The potential of white and soft-rot fungi for biodegradation of multi-walled carbon nanotubes (MWCNTs): characterization and enzyme analysis

ABSTRACT Multi-walled carbon nanotubes (MWCNTs) have significant environmental concerns for soil, water and ecosystems. Since their harmful effects on human health, including respiratory disease, they are considered dangerous substances. Thus, it is required to discover a constructive solution to decrease of MWCNT toxicity. Due to their high degradability, white rot and soft rot fungi have been used in the biodegradation of various pollutants. In this study, we evaluated the ability of native Iranian white and soft rot fungi to degrade MWCNTs mainly by means of dynamic light scattering (DLS) and surface charge measurement. In this regard, growth kinetics, CO2 and protein production, size, surface charge and pH change were measured. Continuous Raman spectroscopy, scanning electron microscope (SEM) and transmission electron microscope (TEM) imaging were carried out to detect the process of degradation. The results showed that the morphology, arrangement, size and diameter of MWCNTs were modified due to fungal degradation. The cytotoxicity of treated MWCNTs was determined by the MTT test. The results indicated that native Iranian white and soft rot fungi have a significant ability for degradation of MWCNTs. Furthermore, it could be concluded that fungal treatment could reduce the toxicity of MWCNTs and the best result was achieved in 500 ppm MWCNTs for Trichoderma sp. The activities of oxidative enzymes such as laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) were measured to determine the mechanism of degradation. Enzyme assays have suggested that these oxidising enzymes especially laccase might play a key role in the degradation of MWCNTs in white rot and soft rot fungi. The overall results confirmed the reproductive ability of Trichoderma sp. WF29 as a soft rot and two white rot fungi Irpex lacteus WF36 and Trametes versicolor to degrade MWCNT.


Introduction
Nanotechnology will change people's lives in addition to revolutionise modern industries and societies.Hence, extensive applications have been characterised by using nanotechnology.These applications are continuously developing.Although the advancement of nanotechnology has made tremendous changes in human life, using of nanomaterials has raised the risk of accidental exposure to humans and organisms in the ecosystems [1].Regarding the enhancement of using nanotechnology in different industries, the concerns about its hazards gradually began to increase [2].
Among the various types of nanomaterials, carbon nanotubes (CNTs) are onedimensional structures categorised into three groups: Single-Walled Carbon Nanotubes (SWCNTs), Double-Walled Carbon Nanotubes (DWCNTs) and Multi-Walled Carbon Nanotubes (MWCNTs).Unique properties of MWCNT such as low weight, electrical conductivity and mechanical strength stem in their structure, small size (1.4 to 100 nm) and high surface to volume ratios.These properties have drawn scientists` attention to consider using MWCNTs in various fields [2].MWCNTs have vast applications in different industries.For instance, in medicine, MWCNTs are used as antibacterial agents [3], drug carriers [4], and vaccine scaffolds [5].MWCNTs have revolutionised food packaging [6], sports industries to make waterproof shoes and lighter clothes [7] electrical [8], petrochemical [6] and agriculture industries [9].Despite the advantages of using MWCNTs in different industries, there is not certain information about the fate of these compounds in the environment.In a few studies, it has been reported that MWCNTs can cause severe problems in the respiratory systems just like asbestos [10,11].Also, because of their small sizes, they can cross the brain-blood barrier (BBB) [12].Moreover, damage to the spleen and liver may occur with long-term exposure to MWCNTs [2].As Ganguly et al. reported the release of non-treated MWCNTs in the environment can affect human health and it might endanger other organisms [13].Regarding these drawbacks, MWCNTs should be eliminated from the environment using physical, photochemical and biological methods [9].Based on previous studies, biological treatment is a more efficient and inexpensive method for removal of organic and inorganic contaminants in comparison to physical and photochemical methods [14].
In the last century, fungi have been considered as a potent agent to decompose various kinds of pollutants, including polycyclic aromatic hydrocarbons (PAH), insecticides, pesticides, and Azo dyes [15].Among all kinds of fungi, white-rot and soft-rot fungi are two groups of wood-rotting fungi with the ability of lignin degradation.Additionally, high potential of these fungi for the production of various non-specific oxidative enzymes including laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) makes them suitable for biotechnological applications [16,17].Several studies demonstrated the potential of white-rot and soft-rot fungi to degrade pollutants such as single-walled carbon nanotubes [18].Berry et al. (2014) found that Trametes versicolor could degrade single-walled carbon nanotubes [19].The ability of microorganism to degrade MWCNTs has been investigated in recent years.Zhang et al. (2013) addressed the ability of a bacterial community, including Burkholderia kuruiensis, Deftia acidovorans and stenotrophomonas maltophilia for degradation of MWCNTs in the presence of an external carbon source via co-metabolism [20].It is supposed that the fungi are more capable for degradation of MWCNTs in comparison to bacteria regarding their tolerance to harsh environmental conditions [16].
Although it is still not clear how fungi are degrading MWCNTs, it seems extracellular enzymes might have a primary role in the degradation of these compounds.Numerous researchers have stated CNT biodegradation by enzymatic catalysis via peroxidase enzymes, such as human myeloperoxidase (MPO), horse-radish peroxidase (HRP), eosinophil peroxidase (EPO), lactoperoxidase (LPO), manganese peroxidase (MnPO), lignin peroxidase (LiPO) and microbial xanthine oxidase (XO) [20].It should be considered that the fate of new synthetic compounds such as nanomaterials in the terrestrial and aquatic environments is important.To our knowledge, there is no comprehensive study in MWCNTs biodegradation by fungi.In this study, the ability of native white-and soft-rot fungi for MWCNTs degradation was studied by means of different approaches such as Dynamic Light Scattering (DLS), surface charge assay, sturm test, growth kinetic assay, Raman spectroscopy, and laccase, manganese peroxidase and lignin peroxidase enzyme assessment.In this study, we demonstrated that white and soft-rot fungi are suitable for biological degradation of MWCNTs.While few studies have been conducted on the biological treatment of nanomaterials, the present study focused on the ability of fungi to the degradation of MWCNTs.For this purpose, native Iranian white-and soft-rot fungi were selected in this study.

Sampling and isolation on of basidiomycetes
Samples of rotten wood of beech and oak trees were collected from the forests of Saravan (37° 1375′ N, 49° 6652′ E), situated in Gilan province and Mount Dena, a sub-range within Zagros Mountain, with 80 Km length and 15 Km average width (30° 57′ N, 51° 26′ E) located in Lorestan province, Iran.The samples quickly were transferred to the laboratory and stored at 4°C.Fungal strains were isolated by cultivating a small piece of stick, aseptically removed from decayed wood on Malt Extract Agar (MEA) 0.06 g/L of Benomyl and 0.01 g/L of streptomycin were added to the medium to prevent the growth of undesirable fast-growing fungi and bacteria, respectively [21].

Screening of white-rot basidiomycetes
he Bavendammn test [22] was carried out by growing the fungi on MEA plates containing 5 g/L gallic acid (Merck, Germany).White-and soft-rot fungi create a brown diffusion zone as a result of gallic acid degradation, while brown-rot fungi create no diffusion zone [21].Then, the isolated fungi were cultured on a two-layer medium containing different concentrations of MWCNTs (0, 200, 500 and 1000 ppm).The diameter of the growth zone was measured and compared to the PDA medium without MWCNTs as a control [18].MWCNTs (CAS Number: 308,068-56-6) was purchased from Neutrino company (Germany).The declared purity of MWCNTs was> 93% with length of >5 μm and diameter of 10-100 nm.

Molecular characterisation of isolated fungi
Molecular methods identified the selected isolates.For this aim, the genomic DNA was extracted using the phenol-chloroform method to determine molecular characterisation.PCR was carried out with ITS1, and ITS4 universal primers using a Multigene TM OptiMax thermal cycler (USA) and the PCR products were analysed by 1% agarose gel [23].The PCR products were sequenced by Macrogen (South Korea).The sequence similarities were attained.Based on the sequences similarities, the isolates WF29, WF31 show 99.48%, and 99.68% similarity to Trichoderma reesei strain SN-14 (MH298761.1)and, Irpex lacteus voucher CLZhao-81 (MG231699.1),respectively.

Assessment of the MWCNTs degradation in broth medium
The ability of the selected isolates for removal of MWCNTs was examined based on the disappearing of MWCNTs dark dye and colour change in minimal Bushnell-Haas (BH) medium including (g/L): MgSO 4 (0.1), CaCl 2 (0.01), NH 4 NO 3 (1), KH 2 PO 4 (1) and K 2 HPO 4 (1), FeCl 3 (0.05) and 200 ppm MWCNTs as a sole carbon source [24].The isolates were cultured on Potato Dextrose Agar (PDA) and incubated at 28°C for one week.When mycelia covered the whole plate, a small piece of PDA was inoculated in 50 ml flasks containing 10 ml BH medium with 200 ppm MWCNTs.Afterwards, these flasks were incubated for 48 hours at 28°C on a shaker operated at 150 rpm.Then, 500 µl of inoculation flasks were transferred to 50 ml flasks containing 10 ml BH medium with 200 ppm MWCNTs and were incubated for 2 weeks at 28°C on a shaker operated at 150 rpm.At the end of the incubation, visual evidence of degradation can be found based on the colour and turbidity change in the culture medium.For each experiment, non-treated MWCNTs were used as negative control and all tests performed in triplicates.

Determination of cell dry weight
For cell dry weight measurement, each sample was prepared, as mentioned above.After 14 days, fungal treated MWCNTs were transferred to the sterile microtubes and centrifuged at 8000 g for 5 min.Then the supernatant was discarded and the pellets were resuspended and washed in sterile normal saline.The washing procedure was repeated three times to make sure that the fungal hyphae were separated from MWCNTs.Then, the pellet was dried at 80°C in the oven for 24 h, and fungal mycelium dry weight was measured [25].

Separation of treated MWCNTs from fungal biomass
To separate the MWCNTs after fermentation, initially, the surface attached MWCNTs were removed from fungal biomass into fermentation solution by sonicating at 40 KHz for 15 min using Elmasonic P sonicator (Elma, Germany).The fungal mycelia pellet was lifted by a tweezer and the MWCNTs precipitated in the medium collected by filtration.To break the residues MWCNTs wrapped in the fungal pellet, the samples were incubated in the solution of 30 g/L SDS and CaCl 2 (10 mM) at 60°C for 12 h.After that, the residues were filtered and incubated in 2.7 mM HCl at 60°C for 24 h.The samples were washed with ethanol and then with water.The MWCNTs were collected for further analysis [26].

Determination of produced CO 2 (Sturm test)
The amount of produced CO 2 can be a suitable indirect sign of microbial growth and active metabolism for MWCNT degradation.For CO 2 measurement the selected isolates were cultured in a BH medium containing 200 ppm MWCNTs in sealed 250 ml flasks.After 14 days of incubation at 28°C and 150 rpm, produced CO 2 was captured by 0.2 M KOH solution.KOH solution was substituted by 1 ml of 0.5 M barium chloride solution periodically.The residual KOH was titrated with 0.1 M HCl.The headspace CO 2 was calculated through the equation.Generated CO 2 = (V B -V A ) × (M CO2 /2)×M HCl Where VB and VA are the volumes of HCl 0.1 M used to titrate the blank and the treated one (ml), respectively.Also, M CO2 is the molar mass of carbon dioxide in g/mol and M HCl is the molar concentration of HCl standard solution (mol/ L), and CF is the correction factor for acid/base molarity (M HCl /M KOH ) [27].Additionally, the amount of CO 2 was measured continuously as previously described at 1, 4,7,11 and 14 days after incubation.

Determination of produced protein
The measurement of total produced protein in the minimal medium can be considered as an indicator of microbial growth.The modified Lowry method was used to measure protein.Different concentrations of Bovine Serum Albumin (BSA) were used for the standard curve preparation [28].

Assessment of the distribution of treated MWCNTs by DLS
Dynamic Light Scattering (DLS) analysis shows the particle size or dispersion of carbon nanotubes at a suspension [28].DLS of fungal treated MWCNTs were measured by a Malvern ZEN 3600 laser irradiation (UK) under 678 nm, before and after fungal degradation.It should be noted that any defect in the structure of MWCNTs could affect the DLS data.Thus, the difference between DLS data before and after biodegradation is an indicator that shows MWCNTs degradation [29].

Assessment of surface load charges by Zeta potential
Degradation of MWCNTs by fungal treatment produces a short-chain tube with additional hydroxyl and carboxyl groups.As a result, MWCNT degradation imposes a more negative charge on the surface of MWCNTs [30].The fungal treated MWCNTs were prepared, as mentioned previously.Then, the surface charge of treated MWCNTs was measured by a Malven ZEN 3600 Zetasizer (UK).The data was analysed by Zetasizer software.

Simultaneous measurement of growth rate, surface charge and pH
Degradation of MWCNTs by fungal treatment produces a short-chain tube with additional hydroxyl and carboxyl groups.As a result, MWCNT degradation imposes a more negative charge on the surface of MWCNTs [30].The fungal treated MWCNTs were prepared, as mentioned previously.Then, the surface charge of treated MWCNTs was measured by a Malven ZEN 3600 Zetasizer (UK).The data was analysed by Zetasizer software.
As previously mentioned, degradation of MWCNTs produced short chains with carboxyl groups.These groups not only induce more negative charge, but also impel the medium turns to acidic pH.Growth rate by measurement of dry cell weight, surface charge and pH changes were monitored every three-day interval during 2 weeks.

Scanning electron microscopy (SEM)
Fungal treatment has influenced the morphology, size and diameter of MWCNTs.For Scanning Electron Microscopy (SEM), 20 μl of treated MWCNTs by fungal strains were fixed on an aluminium sheet [31].The aluminium sheet was used to make more contrast between the sample and the surface.Then, the samples were dried at room temperature for 24 h.After gold coating, microscopic images were obtained by VGOA 3 TESCAN scanning electron microscope (Czech Republic) at 35000, 60000 and 80000 magnifications.These images were compared with non-treated MWCNTs as blank.For each isolate, 5-8 regions were monitored.

Transmission electron microscopy (TEM)
Transmission Electron Microscopy (TEM) was used for measuring the crystal structure, size and length of MWCNTs.The fungal biomass was fixed by glutaraldehyde (2.5%) and osmium tetroxide (1%).The samples were dehydrated by a graded alcohol [32].The powder of the treated MWCNTs was analysed using Philips em208s transmission electron microscope (USA).The accelerating voltage was 100 kV and magnifications were 50-500 times.It is noticeable that the diameter of MWCNTs was measured in this study.

Raman spectroscopy of treated MWCNTs
Raman spectroscopy is a well-known method for investigation of the molecular vibration that can show the defects created by the motion of carbon atoms in the structural body of materials, including MWCNTs [33].TakRam N1-S41 Teksan Co (Iran) Raman spectrophotometer was used to draw a Raman spectrum for treated MWCNTs.The treatment of MWCNTs was explained above.Data were analysed by Origin software (data analysis software).

Evaluation of treated MWCNTs toxicity by MTT assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) cell proliferation assay was used to evaluate the treated MWCNT effect on cell viability [34].The results were compared to non-treated MWCNTs as control and expressed as viable cell count.Fibroblast cell lines were cultured in an RPMI medium supplemented with 10% (v/ v) Foetal Bovine Serum (FBS), 1.0 mM sodium pyruvate and 2 mM L-glutamine at 37°C in the humidified atmosphere of 95% air and 5% CO 2 .After overnight incubation, approximately 2000 fibroblast cells/well were seeded onto a 96-well plate and allowed to adhere for 48 h.Three replicates were incubated with different concentrations (0, 250, 500, 1000 ppm) of fungal treated MWCNTs and the viability was measured after 48 h.MTT assay was run as Abbes et al. (2013) procedure [34].The dye absorbance was measured at 560 nm by ELISA Reader.

Statistical analysis
The experimental results were statistically analysed using SPSS version 24.All the experiments were conducted at least in triplicate.The Kolmogorov-Smirnov test for testing if a variable follows a normal distribution in a population was used to assess the normality of the distribution of investigated parameters.All data in this study were distributed normally.Data in the tables were expressed as mean ± standard deviation (±SD), and error bars in the figures indicate standard deviation.Differences between means were analysed by the ANOVA test followed by the post hoc Tukey's test.A significant difference was considered at the level of p < 0.05.

Isolation of white-rot fungi according to MWCNTs degradation ability
In total, 84 isolates were collected from rotten wood and named WF1-WF84 in addition to Trametes versicolor.Three different tests were done for screening of the best white-and soft-rot fungi based on their ability in gallic acid degradation, growth on the different MWCNTs concentration and disappearing of the MWCNTs dark dye and colour change in minimal medium.
Growth on the different concentration of MWCNTs confirmed that the isolates were grown in the presence of MWCNTs.Colour change of the MWCNTs can qualitatively show that fungi are able to degrade MWCNTs since they were added to the culture medium as a sole carbon source.
It is supposed that the degradative oxidase enzymes such as laccase might play a role in degradation of MWCNTs [16].According to the previous studies [22], the presence of oxidative enzymes primarily assessed using gallic acid as a known phenolic compound [22].
Screening results were presented in the supplementary data.White-and soft-rot isolates were categorised based on their reaction to gallic acid.Images of some potent strains and the score of isolates in the response to gallic acid were shown in Fig. S1 and Table S1 in the supplementary data, respectively.
In the following, white-and soft-rot isolates were cultured on a two-layer medium with different concentrations of MWCNTs to find the most suitable concentration of MWCNTs for further experiments.Based on the results presented in Table S2 in supplementary data, all of the isolates could grow in the presence of 200 ppm of MWCNTs.Four isolates were grown only in the presence of 200 ppm of MWCNTs and eight strains showed a slight decrease in the growth at 500 ppm of MWCNTs.Furthermore, the growth rates of 29 isolates were decreased at 1000 ppm of MWCNTs.Only seven strains were attained that the increase in the concentration of MWCNTs did not impress their growth rate (Table S2 in supplementary data).
The ability of the selected isolates for degradation of MWCNTs was examined based on the colour change in minimal medium [29].Based on the observations, there was a significant change in the colour intensity from the control sample to the treated ones (Data not shown).This changing colour was used as a confirmation test for investigation of the ability of the fungal extracellular oxidative enzyme on biodegradation of SWCNTs based on the study employed by Chandrasekaran et al (2014) [35].Finally, based on the results of these experiments, isolates WF29, WF36 in addition to T. versicolor were shown the best results of MWCNTs degradation and selected for further analyses.

Characterisation of selected isolates
After molecular characterisation, the selected isolates were submitted at the National Center for Biotechnology Information (NCBI) database under the accession numbers MN820451 and MN820450, respectively.The phylogenic trees of the isolates were drawn using MEGA-7 software following the maximum-likehood method.The results for selected isolates were shown in Fig S2 , S3 for WF29 and WF36 isolates, respectively, in the supplementary data.
In the few studies on fungal degradation of carbon nanotubes, Berry et al. (2014) investigated the degradation of SWNCTs by Trametes Versicolor and Phlebia tremellosa, and they found that these fungal strains were competent to degrade SWCNTs in different environmental conditions [19].Also, Parks et al. (2014) showed that the Trametes versicolor could degrade 0.1% of SWNTs [36].There is no evidence about the ability of Trichoderma sp.WF29 and Irpex lacteus for biodegradation of neither MWCNTs nor SWCNTs carbon nanotubes.In our study, MWCNT degradation was considered by isolated indigenous white-and soft-rot fungi and different tests were used to characterise the biological degradation for the first time.

Assessment of fungal growth rate based on the cell dry weight, produced CO 2 and protein
Due to the using of multi-walled carbon nanotubes as an unusual substrate source, the fungal growth rate was investigated by three different methods.Since the fungi are dependent on the carbon source for growth, the MWCNTs play a role as a carbon source.Hence, the estimation of the fungal dry cell weight could demonstrate the MWCNTs degradation.The result of dry cell weights, which was presented in Table 1, indicated that Trichoderma sp.WF29, as a soft-rot fungus, showed the highest amount of biomass production by 2.14 g/L.
In a study conducted by You et al. (2017), the growh rate of Mycobacterium vanbaalenii PYR-1 was investigated to assess the effect of MWCNTs on PYR-1 lag time.According to the results, no significant growth was detected when this bacterium was incubated without glucose or pyrene, whether MWCNTs were present or not due to the insufficiency of MWCNTs to support bacterial proliferation [37].
In addition to dry cell weight, measuring the amount of CO 2 produced at an aerobic-controlled condition could be used as an indirect criterion for fungal respiration activity.In general, the produced CO 2 was captured by NaOH solution to determine respiration through titrimetry [27].The continues measurement of the CO 2 was employed based on the Cerqueira et al (2011).The amount of CO 2 was not detectable until the day 11.After that, the amount of produced CO 2 was increasing.At day 11, the degradation of MWCNTs was almost completed based on the other experiments such as zeta potential, cell dry weight and pH change.Thus, this assessment validates the degradation of MWCNTs.
In this study, the amount of produced CO 2 by selected white-and soft-rot fungi was examined in the presence of 200 ppm MWCNTs and the results are summarised in Table 1.The most and least amount of produced CO 2 was achieved by 2.41 and 1.84 g/L for Trichoderma sp.WF29 and T. versicolor, respectively.Trichoderma sp.WF29 was able to produce CO 2 three times more than the T. versicolor.The CO 2 evaluation was firstly reported by Cerqueira et al. for the investigation of the degradation potential of oily sludge by pure and mixed bacterial culture [27].Allen et al. in 2008 investigated the oxidative degradation of MWCNTs in the presence of horseradish peroxidase (HRP) and H 2 O 2 .Since the degradation of MWCNTs is an oxidation process, the production of CO 2 is expected.The results showed that the total CO 2 in degraded samples were progressively increased in comparison to control [24].In another study by You et al. (2017), the amount of CO 2 was slightly increased in comparison to control during biodegradation [37].Based on the results was reported by xu et al. (2017), MWCNTs converts to different components including CO 2, carboxyl, etc [30].Thus, the amount of produced CO 2 was confirmed the As previously mentioned, the modified Lowry method was used for estimating the amount of produced protein as an indirect criterion for growth rate.This property helped to measure the produced protein during the fungal growth.Estimated total protein for Trichoderma sp.WF29, I. lacteus WF36 and T. versicolor were 12.16, 8.09 and 7.27 g/L, respectively (Table 1).Comparison of the results of cell dry weight, Sturm test and the amount of produced protein indicate that Trichoderma sp.WF29 has the highest amount of cell dry weight as well as the most amounts of produced CO 2 and protein.

Assessment of the distribution of treated MWCNTs by DLS
The disturbance in the SP 2 surface structure of MWCNTs causes an increase in DLS value [29].Moreover, during the degradation of MWCNTs, the arrangements of the sidewalls of MWCNTs have changed.It is indicated that the π-π interaction among sidewalls of MWCNTs increases with DLS rise [29].Then, an increase in the dispersion coefficient estimated by DLS is expected as a result of MWCNTs degradation.The results of the DLS measurement showed that Trichoderma sp.WF29 had a dispersion coefficient, which was 2.40 times greater than of the control.These values were 1.38 and 2.19 times higher than that of the control for I. lacteus WF31 and T. versicolor, respectively.Figure 1 shows the dispersion coefficients of these strains and in comparison to control.
As the results showed, all strains had a dispersion coefficient higher than the control, and all of them were capable of degrading MWCNTs, whereas soft-rot fungus Trichoderma sp.WF29 was the best candidate for MWCNTs degradation.There is no similar study about the relation between fungal degradation of MWCNTs and DLS.Nevertheless, in a study conducted by Zhao et al. (2011), DLS decreased during the enzymatic degradation of MWCNTs [29].This decrease could stem from two reasons: a decrease in the bundling effect of MWCNTs, or a decrease in the actual sizes (i.e.diameters and lengths).Although the obtained data indicated that carbon nanotubes have been degraded; however, their degradation was low.This result was probably due to the small amount of damage that occurred on the surface of MWCNTs during enzymatic degradation, which led to a decrease in DLS [29].On the contrary, in our study, the amount of DLS had increased and showed the efficiency of MWCNTs degradation by white-and soft-rot fungi.Because DLS indicate the number of particles passing in front of the source light in a given time, accordingly, a specific absorption coefficient is defined.This absorption coefficient could determine the degradation degree.In another way, the higher dispersion coefficient in comparison to control shows the biodegradation of MWCNTs [29].However, it should be noted that DLS is inefficient to determine the exact diameter of carbon nanotubes.

Assessment of surface load charges by Zeta potential
For a solid surface, electrical charges will be produced mainly at the surface.Then, the opposite charge at the aqueous surface tries to neutralise polarity at the solid surface.This phenomenon is called the electrical double layer (EDL).The polarity of EDL is indicated as zeta potential [30].In this study, measuring zeta potential was an alternative technique helping the analysis of treated MWCNTs by white-and soft-rot fungi.According to the results, surface charge levels for Trichoderma sp.WF29, I. lacteus WF36 and T. versicolor were negative (Figure 2).
MWCNTs were neutral compounds without surface charges [30].Figure 2 shows the surface charge levels on Trichoderma sp.WF29, I. lacteus WF36 and Trametes versicolor in comparison to control (The zeta potential of control was zero); although their degradation induced the surface charge [30], Trichoderma sp.WF29 had a better function for the degradation of MWCNTs.No significant differences were observed between the two other strains.In a previous study, biotransformation of MWCNTs by resistant soil bacteria was investigated by Chouhan et al. (2016).These bacteria induced the formation of C = O and COOH groups on the outer surface of CNTs [38].Regarding the results of the assessment of zeta potential, we inferred that a different functional group such as carboxyl group produced during the biodegradation of MWCNTs that caused more negative surface charge.It might validate the potential of isolated white-and soft-rot fungi for biodegradation of MWCNTs.

Simultaneous measurement of growth rate, surface charge and pH change
The coincidence measurement of the electrical charge and pH can show MWCNTs degradation, since the degradation of MWCNTs causes an increase in growth rate and at the same time, induce more negative charge and acidic conditions because of carboxyl and hydroxyl groups released during the degradation procedure [30].Then, it was observed that during the fermentation, growth rate and electrical charge of the treated MWCNTs increased and pH decreased, inversely.The kinetic growth rate, electrical charge and pH of the treated MWCNTs were measured for 14 days with three days' intervals.The results are displayed in Figure 3.
The dry weight of Trichoderma sp.WF29 reached the highest amount on day seven, which was 3.8 g/L and was fixed after that (Figure 3).Also, the most amount of dry weight was observed on day 7 for I. lacteus WF36.Although there were a few increases on day 11, the increase in the dry weight initiated at day 7 for T. versicolor and continues until day 11 and 14.The most amount of dry weight was reached after 2 weeks for T. versicolor.
Figure 3 shows that Trichoderma sp.WF29 had the most amount of dry weight.On the other side, the least amount of dry weight belonged to I. lacteus WF36, which was 1.8 g/L on day 7.A comparison of the obtained results of electrical charge, which were introduced as zeta potential in Figure 3, shows that the electrical charge was more negative at day 7 for all strains.Although a proportional increase was observed in I. lacteus WF36 and T. versicolor at day 11, this increase could be due to the interactions between hydroxyl and carboxyl groups that reduced their acidic effect.As previously mentioned, pH decreased during degradation.Surprisingly, no significant pH change was observed during the experimental period [30].Therefore, the comparison of the results shows that Trichoderma sp.WF29 was the most capable isolate at MWCNTs degradation.
In a study by You et al. (2017), the ability of a Mycobacterium vanbaalenii PYR-1 for biodegradation of pristine MWCNTs were investigated in the presence of glucose alone and glucose and pyrene together [37].They found that the rate of degradation in carboxylic MWCNTs was higher than from p-MWCNTs.It is in good agreement with our analysis on the ability of white-and soft-rot fungi for biodegradation of p-MWCNTs based on the results of the kinetic growth rate [37].Also, our results showed that the process of adding a carboxyl group occurred during the biodegradation (Table 1 and Figure 2).

Scanning electron microscopy (SEM)
Scanning Electron Microscopy (SEM) is a well-known method for the determination of morphology and length of MWCNTs.The degradation of MWCNTs could impose a change in arrangement, length and morphology of MWCNTs [31].Figure 4 shows the SEM images of treated MWCNTs by Trichoderma sp.WF29, I. lacteus WF36 and T. versicolor in comparison to control.
In this study, SEM was used to track the morphological changes during the degradation process.The images show that the aggregation state of MWCNTs was decreased due to biodegradation.It is evident that there is a gap in the carbonaceous structure of MWCNTs in Figure 4B.& E Also, the diameters of treated MWCNTs were decreased, as shown in Figure 4. Furthermore, the arrangement of the MWCNTs was modified.A comparison of the images shows that the regular arrangement of MWCNTs was collapsed as a result of biodegradation.This result implies that the degraded MWCNTs had a more irregular arrangement than the control.In treated MWCNTs, the continuous structure was broken down into tubular structures.

Transmission electron microscopy (TEM)
The results of TEM imaging of treated MWCNTs are shown in Figure 5.In the previous study, Ma et al. 2019 showed the TEM images to characterise the diameter of MWCNTs as well as the cross-section interactions of them after thermal construction [32].The obtained results were shown that the reshaping of the layers may lead to the axial defects in MWCNTs.These reshaping might occur during the biological treatment of MWCNTs [32].The morphological changes and the defects of treated MWCNTs in the structure of MWCNTs were shown in Figure 5.Our results were confirmed that the white-and soft-rot fungi were able to degrade MWCNTs and made axial and lateral defects in their structures (Figure 5C-H).In the obtained images, the distance between dark lines in Trichoderma sp.WF29 was shorter than the two other strains (Figure 5C,D).Thus, it could be concluded that the treated MWCNTs using of Trichoderma sp.WF29 were smaller than the other.In addition, I. lacteus WF36 and T. versicolor could break the MWCNTs and Shortened MWCNTs are circled in red arrow.

Raman spectroscopy of treated MWCNTs
Raman spectroscopy is used as a powerful technique for the determination of the molecular morphology of carbon materials [39].Raman spectrum for MWCNTs has two main peaks in two different areas.The first peak observes at 1500-1600 (cm −1 ) call G band taken from graphite which is related to vibration at graphitic planes.The second peak appears at 1300-1350 (cm −1 ) call D bands retrieved from defect band, which shows defects in lateral layers.The appearance of this peak depends on the electron excitation that has arisen from MWCNTs degradation.Besides, D/G ratio (D band to G band ratio) could provide a good criterion for estimating the number of defects at MWCNTs structure [40].MWCNTs have a multi-layer structure with numerous internal layers.During the degradation process, the oxidation procedure can create defects within these internal layers of sidewalls and also both ends.When the outer layer undergoes degradation, the graphitic structures are further oxidised, and thus, the D to G ratio increased [29].In this study, the spectra were set up in the G band at around 1570 cm −1 to compare the change in the D band at around 1323 cm −1 .Raman spectrum of treated MWCNTs by Trichoderma sp.WF29, I. lacteus WF36 and T. versicolor were shown in Figure 6 in comparison to the control sample.The D band to G band ratio (I D /I G ) exhibits a lower value for Trichoderma sp.WF29, I. lacteus WF36 and T. versicolor with an average of ~ 0.450, 0.658 and 0.652, respectively.This value was ~0.844 for control.It shows that the Trichoderma sp.WF29 as a soft-rot fungus was more efficient in biodegradation of MWCNTs.
Previously, Osswald et al. (2007) used Raman spectroscopy to observe the structural changes in MWCNTs due to chemical oxidation.They found that the integrity ratio (I D /I G ) was ~1.8 for pristine MWCNTs, while oxidised MWCNTs showed a lower value at around ~1.1 [41].In a previous study devoted by You et al. (2017) Raman spectra were provided for treated MWCNTs with Mycobacterium vanbaalenii PYR-1.The I D /I G was higher in carboxylic MWCNTs than the pristine MWCNTs [37].This result was due to better dispersion and bioaccessibility of carboxylic groups on MWCNTs.In our study, the significant change was shown in the intensity ratio of treated MWCNTs by selected isolates.It means that the white-and soft-rot fungi might be capable of biodegradation of MWCNTs.

Evaluation of toxicity of fungal treated MWCNTs
The cell viability in different concentrations of treated MWCNTs was shown in Table 2.The amount of cell viability was increased in the presence of 250 and 500 mg of MWCNTs and decreased in the presence of 1000 mg.However increasing the concentration of treated MWCNTs to 2000 mg indicated a toxic effect on the cell lines regarding the decrease in cell viability.
The results show that the degradation of MWCNTs leads to a decrease in the toxicity effect.The most amount of reduction in toxicity was demonstrated in 500 ppm for Trichoderma sp.WF29 as well as the significant decrease in toxicity in other concentrations.The comparison of the results in other concentrations of treated MWCNTs by fungal isolates shows no significant effect on the reduction of toxicity.The toxicity of MWCNTs depends on different parameters such as geometric structure, agglomeration state, concentration and the number of layers [42].In a published review article Francis and Devasena (2018) reported that the dispersion of MWCNTs in surfactant was less toxic compared to the agglomerated ones.The results of the cell viability of treated MWCNTs showed a decrease in the toxicity of MWCNTs.On the other side, the SEM and TEM images (Figures 4 and 5) indicate that the agglomeration state of MWCTNs was modified due to fungal treatment.Therefore, it might be possible that the change in the agglomeration state of MWCNTs leads to a decrease in the toxicity of MWCNTs [42].
The diameter of MWCNTs has a substantial effect on the toxicity of MWCNTs.No destructive effects were reported for MWCNTs of diameter wider than 40 nm, whereas mild toxicity was observed for diameters between 15 and 40 nm.The results of this study indicated that the decrease in MWCNTs diameter led to a reduction in toxicity.Moreover, it was reported that the MWCNTs with carboxyl group had a reducing effect on the cytotoxicity of MWCNTs.The reason for this reducing effect has not been clarified yet [43].Biodegradation of MWCNTs by selected isolates induces functional groups such as carboxyl groups [30].These functional groups might play a role in the reduction of the toxicity.Further studies will be required to clarify this relationship between functionalization and toxicity reduction.

Assessment of oxidase Enzymes activity
The results of laccase enzyme activities for 14 days are shown in Figure 7a.According to the results, the evaluation of laccase enzyme activities revealed that the maximum activity was achieved at 11 days after incubation and after that, a decrease was observed, particularly in Trichoderma sp.WF29.It was reported that the lignolytic enzymes of fungi have a critical role in the degradation of PAHs and Crude oil, as reported by Moghimi et al [16].Moreover, Hidayat and Tachibana (2013) reported that laccase enzyme activity in Fusarium sp.used to degrade aliphatic hydrocarbons [44].In this study, we found that the white-and soft-rot fungi produce laccase during the MWCNTs removal that might play a role in the biodegradation of MWCNTs for the first time.The results showed that the most amount of enzyme production occurred on day 11.It seems reasonable in comparison to the kinetic growth rate and dry weight of the isolates.The simultaneous increase in enzymatic activity and growth rate suggested that laccase might be involved in the degradation of carbon nanotubes as a result of using these nanotubes as carbon sources.
The results of the assessment of manganese peroxidase (MnP) activity showed that the highest MnP activity found in Trichoderma sp.WF29, I. lacteus WF36, and T. versicolor that were 0.93, 0.78 and 0.46 U/mg, respectively, in the presence of 200 ppm MWCNTs, while no LiP activity was detected.These results were shown in Figure 7b.
Manganese peroxidase can generate free Mn (III), which can oxidise the terminal phenolic substrates.Thus, polyphenolic compounds bind to the fungal mycelium, and these bounded enzymes could be more active than the other extracellular ones like LiP [28].There is a report about the ability of partially purified lignin peroxidase enzyme of Sparassis latifolia for degradation of SWCNTs although there is no report about the role of MnP in their experiment.In contrast, our results indicated that the activity of MnP enzyme was more considerable than the LiP enzyme.
Our results suggested that the oxidative enzymatic system probably plays a significant role in the process of biodegradation.It should be reminded that the exact mechanism of fungal enzymes for biodegradation of MWCNTs has not been precisely explained yet.
Recently, wang et al. (2021) used Labrys sp.bacteria for degradation of MWCNTs.They found that a fenton-like reaction drives the degradation of MWCNTs.In this reaction, reduction of H 2 O 2 involving a continuous cycle of Fe(II)/Fe(III) leads to the hydroxyl (─OH) production [45].Clearly, the degradation mechanism of fungi is different from the bacteria.Thus, more techniques should be applied to determine the mediators and mechanisms of biodegradation.It would provide more information about the optimisation of the degradation process.
It should be noted that all the results were normalised by SPSS version 24 based on Kolmogorov-Smirnov test.The results were shown that there is a significant difference on the ability of MWCNTs biodegradation among the fungi including Trichoderma sp.WF29, I. lacteus WF36 and T. versicolor which were employed in this study.
Based on the previous study, the fate of MWCNTs is unclear after releasing it into the environment.Fortunately, in recent years, understanding the future of MWCNTs in the environment and the potential of the biodegradation of them was initiated.It shows the importance of this topic, although it is worth mentioning that the number of these studies is limited.Upon examination of some native isolates, we introduced three white-and softrot fungi for biodegradation of MWCNTs.As mentioned above, it is claimed that a group of bacteria can degrade CNTs.However, our study is focused on the ability of white-and soft-rot fungi for biodegradation of MWCNTs.
In the past studies, some kinds of white-rot fungi were introduced as an important candidate for biodegradation of CNTs while there were few data about the soft-rot fungi.Surprisingly, in this study, Trichoderma sp.WF29 as a soft-rot fungus has more ability for biodegradation of MWCNTs in comparison to white-rot fungi.The results of DLS and Raman spectroscopy were confirmed these observations.In addition to this result, the extracellular enzymes had more activity in Trichoderma sp.WF29 compared to I. lacteus WF36 and Trametes versicolor.Furthermore, Trichoderma sp.WF29 made more change in the morphology of the treated MWCNTs than the other fungi according to the SEM and TEM images.

Conclusion
MWCNTs should be removed from the environment due to the long-term stability and agglomeration of these materials, which could be harmful to different organisms and human-being.Thus, finding effective ways for mitigation of their impact is a necessity.This study aimed to assess the capabilities of Iranian native white and soft-rot fungi for degradation of MWCNTs.In Summary, the results of the study showed that Trichoderma sp.WF29 as a soft-rot fungus and Irpex lacteus WF36 and Trametes versicolor as white-rot fungi could be promising candidates to degrade MWCNTs, and could provide a possible alternative for chemical and physical degradation methods used for degradation of MWCNTs.It seems that the extracellular enzymes of white and soft-rot fungi might play a role in the degradation of MWCNTs.However, more experiments are required to confirm this hypothesis and dissect the mechanisms involved.

Figure 2 .
Figure 2. Zeta potential levels (mV) of T. longibrachiatum WF29, I. lacteus WF36 and T. versicolor.The zeta potential of control was zero.Data presented as means SD (n = 3).Different alphabets represent significance at P < 0.05 after applying Post Hoc Tukey's test.

Figure 4 .
Figure 4. SEM images of treated MWCNTs.Images A, B and C indicate the treated MWCNTs by Trichoderma sp., D, E, F are related to treated MWCNTs by I. lacteus, G, H and I indicate the treated MWCNTs by T. versicolor and J, K and L showed control sample (without fungal treatment).Each triple image showed at ×35000, ×60000 and ×80000 magnifications, respectively.

Figure 5 .
Figure 5. TEM images of fungal treated MWCNTs.A and B showed control sample (without fungal treatment), C and D indicate the treated MWCNTs by Trichoderma sp., E, F are related to treated MWCNTs by I. lacteus, G, H indicate the treated MWCNTs by T. versicolor.Shortened MWCNTs are circled in red arrow.

Figure 6 .
Figure 6.Raman Spectrum of non-treated MWCNTs.The spectrums of control, Trichoderma sp.WF29, I. lacteus WF36.and T. versicolor were shown by black, blue, green and red curves, respectively.
[35].The results showed that the LiP might play a significant role in the biodegradation of SWCNTs [35],

Table 1 .
The results of dry cell weight produced protein and CO 2.

Table 2 .
Amount of cell viability at different concentrations of treated MWCNTs.The cell viability of Trichoderma sp.WF29, I. lacteus WF36, T. versicolor in comparison to control.