2.1 Culture establishment and generation of fungal biomass

The ELF Pseudopestalotiopsis theae (PT, accession number MG881833) and Astrocystis bambusae (AB, accession number MH370741) were obtained as pure stock cultures. These isolates were isolated from the lichen Usnea pectinata (Sagada Mountain Province, Philippines) and U. bismolliuscula (Bukit Larut, Perak, Malaysia), respectively. The non-endolichenic fungus Trichoderma asperellum (TA, accession number KP792512) was also obtained as stock culture, previously been isolated from Phragmites sp., a phytoremediator plant (Sim et al., 2016). The isolates were cultured on potato dextrose agar (PDA, Friendemann Schmidt) and incubated at room temperature (RT, 25 ± 2 °C). To generate biomass, ten mycelial plugs (5 mm diameter) of each isolate were inoculated into 250 mL potato dextrose broth (PDB, Friendemann Schmidt) and incubated (100 rpm, RT) (Innova 44 stackable incubator shaker, New Brunswick Scientific) for 5 days or until sufficient biomass is attained (Chew and Ting, 2016). The mycelium was then harvested via filtration through sterile cheesecloth, rinsed with sufficient amount of distilled water, and weighed to 0.5 g fresh weight (Macharchand and Ting, 2017). The fresh biomass was used for subsequent experiments.

2.2 Dye decolorization activities

Malachite Green (MG) (CAS-No.2437–29-8, Friendemann Schmidt) dye was added into 25 mL autoclaved MilliQ water (18.2 MΩ, Sartorius, Malaysia) to produce the dye solution (100 ppm concentration). The fresh fungal biomass (0.5 g) (from section 2.1) was then inoculated into the dye solution and incubated for 14 days (25 ± 2 °C, 120 rpm). Non-inoculated (untreated) MG solution was assigned as negative control and incubated similarly. For every 24 h-interval (until day 14), 2 mL of dye solution was pipetted and centrifuged (10,000 rpm, 10 min), and the absorbance of the supernatant read at 617 nm (Tecan Spark M10 plate reader). The pellet was collected for FTIR analysis. The procedure was repeated for all isolates and the decolorization efficiency (DE, %) (Equation 1) was determined as follows: $$\text{Decolorization}\text{efficiency}(\text{DE},\%)=\left[\left({\text{A}}_{\text{i}}-{\text{A}}_{\text{o}}\right)/{\text{A}}_{\text{i}}\right]\times 100\%$$ where A_i represented initial absorbance and A_o for observed absorbance upon treatment with the isolates (Macharchand and Ting, 2017).

2.3 Ultraviolet–visible (UV–vis) spectral analysis for detection of biodegradation potential

Fresh fungal biomass (0.5 g) was introduced into 25 mL of MG solution, and incubated for 14 days (100 rpm, RT). Non-treated dye solution was assigned as negative control and incubated similarly. Two mL of aliquot was withdrawn every 24-h interval for the next 14 days. The suspension was centrifuged (10,000 rpm, 10 min) and the absorbance of the supernatant was measured (300 to 800 nm) with a UV–vis spectrophotometer (UviLine 9400, Secomam). The UV–vis spectra peaks (day 1–14) for treated and non-treated (control) MG solutions were then plotted and compared (Macharchand and Ting, 2017).

2.4 FTIR-analysis of fungal biomass in Malachite Green solution

The biomass of AB, PT and TA were collected at day 1 and 8 from treated solutions (Section 2.2). The pellet obtained was oven-dried (50 °C) overnight, and ground into powder form using pestle and mortar. Single-reflection attenuated total reflection spectra (within 4000–400 cm⁻¹) was obtained using the FTIR spectrometer conducted under ambient temperature. Data were collected within the mid-infrared region from 4000 to 700 cm⁻¹ (50-scan speed) to exclude interference (Chew and Ting, 2016).

2.5 Statistical analysis

Each experiment was conducted in triplicates, and the data analysed with One-way Analysis of Variance (ANOVA) using Statistical Package for the Social Science (SPSS) version 22.0 (IBM). Means were compared using Tukey’s Test (Honestly Significant Difference, HSD_(0.05)).

3.1 Decolorization of Malachite Green dye

All three isolates decolorized MG, resulting in hues of bluish green. The isolate P. theae (PT) was most effective with significantly highest DE (71.42%), compared to T. asperellum (TA) (64.16% DE) and A. bambusae (AB) (55.39% DE) (Fig. 1). A difference of 7.26% in DE was observed for PT and TA. Decolorization of MG by PT was also more effective compared to AB, resulting in a difference of 16.03% in DE.

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Fig. 1

Decolourisation efficiency (DE%) of T. asperellum (TA), P. theae (PT) and A. bambusae (AB) on Malachite Green (MG) (100 mg L^-1). Mean comparisons with the same letter and font type are not significantly different according to Tukey’s Test (HSD_0.05). Error bars indicate standard deviation of means.

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The dye decolorization rate was, however, more rapidly observed when TA was used for dye treatment. The DE was increasing significantly for the first 4 days, with maximum DE attained by day 4 (66.77%). On the contrary, dye decolorization by both ELF (PT, AB) were less rapid than TA. Decolorization activities peaked at day 5 for both isolates (79.27% and 55.14%, for PT and AB, respectively) (Fig. 2). By day 14, PT demonstrated the highest DE with 89.22% DE, compared to TA (76.19% DE) and AB (67.69% DE) (Fig. 2).

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Fig. 2

Decolourisation efficiency (DE%) of T. asperellum (TA), P. theae (PT) and A. bambusae (AB) on Malachite Green (MG) (100 mg L^-1). Mean comparisons with the same letter and font type are not significantly different according to Tukey’s Test (HSD_0.05). Comparisons are made across time (days) within the same isolate. Error bars indicate standard deviation of means.

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3.2 UV–vis spectra

By day 1, the spectra peak for MG (at 617 nm) treated with TA (absorbance = 2.008) had the lowest absorbance compared to AB (absorbance = 2.099) and PT (absorbance = 2.381). Spectrum for negative control (untreated MG) remained the highest (absorbance = 2.568) (Fig. 3). By day 14, the untreated MG solution retained the absorbance peak at 617 nm (absorbance = 2.499), but treatment with PT (absorbance = 0.271), TA (absorbance = 0.581) and AB (absorbance = 0.753) showed diminished peaks (Fig. 3). The UV–vis spectra suggested possible degradation of MG as the dye chromophore was no longer detected. PT was concluded to have the most potential for biodegradation of MG dye (Fig. 3).

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Fig. 3

UV–vis spectra of Malachite Green (MG) (100 mg L^-1) at (A) day 1 and (B) day 14 after treatment with T. asperellum (TA), P. theae (PT) and A. bambusae (AB). Spectra for control (non-inoculated/untreated) MG solution is included.

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3.3 FTIR spectra

The FTIR spectra for TA, PT and AB revealed the presence of additional functional groups (1000–2000 cm⁻¹) detected post-treatment in MG (Figs. 4–6). The major functional groups detected were hydroxyl (—OH), carboxyl (—COOH), amine (—NH) and alkane (—CH). Other functional groups observed include the stretching of C⚌O in esters (peaks at 1745.26 cm⁻¹) and ⚌C—H bending in aromatic rings (peaks at 815.49 cm⁻¹) (Supplementary File-Table S1-S6). The FTIR spectra also revealed that AB in natural form (pre-treatment with MG), have lesser functional groups (∼4 major groups) (Fig. 4) compared to TA (∼6 major groups) (Fig. 5) and PT (∼6 major groups) (Fig. 6). The number of major functional groups for AB remained the lowest of the three isolates, even post-treatment, with only ∼ 5 major groups detected compared to ∼ 8 major groups for both TA and PT (Figs. 4–6).

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Fig. 4

FTIR spectra analysis of A. bambusae (AB) (A) at day 1 and (B) day 14 after inoculation for the treatment of Malachite Green (MG) solutions. For each of the peaks, the following primary functional groups are detected; (a) hydroxyl, (b) amine and (c) alkanes.

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Fig. 5

FTIR spectra analysis of T. asperellum (TA) (A) at day 1 and (B) day 14 after inoculation for the treatment of Malachite Green (MG solutions). For each of the peaks, the following primary functional groups are detected; (a) hydroxyl, (b) amine, (c) alkanes and (d) other groups.

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Fig. 6

FTIR spectra analysis of P. theae (PT) (A) at day 1 and (B) day 14 after inoculation for the treatment of Malachite Green (MG solutions). For each of the peaks, the following primary functional groups are detected; (a) hydroxyl, (b) amine, (c) alkanes and (d) other groups.

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For AB, the bands representing the stretching of C⚌O in esters and ⚌C—H bending in aromatic rings were absent (Fig. 4). Binding of MG to AB is ascribed to —OH and —NH (shifts from 3280.31 cm⁻¹ to 3281.49 cm⁻¹), asymmetric CH₂— and CH₃— stretching vibrations (shifts from 2924.28 cm⁻¹ to 2923.06 cm⁻¹) and CH₃ deformations (shifts from 1373.16 cm⁻¹ to 1362.90 cm⁻¹) (Fig. 4). Bands at 1633.82 cm⁻¹ (C⚌N and C⚌O stretching within the aromatic ring) shifted to 1634.77 cm⁻¹. A new peak observed at 1518.79 cm⁻¹ (C—H bending in aromatic ring), suggested the possible binding of the MG dye to AB (Fig. 4).

The FTIR spectra for both PT and TA were similar. For PT, binding of MG was detected by shifts in peaks from 3278.01 cm⁻¹ to 3275.21 cm⁻¹ (hydroxyl (—OH) and amine (—NH)), from 2922.95 cm⁻¹ to 2923.28 cm⁻¹, 2853.24 cm⁻¹ to 2858.02 cm⁻¹ (symmetric and asymmetric CH₂— and CH₃— stretching vibration), from 1374.26 cm⁻¹ to 1372.96 cm⁻¹ (CH₃ deformations) and emergence of peaks at 1518.20 cm⁻¹ (C—H bending in aromatic ring) and 813.66 cm⁻¹ (⚌C—H bending in aromatic ring) (Fig. 6). For TA, exposure to MG resulted in shifts for the hydroxyl (—OH) and amine (—NH) groups (from 3269.18 cm⁻¹ to 3272.13 cm⁻¹) and for symmetric and asymmetric CH₂— and CH₃— stretching vibrations and CH₃ deformations from 2923.23 cm⁻¹, 2853.54 cm⁻¹ and 1375.91 cm⁻¹ to 2923.02 cm⁻¹, 2854.01 cm⁻¹ and 1374.49 cm⁻¹, respectively (Fig. 5). New peaks were detected at 814.81 cm⁻¹ (C—H bending in aromatic ring) after 14 days of treatment in MG.

To summarize, the FTIR spectra revealed that PT and AB shared similar major functional groups as the non-endolichenic TA. The number of functional groups may have a predisposition on the dye decolourization activities, as AB, which has the lowest number of functional groups, demonstrated lower dye decolourization efficiency and vice versa. Another critical observation is that new peaks were detected in all isolates post-treatment of MG, i.e. ⚌C—H bending in aromatic ring (814.81 cm⁻¹ or 813.66 cm⁻¹) for TA and PT, and C⚌C stretching vibration in the benzene ring (1518.20 cm⁻¹ or 1518.19 cm⁻¹) for PT and AB. Isolate PT, with the highest decolourization activities, was discovered to have both these new peaks.