*3.4. SEM and FTIR Analysis*

The morphological changes before and after the Cd2<sup>+</sup> ions accumulation in the fungal isolates, i.e., *T. fasciculatum* and *T. longibrachiatum*, were further analyzed by scanning electron microscopy (SEM), and the results are presented in Figure 3.

**Figure 3.** SEM images of (**a**) *T. fasciculatum* control and (**b**) after treatment with Cd2+, (**c**) *T. longibrachiatum* control and (**d**) after treatment with Cd2+.

The observation revealed that after 120 h, the hyphae of both the fungi was tube shaped, septate, and diverged with no metal treatment. However, after treatment with 20 mg/L of Cd2<sup>+</sup> after 120 h, there was a thorough disruption and dissolution of mycelium of *T. fasciculatum* and *T. longibrachiatum*. These metals were consistently intact to the fungal mycelium, and a higher absorption of Cd2<sup>+</sup> ions along with flocculation in mycelium was seen. The formation of such complexes was most probably because of the detoxification mechanism, in which normally fungal isolates are used in order to manage the lethal concentrations of heavy metal ions [48].

To understand the metal ions bindings, the attachment of various functional groups on the fungal surface are important to study. To examine the functional groups available on the surfaces of fungal isolates, FTIR studies were performed for both the fungal biomass, i.e., *T. fasciculatum* and *T. longibrachiatum* before and after Cd2<sup>+</sup> ions treatment. Figure 4 exhibits the typical FTIR spectra in the range of 450–4000 cm−<sup>1</sup> for both the fungal biomass, i.e., *T. fasciculatum* and *T. longibrachiatum* before and after Cd2<sup>+</sup> ions treatment. The Cd2<sup>+</sup> free biomass of *T. fasciculatum* and *T. longibrachiatum* exhibited several absorption peaks, which show the complex nature of the biomass. The peak that appeared in the range of 3500–3200 cm−<sup>1</sup> was due to stretching of the N–H bond of the amino groups and the O–H bond of the hydroxyl group. Upon Cd2<sup>+</sup> loading to the biomass of *T. fasciculatum* and *T. longibrachiatum*, a significant change in the peak positions was seen, which confirmed the Cd2<sup>+</sup> binding with hydroxyl and amino groups (Table 3). The peaks that appeared at 2853 cm−<sup>1</sup> and 2922 cm-1 exhibited the presence of C–H methyl groups stretching. Furthermore, the peak shown at 1747 cm−<sup>1</sup> represents the native carbonyl stretching, while the peak at 1373 cm−<sup>1</sup> represented as CH symmetric/symmetric band. The peaks that appeared at 2853 cm−<sup>1</sup> , 2922 cm−<sup>1</sup> , and 1747 cm−1did not show any significant change in samples exposed to Cd2<sup>+</sup> except for a peak exhibited at 1373 cm−<sup>1</sup> , which showed a slight change from its original position.

**Figure 4.** Fourier transform infrared spectroscopy (FTIR) spectra of (**a**) *T. fasciculatum* and (**b**) *T. longibrachiatum* fungal biomass before and after treatment with Cd2+.


**Table 3.** FTIR peaks and their corresponding functional groups of *T. fasciculatum* and *T. longibrachiatum* before and after the addition of Cd2+.

In case of Cd2+-unloaded spectra of *T. Fasciculatum* and *T. longibrachiatum*, the peaks appeared at 1650 and 1544 cm−<sup>1</sup> are related with the C=O of amide I and the NH/C=O blend of the amide II bond, respectively confirming the availability of the carboxyl groups [49,50]. It is interesting to see that the peak positioned at 1544 cm−<sup>1</sup> expanded in the presence of Cd2<sup>+</sup> ions, which confirms the interaction of Cd2<sup>+</sup> with carboxyl groups. The above observations revealed the presence of various functional groups such as -CH, -OH, –C=O, and –NH in the binding of Cd2+ions with the fungal biomass, i.e., *T. fasciculatum* and *T. longibrachiatum*. The observed results are in agreement with the reported literature [49–51].
