Introduction

The textile industry is one of the sectors in the world economy with the highest growth in recent years. The discovery of mauve synthetic dye promoted the marketing of many new dyes replacing natural pigments (Cañamares et al. 2014). There are several industries that use synthetic dyes, and it is estimated that there are over 2000 types of these xenobiotics available for the textile industry (Banat et al. 1996; Verma et al. 2015). The dyes do not have a defined chemical structure and, for this reason, are commercially named according to the two main groups, chromophores and auxochromes, present in their molecules which are responsible for giving colour and fixing the colour on the fibre, respectively (Banat et al. 1996; Welham 2000).

A significant amount of the world’s dye production is dispersed in the environment, and these effluents are not treated properly, causing damage to the environment and human health (Banat et al. 1996; Bisschops and Spanjers 2003; Chung 2016; Meehan et al. 2006; Urra et al. 2006).

Bioremediation processes, particularly those using microorganisms as biodegradation or biotransformation agents of pollutants by non-hazardous or less hazardous chemicals, are presented as one of the most promising alternatives for reduction and/or elimination of toxic effluents, aiming at low environmental impact and financial cost (Mcmullan et al. 2001; Singh et al. 2014).

The bioprospecting of microorganisms isolated from different habitats has become a source of biotechnological resources for these and other possible applications, since these microorganisms can have unique metabolic characteristics. In this context, endophytic microorganisms, applied with or without a plant, have been presented as good degraders or stimulants of bioremediation of textile dyes and as future alternatives for the treatment of textile effluents (Afzal et al. 2014; Lima et al. 2007). These microorganisms, usually bacteria and fungi, all inhabit the interior of tissues and plant organs in an asymptomatic way, carrying out important roles in adapting plant processes to the environment (Azevedo and Araujo 2007; Ma et al. 2016; Rhoden et al. 2012), and are often involved in complex chemical and genetic relationships resulting in the synthesis, accumulation and degradation of secondary metabolites and other organic molecules (Kusari et al. 2012; Polonio et al. 2016).

Studies have shown that wastewater can contain mutagenic or carcinogenic substances. To analyse the toxic potential of these effluents, fish are good organisms on which to test the toxicity of these compounds in the laboratory aiming the direct exposure of these animals in contaminated environments (Avni and Jagruti 2016; Kaur and Dua 2016). Micronucleus assays make it possible to detect the genotoxic effects of toxic agents present in industrial effluents and may be used in the evaluation of environmental conditions (Kaur and Dua 2016; Von Ledebur and Schmid 1973).

Aiming at investigating the potential of endophytes as bioremediators, this work demonstrated the capacity for discolouration/remediation of Reactive Black 5 (RB5) and Reactive Blue 19 (RB19) textile dyes using two endophytic strains: strain IG03 isolated from Helianthus annuus L., identified in this paper as Paecilomyces formosus, and Phlebia sp. (JF766997.1) isolated from Piper hispidum Sw. (Orlandelli et al. 2012). The cytotoxicity of dyes and post-treatment dyes were evaluated by micronucleus frequency analysis in fish erythrocyte cells.

Materials and methods

Dyes and chemicals

The textile dyes RB5 and RB19, Protein Quantification Kit-Rapid (Bradford), bovine catalase and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich®.

Endophytic fungi and culture conditions

The endophytic strains Phlebia sp. (JF766997.1) isolated from P. hispidum Sw. (Orlandelli et al. 2012) and P. formosus, initially identified as IG03, isolated from H. annuus L. belong to the microorganism collection of the Microbial Biotechnology Laboratory (LBIOMIC) of the State University of Maringa in Maringa, State of Parana, Brazil.

The endophytes were grown on plates containing potato dextrose agar (PDA) (HiMedia) at pH 6.8 and potato dextrose (PD) broth (HiMedia). For treatments containing the dyes, 0.1 g L−1 was added to the culture medium. All assays were performed after a previous reactivation of fungal strains for 7 days at 28 °C on PDA.

Three controls were performed for the tests: (1) Erlenmeyer flasks containing only medium (PDA and/or PD broth), (2) Erlenmeyer flasks containing medium with inoculum but without dyes and (3) Erlenmeyer flasks containing medium with 0.1 g L−1 of the dyes added, but without fungal inoculum. All controls were performed under the same cultivation treatment conditions.

Microscopic characterisation and molecular identification of the IG03 strain (P. formosus)

Microscopic characterisation was performed by the microcultivation technique. After the procedure, microscope slides were viewed under a light microscope with an image capture system. The results of macroscopic and microscopic evaluations were compared with features available in the literature (Samsin et al. 2009).

For molecular identification, genomic DNA was extracted according to the methodology described by Raeder and Broda (1985) and modified by Pamphile and Azevedo (2002), except that the endophytes were previously grown for 7 days on petri plates with PD broth. DNA integrity was checked by electrophoresis in 1% agarose gel with the use of 1 kb DNA Ladder (RTU Kasvi®) as the molecular weight standard.

Amplification of the ITS1-5.8S-ITS2 regions of the rDNA was performed using V9G (5′-TTACGTCCCTGCCCTTTGTA-3′) (Van Den Ende and De Hoog 1999) and ITS4 (5′-TCCCCGCTTATTGATATGC-3′) primers (White et al. 1990). The PCR was performed under the conditions: 50 ng of DNA, 1× PCR buffer (50 mM of KCl, 200 mM of Tris-HCl, pH 8.4), 1.5 U of Taq polymerase (Invitrogen, Carlsbad, CA, USA), 0.25 μM of each primer, 0.2 mM of each dNTP (Amersham Bioscience, Freiburg, Germany) and 3 mM of MgCl2 in a total volume of 25 μL. The amplification was performed in an Eppendorf® thermocycler (model: Mastercycler Gradient) using the following conditions: initial denaturation at 94 °C for 5 min; 35 cycles of 30 s at 94 °C, 50 s at 48 °C and 2 min at 72 °C and a final extension of 5 min at 72 °C.

All amplification products were purified using Exo1 and FastAP™ enzymes (GE Healthcare, USA). The product from PCR (10 ng of DNA template) was sequenced using a BigDye Terminator Cycle Sequencing Kit v.3.1 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions, using the same set of primers (V9G and ITS4), and the following conditions: initial denaturation at 96 °C for 1 min and 25 cycles of 15 s at 96 °C, 15 s at 50 °C and 4 min at 60 °C. The sequences were analysed on an ABI3500 DNA Sequencer (Applied Biosystems, Foster City, CA, USA).

The sequences obtained were analysed using BioEdit software v.7.2.2 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html), subsequently were aligned with other type sequences deposited in the GenBank database of NCBI (http://www.ncbi.nlm.nih.gov/) and performed the phylogenetic analyses according Bongiorno et al. (2016).

After molecular and microscopic identification, the sequence was submitted to GenBank under accession number KT781078.

Spectrophotometry of textile dyes

Scanning spectrophotometric analysis was performed in aqueous solutions containing 0.1 g L−1 of each textile dye in the region 200 to 800 nm, in quartz cuvettes with 1-cm optical path, using a Biochrom Libra S60PC spectrophotometer.

The analysis of absorption spectra allowed us to determine the wavelength of maximum absorption for each dye tested. Based on the results obtained, the wavelengths used for remaining analysis were selected.

Evaluation of the decolouration potential of Phlebia sp. and P. formosus

An initial assessment of dye decolouration potential using endophytes was performed at the wavelength established for each dye (593 nm for dye RB19 and 597 nm for RB5). Discs of 6-mm diameter of mycelial fungi were inoculated on petri plates containing PDA medium and in Erlenmeyer flasks with 50 mL PD broth, both with 0.1 g L−1 of the dyes. Assays were performed in triplicate and incubated for 15 days at 28 °C.

To evaluate the decolouration potential of the samples in PD broth, the medium was centrifuged at 4200×g for 10 min to obtain the supernatants. For samples grown on PDA in petri dishes, the materials secreted by fungi were extracted by adding 5 mL of PD broth and stirring at 175 rpm for 30 min. Samples were measured spectrophotometrically with a specific wavelength for each dye.

For calculation of the dye discolouration percentage (D%), Eq. (1) was used:

$$ D\%=\left(1-\frac{At}{Ac}\right)\times 100 $$
(1)

where At corresponds to the absorbance value of treatments and Ac the absorbance value of controls.

Decolouration activity and protein content over 30 days

The endophytic fungi were inoculated into Erlenmeyer flasks containing 50 mL of BD broth with 0.1 g L−1 of dye and incubated at 28 °C. Upon reaching 3, 7, 15 and 30 days of incubation, the broths were transferred to 50-mL centrifuge tubes and centrifuged at 16,800×g for 5 min. The supernatants were analysed in a Biochrom Libra S60PC spectrophotometer.

The mycelia were separated and dried at 50 °C for 24 h. The dried mycelia were macerated with 250 μL NaOH (1 M) and brought to boiling point in a bath for 5 min. Finally, the samples were centrifuged for 5 min at 16,800×g.

The PD and PDA supernatant media were measured for total protein using the Bradford method (Bradford 1976). All samples were performed in triplicate.

Enzymatic activity

The total enzyme, laccase and peroxidase activity was evaluated by monitoring oxidation of ABTS at 420 nm at intervals of 30 s for 20 min, according to the methodology described by Machado and Matheus (2006), with modifications.

To calculate the enzyme activity, an enzyme unit (EU) was considered to correspond to the amount of enzyme capable of oxidising 1 μmol of substrate per minute, using the molar extinction coefficient for oxidised ABTS (ε = 36,000 M−1 cm−1).

For calculation of enzyme activity, values were calculated using Eq. (2):

$$ mEU=\frac{\left(\frac{A\times L}{\varepsilon}\right)\times d\times {10}^4}{t} $$
(2)

where A is the absorbance value at 10 min, L is the optical path (1 cm), ε the molar extinction coefficient (36,000 M−1 cm−1), d is the sample dilution value (1:10), 104 corresponds to the conversion factor and t is the time for measuring the absorbance.

Total enzyme activity

For total enzyme activity, solutions were prepared containing 1.2 mL of enzyme extract, 0.5 mL citrate-phosphate buffer (pH 4.0), 0.1 mL of 2 mM hydrogen peroxide and 0.2 mL of 5 mM ABTS.

Laccase

The laccase activity was determined by oxidation of ABTS (Machado and Matheus 2006) with addition of bovine catalase (with 15-min pre-incubation).

Total peroxidase

For the determination of total peroxidase, we calculated the difference between the total oxidation of ABTS and laccase activity (Eggert et al. 1996).

Micronucleus test

For micronucleus (MN) analysis, 20 Astyanax sp. (Lambeth) fish were used. The fish were submitted to different treatments: with dyes, with dyes treated by endophyte, with endophyte broth and without treatment. The aquariums were divided into 10 tests, with two fish in each.

The first tank contained 10 L of water (negative control); the second contained 0.01 g L−1 PD broth (PD control); in the third and fourth tanks, the fish were exposed to 0.01 g L−1 blue dye and black dye, respectively; in the fifth aquarium, the fish were exposed to P. formosus fungus fermentation broth; in the sixth and seventh aquariums, the fish were exposed to blue and black dye biodegraded by P. formosus, respectively; in the eighth aquarium, the fish were exposed to Phlebia sp. fermentation broth; in the ninth and tenth aquariums, the fish were exposed to blue and black dyes biodegraded by Phlebia sp., respectively.

Peripheral blood obtained from tail vein puncture was collected from each fish for preparation of slides to evaluate the presence of MN; then, smear slides were prepared with a drop of blood (approximately 50 μL) and dried for 24 h at room temperature. The samples were fixed in absolute methanol for 15 min and stained with 5% Giemsa solution for 10 min. A light microscope at 100× objective lens was used to analyse 1000 erythrocytes per treatment (Hooftman and De Raat 1982).

Statistical analysis

The data from the experiments were analysed using SISVAR v.5.4 software and by analysis of variance (ANOVA), followed by application of Tukey’s test for comparison of the means of fungal discolouration potential assessment, colour discolouration activity range up to 30 days, and MN test and the Scott-Knott test for comparison of means, to avoid ambiguity in the multiple comparison procedures for the protein content test by the Bradford method over time. For all tests, were considered p < 0.05.

Results

Microscopic characterisation and molecular identification of fungus strain IG03 (P. formosus)

The fungus strain IG03 is composed of long, branched filamentous hyphae (Fig. 1c). The conidiophores are irregularly branched, with brown-olive conidia (Fig. 1b). Chlamydospores present (often in small stalks) with smooth surfaces are globular and weakly pigmented. Conidia of this species can range from ellipsoidal to cylindrical, all with truncated ends (Samsin et al. 2009).

Fig. 1
figure 1

Identification of the IG03 strain (Paecilomyces formosus). a Dendogram constructed using the neighbour-joining method with p-distance for nucleotides, pairwise deletion gap and bootstrap with 10,000 repetitions. b Macroscopic view of P. formosus IG03 strain. c Microscopic structures of the endophytic fungus P. formosus. The arrows correspond to typical conidiophores

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Alignment of the DNA sequence of the IG03 strain showed 99% identity to P. formosus CBS 372.70 (FJ389926.1) (Fig. 1a). Phylogenetic analysis showed high evolutionary similarity between these two strains, these being grouped with two other strains of the same species with 99% bootstrap. Despite the genetic and morphological similarity between P. formosus and P. variotii, these remained isolated separately on another branch in the cladogram. Accordingly, the microscopic and molecular analysis corroborates the identification of the endophytic strain IG03 as P. formosus. The sequence is available for access in GenBank under number KT781078.

Textile dye absorption spectra

UV-Vis light absorption spectra from aqueous solutions of RB5 and RB19 dyes (0.1 g L−1) were performed in order to select the best wavelengths to be used in the other tests.

Two maximum peaks were observed for each dye and were selected for the other tests. The spectrum peaks were present in the visible wavelength region: 593 nm for the blue dye and 597 nm for the black dye.

Fungal decolouration potential

Considering the graph shown in Fig. 2, we can conclude that both Phlebia sp. and P. formosus possess the ability to bleach the dyes when inoculated on solid medium; the percentage decolouration for Phlebia sp. was 91% for the blue dye and 100% for the black dye, while for P. formosus, it was 75 and 97%, respectively.

Fig. 2
figure 2

Percentage dye decolouration by Phlebia sp. and P. formosus fungi in liquid and solid mediums

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In liquid medium, Phlebia sp. presented a great potential for decolouration of both dyes, with 79% for blue and 91% for black dye. Since P. formosus showed significant decolouration of the black colour, with an average rate of 92%, these strains present less effectiveness for blue dye in liquid medium (15% decolouration).

Decolouration activity over time

The graph in Fig. 3 shows linear growth of decolouration of the textile dyes for Phlebia sp. until the 15th day. After this period, the treatment with the blue dye remained constant in the range of 70%, while the treatment with the black dye achieved 90% decolouration within 30 days.

Fig. 3
figure 3

Decolourisation of dyes by Phlebia sp. and P. formosus fungi over 30 days

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For P. formosus, the maximum point of decolouration for both dyes was observed at 7 days of incubation. For decolouration of black dye, the maximum value was 90% in this period. However, less decolouration was observed for the blue dye (20%). After 7 days, a reduction of the decolouration percentage was observed.

Considering the treatment with black dye, a reduction to 70% was observed, following a linear trend until the last day of analysis. The treatment with the blue dye showed no decolouration after this period.

In Fig. 4a, it can be observed that at 7 days, the treatment with the P. formosus strain showed little difference in staining compared to the blue control, and after 15 days, under the same conditions, the broths presented a darker colour, possibly because by-products released by fungal activity interfered with the absorbance analysis. In Fig. 4c, which shows the treatment of the black dye, degradation is visible mainly after 7 days of incubation. With regard to treatment with Phlebia sp., when analysing Fig. 4b, decolouration of the blue dye over time can be seen, with the black dye decolouration (Fig. 4d) more visible from the 15th day of treatment.

Fig. 4
figure 4

Decolouration of the textile dyes over 30 days. a Treatment of blue dye with P. formosus. b Treatment of blue dye with Phlebia sp. c Treatment of black dye with P. formosus. d Treatment of black dye with Phlebia sp.

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Protein content at intervals up to 30 days

In both analyses of protein concentration (intracellular and extracellular proteins), there was a higher concentration of total protein produced by the P. formosus strain. In Fig. 5a, at 15 days of incubation, it can be observed that concentrations of total protein present in the mycelia were statistically similar for both treatments (black dye and blue dye).

Fig. 5
figure 5

Protein content measured by the Bradford method over 30 days. a Evaluation of protein in the mycelium. b Evaluation of protein in the supernatant

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At 30 days, treatments with Phlebia sp. had decreased intracellular total protein, with 238 μg mL−1 with blue dye and 380 μg mL−1 with black dye, when compared to 15 days (850 and 1197 μg mL−1, respectively). P. formosus maintained a relatively constant mycelial protein content compared to the values obtained in 15 days, varying in the range between 892 and 1281 μg mL−1 in this period.

In Fig. 5b, which represents the values obtained in the extracellular medium, it can be observed that the protein levels from both fungi presented at similar concentrations until the seventh day. The concentrations showed an increase between 15 and 30 days of incubation, but it can be observed that concentrations of extracellular proteins are higher for P. formosus compared to Phlebia sp. The highest extracellular protein concentration was observed at 15 days for the P. formosus strain in the presence of blue dye, with accumulation of 122 μg mL−1, while for Phlebia sp., the measure averaged 60 μg mL−1 in the presence of black dye. At 30 days of treatment under the same conditions, it was quantified as 227 and 93 μg mL−1, respectively.

In both evaluations, protein concentrations in the presence of dyes were reduced at the end of 30 days when compared with the control treatments containing only fungi in the absence of dyes.

Enzymatic activity

The results of oxidative capacity for quantifying the activity of extracellular lignolytic enzymes were positive only for the Phlebia sp. endophytic strain. Laccase enzymatic activity appeared to be primarily responsible for the ABTS oxidation activity by the Phlebia sp. enzyme extract, whereas for total peroxidase, there was low or no oxidative activity (Table 1).

Table 1 Values observed for enzymatic activity at 10-min incubation for the fungus Phlebia sp.
Full size table

With 10 min of reaction, the Phlebia sp. presented an ABTS oxidation value of 0.17 mEU, 0.135 mEU in the determination of the laccase enzyme and 0.035 mEU for total peroxidase. The ABTS oxidation values obtained on the presence for both blue and black dyes were 0.16 mEU. There was little difference in the levels of laccase and peroxidase determined under dye exposures: It yielded 0.01 mEU of peroxidase in the blue dye and no peroxidase enzyme units for the black dye.

Micronucleus test

The frequency of MN in fish erythrocytes is shown in Table 2. It was observed that, in aquariums with dyes, the frequency of MN was higher than for other treatments (Fig. 6), with 0.75% for the blue dye and 0.85% for the black dye. The fungal treatments showed a reduction in the frequency of MN, with the highest rate of 0.65% for aquariums containing Phlebia sp. with the black dye and the same percentage for P. formosus with the blue dye. These results are statistically classified in the same groups as the negative controls in water and PD broth with 0.25% MN frequency. However, for the RB19 treatments, statistical differences from the positive control were not observed.

Table 2 Frequency of micronuclei observed in fish erythrocytes in the presence of RB19 and RB5 textile dyes
Full size table
Fig. 6
figure 6

Erythrocytes of Astyanax sp. fish. a Normal erythrocyte. b Micronucleus

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Discussion

In the analysis of textile dye degradation, the wavelength, on the visible light spectrum, was selected for each dye where absorption peaks were observed around 600 nm. That absorption region corresponds to the presence of two azo groups (–N = N–) attached to aromatic systems for the black dye (Bisschops and Spanjers 2003; Wang et al. 2003), and for the blue dye, the absorbance peak represents aromatic ring conjugation with the anthraquinone group (Hunger et al. 2004). These chemical groups are the respective chromophores responsible for the colour of these molecules.

Many organisms are used to assist in removing recalcitrant compounds originating from discharges from industry. Some fungi are being studied for this purpose. The potential of endophytic fungi for bioremediation of many compounds such as industrial slurry, diesel and herbicides has been demonstrated, and regarding effluents from the textile industry, microorganisms, especially fungi, are being studied as biodegraders/bioremediator agents (Kaushik and Malik 2009; Manai et al. 2016a; Solís et al. 2012; Wesenberg et al. 2003).

The results for the decolouration potential of Phlebia sp. and P. formosus demonstrate that both were initially effective in decolouration of the blue and black dyes (Fig. 3), and to verify this potential over time, it was observed that Phlebia sp. degraded 90% of the black dye and 70% of the blue dye with 30 days of treatment (Fig. 4). Treatment with P. formosus after 7 days resulted in a reduction of 90% of the black dye, which could be explained by the possible production of by-products of the remediation process or other extracellular compounds which can absorb light in the visible spectrum used for monitoring the dye degradation. Niebisch et al. (2010) described Lentinus crinitus as a fungus with decolouration potential for Reactive Blue 220 dye (RB220), with 90% bleaching at a concentration of 0.1 g L−1 in 10 days.

In relation to total protein concentration, values were opposed to discolouration, whereupon P. formosus showed higher levels of both mycelial extracellular proteins compared to Phlebia sp. at the end of 30 days (Fig. 5).

It is known that there are various ways to treat textile effluents, and research using microorganisms, principally fungi that secrete lignolytic enzymes, has shown that they are efficient in removing recalcitrant compounds (Manai et al. 2016a; Silva et al. 2012). The evaluation of lignolytic enzyme activity indicates the presence only for Phlebia sp. wherein the laccase enzyme is primarily responsible for substrate oxidation activity (Table 1). Gill et al. (2002) related that the use of Phlebia sp. for dye decolourisation with Phlebia bervispora discolours 60% of the crystal violet dye in only 2 days and Phlebia fascicularia and Phlebia flavus 100% of decolourisation of various dyes at 5 days of treatment. This technique using fungal enzyme systems in environmental decontamination is promising because they are great for degradation of compounds in effluents such as dyes (Kamida and Durrant 2005; Manai et al. 2016b).

We can consider that lignolytic enzymes may be related to decolouration of the dyes tested by Phlebia sp. In contrast, the decolouration activity of P. formosus may be related to bioabsorption of dyes, since it showed no enzyme activity.

Knowing that the textile industry has significantly contributed to environmental pollution due to high production of waste, including dyes which have mutagenic and carcinogenic activity and possible toxic by-products from waste treatment (Khatri et al. 2015; Mcmullan et al. 2001), cytotoxicity analysis is necessary to assess the possible impact of future treatments at larger scales.

For this reason, a toxicity assessment test was employed for the dyes treated by endophytes. That test positively pointed to consideration of the use of this remediation process with endophytic P. formosus and Phlebia sp. strains.

The presence of MN in fish erythrocytes at low levels of the dyes (0.01 μg mL−1) was observed. There was an increase in MN rate in the positive controls, where the fish were exposed only to the dyes, with 0.75% MN with the blue dye and 0.85% with the black dye (Table 2). After treatment with the fungi, there was a MN reduction of approximately 20%.

Studies using fish as bioindicators of recalcitrant compounds in effluent have shown that discharge of industrial waste leads to an increase in the frequency of MN (Avni and Jagruti 2016; Kaur and Dua 2016). Grisolia et al. (2009) evaluated fish cells collected in five points from five Paranoá lake located in Brasilia, Brazil, and noted that among the species collected, the highest frequency of MN was 1863%. Saotome and Hayash (2003) also showed positive results for MN frequency when evaluating industrial effluents. The induction of micronucleus frequency by dyes in fishes can be dose and time dependent (Avni and Jagruti 2016). The analysis of DNA abnormalities, such as cells containing MN, using fish for effluent monitoring, can provide an alert for genotoxic threats to the ecosystem (Al-Sabti 2000). It is important for validating experiments on biodegradation/bioabsorption/decolourisation treatments.