 99.5% with Trichoderma atroviride (MN341302), Trichoderma petersenii NFCF133 (KT852818). In the same way, FM16 showing 99% sequence similarity with Penicillium ochrochloron strain PFR8 (KF358720), Penicillium simplicissimum CBS:280.39 (MH856014), Paecilomyces victoriae CBS:469.70 (MH859799), and 98.5% with Penicillium pulvillorum CBS:132,165 (MH865967). The same genera that showed biotechnological potential in the present study have already been found in the same marine substrates used here. Filamentous fungi strains belonging to the genus Penicillium have already been isolated from samples of sea urchin Scaphechinus mirabilis [55], Brisaster latifrons [56], and from deep-sea coral [57, 58]. Likewise, Trichoderma species have been isolated from coral samples recovered from the marine environment [59, 60].

Fig. 4.

Phylogenetic analysis based on partial fungal ITS sequences of isolates FM14 (a) and FM02 e FM16 (b). Bootstrap values (1000 replicate runs) greater than 70% are listed. GenBank accession numbers: FM02 (PQ388185); FM14 (PQ388186) and FM16 (PQ388187)

Coral reefs are complex ecosystems that harbor a series of microorganisms, called holobiont coral. Filamentous fungi play an important role in converting biomass to nutrients in a coral reef ecosystem [61]. Although other groups may also exist in these ecosystems such as endoliths, endobionts and saprotrophs, which may be involved in microperforation, carbonate alteration, decolorization and symbiotic or parasitic association [44]. Some fungi have been causing massive destruction of some soft corals around the world [62]. Sea urchins inhabit all marine ecosystems, are present in coral reefs and can influence this ecosystem as they alter the properties of the substrate through the bioturbation of sediments [63]. Species of the genera Penicillium and Aspergillus are two of the most widespread fungi on Earth, being permanent components of marine microbial communities, being studied as promising strains producing metabolites with a wide range of biological activities [55]. Thus, the results of the present work demonstrated the biotechnological potential that filamentous fungi recovered from marine environments can present. The fungal strains evaluated in this work can be used in bioremediation processes in marine environments contaminated with petroleum-derived compounds. More research is needed to characterize the possible enzymes involved in pyrene degradation as well as to identify the possible metabolites formed.

Author contributions

Osvaldo Manuel Núñez Nogueira: Methodology, formal analysis. Suzan Prado Fernandes Bernal: formal analysis. Cleto Kaveski Peres: sample collection. Marcela Boroski: Writing– original draft, Data curation, Validation, Supervision. Michel Rodrigo Zambrano Passarini: Writing– original draft, Data curation, Validation, Supervision, sample collection.

Funding information

This work was supported by the Institutional Program to Support Research Groups EDITAL PRPPG Nº 205/2021 - Institutional Program Triple Agenda– UNILA.

Data availability

In this manuscript, there is no raw data such as nucleotide acid sequences, protein sequences, genetic maps, SSR, expression data, etc. Nevertheless, all the original metaphases pictures are in possession of the authors and available for the reviewers or for submission in my database if necessary.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.