Trichoderma samples were collected from the Biotechnology Research Institute (IPB), Universiti Malaysia Sabah (UMS), which were morphologically and genetically previously identified by Siddiquee et al. [20-22].

The Cu²⁺, Pb²⁺, Zn²⁺, and Ni³⁺ stock solutions were prepared by dissolving copper sulphate pentahydrate (CuSO₄·5H₂O), lead nitrate (Pb(NO₃)₂), Zinc sulphate (ZnSO₄·7H₂O), and nickel oxide (Ni₂O₃) (analytical grade, Merck) in distilled water. The solid medium was prepared by pouring 4 mL of stock solution of Cu²⁺/Pb²⁺/ Zn²⁺/Ni³⁺, followed by 16 mL of sterilized potato dextrose agar (PDA) into a universal bottle to obtain the desired heavy metal concentrations of 100 mg/L, 300 mg/L, 500 mg/L, 700 mg/L, 900 mg/L, and 1200 mg/L, respectively. The universal bottle was then gently shaken to homogenize the solution. The PDA incorporated with the above-mentioned heavy metal solutions were then immediately poured into the petri dishes and swirled gently. The medium was inoculated with 3 mm diameter of agar disc cut from the edge side of an actively growing pure culture of Trichoderma species samples and were observed daily until mycelia growth covered the whole petri dish. Three replicate plates were done for each treatment, and the experiment was repeated thrice under room temperature conditions (28˚C ± 2˚C, 12 h daylight and 12 h darkness).

Potato dextrose broth (PDB) (Difco, USA) was prepared as per the manufacturer’s instructions (10 g of PDB media were decanted into individual 500 mL Erlehnmeyer flasks with cotton wool stoppers placed over the flask mouths and then autoclaved at 121˚C, 1.4 kg·cm⁻¹ for 15 min). The used stock solution of either Cu²⁺ or Pb²⁺ or Zn²⁺ or Ni³⁺ (10 mL) was added to the medium in each 250 mL conical flask separately to reach the required concentrations (100, 200, 300, 400 and 500 mg/L) in a volume of 100 mL. The medium was later inoculated with six disks of pure culture of the selected Trichoderma samples taken from the edge side of an actively growing PDA culture [23]. The initial concentrations of each heavy metal in each conical flask were checked using an inductive coupled plasma spectrometer (ICPOES Model Optima 2000 DV, PerkinElmer, United States) before fungal inoculation [10]. Cultures were incubated under room temperature conditions (28˚C ± 2˚C, 12 h daylight and 12 h darkness). Three replicates were done for each heavy metal, and the experimental works were repeated three times.

After seven days of incubation, flasks containing fungal biomass at different concentrations of heavy metals were harvested and filtered through Whatman No. 1 filter paper. Biomass samples were rinsed several times with distilled water and left in an oven at 50˚C until a constant weight was achieved and defined as dry biomass (g/L) [6].

According to the method proposed by Lopez and Vazquez [10], concentrations of heavy metals in liquid cultures were measured with ICP-OES before fungal inoculation. The amount of heavy metal uptake (Q, mg/g) was calculated by using the following equation [3,10,24]:

where Q (mg/g) = mg of metal ions uptake per gram biomass Ci (mg/L) = initial metals concentration Cf (mg/L) = final metals concentration m (g) = amount of dry biomass V (L) = volume of the medium

Statistical analyses of data were performed by using the Statistical Package for the Social Science (SPSS), version 21.0. One way analysis of variance (ANOVA) was carried out to show the significance difference at p ≤ 0.05. Tukey test was used to compare the means.

This research work was designed to determine the capability and absorption methods of the selected Trichoderma species for the different concentrations of heavy metals. According to Gadd [25], it has been reported that exposure of filamentous fungi to heavy metals can lead to physiological adaptation or the selection of mutants and such changes may be associated with increased metal absorption capacity. Filamentous fungi are able to grow in the presence of heavy metals even at higher concentrations. The selected fungi that were used belong to the genera Trichoderma which consisted of T. harzianum, T. aureoviride, and T. virens. In the present study, the selected fungi were observed at different concentrations of 100 mg/L, 300 mg/L, 500 mg/L, 700 mg/L, 900 mg/L, and 1200 mg/L in Cu²⁺, Zn²⁺, Pb²⁺, and Ni³⁺ heavy metals on PDA. From the analysis of Cu²⁺ ion in different concentrations, it was found that T. harzianum strain T32 obtained the highest mycelia growth values, followed by T. harzianum strain T30, T. virens strain T128, and T. aureoviride strain T121 within 3 days of incubation. At 100 mg/L-1200 mg/L concentration of Cu²⁺, the highest mycelia growth values were 7.5 cm for 100 mg/L, 7.2 cm for 300 mg/L, 6.2 cm for 500 mg/L, 5.4 cm for 700 mg/L, 2.9 cm for 900 mg/L, and 2.1 cm for 1200 mg/L, respectively, by using T. harzianum strain T32. When comparing with other fungi, T. harzianum strain FA30 recorded the lowest growth rate. Even though the same species of T. harzianum was used, a different strain exhibited different ability and biosorption capacity with different concentrations of Cu²⁺. This result suggests that different strains of even the same species do not exhibit the same tolerance capacity. After five days, all fungi fully covered the whole petri dish at concentration ranges of 100 mg/L - 700 mg/L. Concentrations of 900 mg/L and 1200 mg/L of Cu²⁺ showed completely grown mycelia within 20 days. Nur Liyana et al. [2] reported that Aspergillus niger has a high tolerance ability in the presence of Cu²⁺ than the P. simplicissimum species with a concentration of 1000 mg/L. Zapotoczny et al. [23] also reported that Acremonium pinkertoniae could tolerate up to 600 mg/L concentration of Cu²⁺ in malt agar. Anand et al. [6] reported that there is no growth at 300 mg/L of Cu²⁺ using T. viride species. Tsekova and Todorova [26] also reported similar level of tolerance to Cu²⁺ ions by A. niger B-77 strain where 300 mg/L of the Cu²⁺ ion was inhibitory to the growth of the organism. In comparison with previous research results, we first reported up to 1200 mg/L concentration of maximum tolerance on PDA.

By increasing the initial concentration of Zn²⁺ up to 1200 mg/L in the PDA culture medium, significantly different mycelia growth rate was found for those fungi. In Table 1, T. virens strain T128 showed the highest growth rate value compared to other fungi. Even though other fungi had a slow growth rate, they still showed tolerance with a high Zn²⁺ concentrations. Maximum concentrations (1200 mg/L) of Zn²⁺ with a value of 4.40 mg/L of T. harzianum strain FA30, 2.70 mg/L of T. harzianum strain T32, 4.70 mg/L of T. aureoviride, and 4.81 mg/L of T. virens strain 128 were found. In 100 mg/L 500 mg/L concentrations of Zn²⁺, the mycelia growth of T. harzianum strain T32 was higher than that of T. aureoviride strain T121; after that, with increasing concentrations of 700 mg/L - 1200 mg/L, the mycelia growth decreased. Based on the heavy metal study of Zn²⁺, the T. virens strain T128 showed the best selected filamentous fungi to tolerate and reduce the toxicity at low to high concentrations of metal solutions.

With increasing concentrations of Pb²⁺ from 0 mg/L - 1200 mg/L, the mycelia growth of the selected fungi in the PDA media decreased. The highest mycelia growth was recorded for the T. harzianum strain T32 at different concentrations for 3 days whereas compared to other fungi, almost the same growth rates were recorded. These fungi were determined as being the most tolerant fungi for Pb²⁺ when compared to other heavy metal ion concentrations. The maximum tolerance of Pb²⁺ for metal solution occurred at 1200 mg/L concentration with a value of 6.10 mg/L for T. harzianum strain FA30, 6.90 mg/L for T. harzianum strain T32, 6.71 mg/L for T. aureoviride, and 6.60 mg/L for T. virens, respectively.

There was fluctuation of the data as the fungal mycelia growth of T. harzianum decreased with the increasing concentration of Ni³⁺ when compared to T. virens. This result indicated that the T. virens is the most suitable fungus for biosorption and tolerance to Ni³⁺ pollutants because it can be adapted to such environments, followed by other fungal species.

According to Kapoor et al. [9], reported fungi are well-known to be tolerant and even able to accumulate Pb²⁺, Cu²⁺, Zn²⁺, Ni³⁺, and Cd²⁺ concentrations. Some species of fungi can accumulate a broad range of heavy metal contaminations, while others are specific for certain types of heavy metals. Different types of fungi, such as Trichoderma species that show high tolerance to toxic metals, may be useful in metal recovery systems.

This study showed that the T. virens strain T128 had the highest tolerance toward Ni³⁺ and Pb²⁺ concentrations when compared to other heavy metals, even at the maximum concentration of 1200 mg/L. For Cu²⁺ heavy metal ion solutions, T. harzianum recorded the highest average growth rate compared to other fungi. The colony growth rate observed in the T. aureoviride strain T121 after 5 day cultures on a solid medium containing Cu²⁺ solutions at low to high concentrations also supported fungal growth. According to Baldrian et al. [27], different ranges of heavy metals tolerance on solid medium may be caused by the metal binding property of various agar medium or the diverse ability of different fungi.

T. harzianum strain FA30 showed the most tolerant and

Table 1. Growth rate of fungi on PDA at different concentrations of Cu²⁺, Zn²⁺, Pb²⁺ and Ni³⁺.

The growth rate of fungi were compared by using one-way ANOVA and Tukey test for different concentrations of Cu²⁺, Zn²⁺, Pb²⁺ and Ni³⁺. Means with different letters in each column of Cu²⁺, Zn²⁺, Pb²⁺ and Ni³⁺ concentrations were significantly different at p ≤ 0.05.

higher absorption skill with Ni³⁺ and Pb²⁺ concentrations, as shown in supplementary Figure S1. The increasing growth rate was found for Pb²⁺ and Ni³⁺ when compared to Cu²⁺ and Zn²⁺ concentrations, and no visible appearance changes occurred in the mycelium or sporulation. Subheading on screening for the most tolerant fungi results also indicated that T. harzianum strain T32 showed high ability and tolerance skills to absorb the toxicity of heavy metals when compared to T. harzianum strain FA30. T. harzianum strain T32 always recorded faster growth in the heavy metals consisting of Ni³⁺, Pb²⁺, and Zn²⁺ concentrations (supplementary Figure S2).

T. aureoviride strain T121 showed the highest tolerance toward the concentrations of Pb²⁺ and Ni³⁺, as shown in supplementary Figure S3. This species strain was fully grown at low concentrations of 100 mg/L - 500 mg/L, but no change occurred in physical appearances. Based on Figure 1, the T. virens strain T128 showed almost the same growth rates at low concentration when the concentration increased for Cu²⁺ and Zn²⁺, the growth rate of mycelia were decreased compared to Pb²⁺ and Ni³⁺ concentrations. The concentrations ranged from 300 mg/L to 1200 mg/L of Cu²⁺, the mycelia physical appearance changed as it turned from green to a darker colour. These similar changed results were also observed by Venkateswerlu et al. [28], who determined that there are blue colored particles in the presence of Cu²⁺ in Neurospora crassa and Cunninghamella blackesleeana and they proposed that this was caused by the binding of the Cu²⁺ ions to the protein in the cell wall of the mycelium. Anand et al. [6] tested in agar medium at concentration ranges of 1000 mg/L - 5000 mg/L of Cu²⁺; the mycelia turned blue both on the agar media and in broth culture at all concentrations of copper, which is due to the binding of Cu²⁺ to the fungal cell wall.

Dry biomass of T. harzianum, T. virens, and T. aureo-

viride decreased with increasing initial concentrations of heavy metals ( Ni³⁺, Zn²⁺, Pb²⁺, and Cu²⁺) for 7 days at room temperature conditions of 28˚C ± 2˚C. With increasing concentrations of Ni³⁺, the dry biomass values of those fungi decreased. The result indicated that the highest biomass values of those fungi were at the control concentration level while the lowest biomass values were recorded at 500 mg/L of Ni³⁺ (Table 2). It was found that the T. aureoviride strain T121 obtained the highest dry biomass values of 0.3518 g/L, followed by the T. virens strain T128 (0.1963 g/L) and T. harzianum strain T32 (0.137 g/L) at 500 mg/L concentration. The lowest dry biomass of T. harzianum strain T32 compared to the other fungi, however, gave the highest Ni³⁺ uptake capacity (Figure 2), even though the dry biomass of the T. aureoviride strain T121 recorded the highest values, but gave the lowest Ni³⁺ uptake capacity.

By increasing the initial concentration (0 mg/L) of Zn²⁺ up to 500 mg/L in the liquid culture medium using selected fungi, the level of dry biomasses decreased. A remarkable decrease of the biomass values was observed in the T. harzianum strain T32 while compared with the T. virens strain T128 and the T. aureoviride strain T121. From 0 mg/L - 500 mg/L concentration of Zn²⁺, the highest biomass values occurred between 0.5 g/L - 0.3 g/L by using T. aureoviride, followed by T. harzianum and T. virens (Table 2). With the increase of Zn²⁺, the metal uptake capacity by T. harzianum, T. virens, and T. aureoviride were always increasing. The maximum uptake of Zn²⁺ from the metal solution occurred at 500 mg/L concentration with a value of 3.1789 g/g for T. harzianum, 2.1719 g/g for T. virens, and 1.5065 g/g for T. aureoviride, as shown in Figure 3.

With different concentrations of Pb²⁺, dry biomass values of T. aureoviride were higher compared to T. virens and T. harzianum (Table 2). The dry biomass obtained for T. aureoviride was 0.1078 g/L and followed by 0.0981 g/L for T. virens and 0.0244 g/L for T. harzianum at 500 mg/L Pb²⁺ concentration. The noticeable decreased dry biomass value was found for T. harzianum at different concentrations of Pb²⁺ (0 mg/L - 500 mg/L). The growth and metal removal properties of the three fungi were highly affected by initial metal ion concentrations. The highest Pb²⁺ uptake (13.48 g/g) at 500 mg/L of the initial Pb²⁺ concentration was obtained for T. harzianum, whereas T. virens and T. aureoviride were only able to remove 7.9126 g/g and 4.6683 g/g of Pb²⁺, respectively (Figure 4).

Based on Table 2, dry biomass of T. virens, T. aureoviride, and T. harzianum decreased with increasing initial concentrations of Cu²⁺. The result indicated that the highest biomass values of those fungi were at the control concentration while the lowest biomass values was recorded at 500 mg/L of Cu²⁺. It was found that at 500 mg/L of Cu²⁺, the maximum Cu²⁺ uptake occurred and the highest dry biomass was obtained by T. aureoviride (0.3505 g/g), followed by T. harzianum (0.1678 g/g), and T. virens (0.1552 g/g) (Figure 5).

These three species of fungi are tested to determine the Minimum Inhibitory Concentration (MIC) for the different types of heavy metals. The following order of toxicity found Cu²⁺ > Zn²⁺ > Ni²⁺ > Pb²⁺ by the selected fungi. The MIC values suggest that the resistance level against individual metals was dependent on the fungi.

Table 2. Dry biomass values of fungi on PDB at different concentrations of Cu²⁺, Zn²⁺, Pb²⁺ and Ni³⁺.

Dry biomass values of fungi were compared by using one-way ANOVA and Tukey test for different concentrations of Cu²⁺, Zn²⁺, Pb²⁺ and Ni³⁺. Means with different letters in each column of Cu²⁺, Zn²⁺, Pb²⁺ and Ni³⁺ concentrations were significantly different at p ≤ 0.05.

The growth and metal removal properties of these fungi were highly affected by the initial concentration to increase different heavy metals concentrations. Based on the results, increasing Pb²⁺ concentrations may lead to the decrease of microbial growth biomass. At 500 mg/L, where the highest Pb²⁺ uptake occurred, the dry biomass obtained for T. aureoviride was 0.13 g/L and the uptake capacity of Pb²⁺ concentrations by T. aureoviride was the

lowest at 4.7 g/g compared to other fungi.

According to Deshmokh and Rai [29], the increase of the uptake capacity of metals may be often associated with toxicity or the increasing permeability of cell membrane on account of further binding of the metal to exposed intracellular sites, causing the biomass to decrease with elevated metal exposure. Kapoor et al. [9] showed that the lower uptake at higher biomass concentrations can be attributed to the electrostatic interactions of the functional groups at the cell surfaces. The cells at higher concentrations in suspension attach to each other, thus lowering the cell surface area in contact with the solution.

Among the fungi, T. harzianum are able to absorb more amounts of Ni³⁺ metal concentrations, especially at lower concentrations between 300 mg/L - 500 mg/L. The differences may be due to the larger surface area of T. harzianum biomass for the adsorption processes. Zafar et al. [3] suggested that the main mechanism of heavy metal removal from aqueous solution was a redox reaction between the heavy metal ion concentrations and the fungal biomass. In addition to the removal of metals from solution during growth, the grown biomass cells can absorb metals from solution. The grown biomass cells can absorbs the metals at a high concentration especially where tolerance to metals during growth is fairly low. Kacprzak and Malina [11] reported that due to high growth, more ligands and negative charge are exposed, which in turn attract more positively charged metal ions from the aqueous solution.

Our result also proved that metal uptake occurred as a consequence of physical binding to cell surfaces rather than as an active process, since the mycelia appeared to be partially degraded and the fungal cell wall plays an important role in metal uptake (biosorption). The ability of the Trichoderma species is to tolerate and undergo the biosorption process of toxic metals based on ionic species associating with the cell surface or extra cellular polysaccharide, protein, and chitin. Usually, fungal cell walls contain chitin and chitosan. Chitin and chitosan contents of the fungal cell wall can change during the growth of mycelia and this can account for the variations in the metal-uptake capacity with the fungi [30].

The accumulation and uptake capacity of T. harzianum for Ni³⁺, Pb²⁺, and Cu²⁺ concentrations in liquid medium were found to be highest when compared to T. virens and T. aureoviride, while for Zn²⁺, the highest tolerance and uptake capacity of metals were recorded by T. virens. It was also showed that these fungi tested above had their own ability to absorb different types of heavy metals depending on the toxicity of each heavy metal. Based on this study, there is a possibility that by increasing the initial concentration, the removal of metals solution also increases due to the increase of metal uptake. These results are in agreement with those of Yalcin et al. [31], who reported that a higher initial concentration provided an important driving force to overcome all mass transfer resistances between the heavy metals solution and the fungi cell wall.