*2.3. Screening and Identification of E*ffi*cient Cd2*+*-Tolerant Fungal Isolate*

The isolated fungal isolates were tested for their potential to remove Cd2<sup>+</sup> at 25, 50, and 75 mg/L concentrations. The concentrations were prepared from a sterilized 100 mg/L solution of Cd(NO3)<sup>2</sup> containing PDA. A loop full of purified isolates was positioned at the Petri plates' center comprising of PDA (pH 5.0) and the above-mentioned Cd2<sup>+</sup> concentrations. The growth of fungal isolates on the pure PDA medium was labeled 'Control'. All plates were sealed with parafilm and subjected to incubation of 5 days. Based on the visual observation, the growth of fungal isolates was labeled as absent growth (−), least growth (+), less than normal growth (++), slightly less than normal growth (+++), and normal growth (++++). The highly efficient Cd2+-tolerant fungal isolates were sent for identification to the Indian Agricultural Research Institute, New Delhi, India.

#### *2.4. Optimization of Batch Cultures*

The highly efficient Cd2+-tolerant fungal culture was tested at different pH, i.e., 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 for uptake and percentage removal of Cd2<sup>+</sup> from PDA containing 20 mg/L of Cd2+. First, 1M HCl and 1M NaOH were used to adjust the pH of PDB. Then, 1mL spore suspension of efficient fungal culture containing 10<sup>4</sup> spores (1%) was inoculated into 250 mL Erlenmeyer conical flasks containing 100 mL of PDB enriched with 20 mg/L of Cd2<sup>+</sup> followed by shaking these flasks for 120 h at 30 ◦C. For the control, a non-inoculated flask having 20 mg/L of Cd2<sup>+</sup> and PDB was used. After 120h, fungal growth was collected through filtration using Whatman Filter Paper No. 1, which was later dried for 48 h at 80 ◦C in an oven and then weighed. Atomic absorption spectrophotometer (AAS, GBC 932, Dandenong, Australia) was used to evaluate the Cd2<sup>+</sup> concentration left in the filtrate. To evaluate the incubation time effect, the incubation time was varied between 24 and 144h under optimum pH. Under optimum pH and incubation time, the effect of inoculum size was investigated by performing batch experiments at different inoculum sizes i.e., 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3.0%. Temperatures of 20, 25, 30, and 35 ◦C were studied to examine the temperature effect at optimum pH, incubation time, and inoculum size. Under optimum pH, the effect of initial Cd2<sup>+</sup> concentration, inoculum size, and incubation time was investigated by changing the concentrations of Cd2<sup>+</sup> ions from 5 to 50 mg/L. The process to perform all the experiments was the same as mentioned above. All experiments were performed in triplicate, and data were statistically examined by *t*-test with the help of SPSS 11.5.

The Cd2<sup>+</sup> uptake by fungal biomass was calculated as follows:

$$\mathbf{Q} = \frac{\mathbf{v} \mathbf{(C\_0 - C\_f)} \,\mathrm{s}}{\mathbf{M}} \tag{1}$$

where Q is the Cd2<sup>+</sup> uptake (mg/g); C<sup>0</sup> and C<sup>f</sup> are the initial and leftover concentrations of Cd2<sup>+</sup> (mg/L), respectively; v is the total volume of the solution (L); and M the dried biomass of fungus (g).

The removal of Cd2<sup>+</sup> (%) was calculated using following equation:

$$\% \text{ Removal of } \text{Cd}^{2+} = \frac{\text{C}(initial) - \text{C}(final)}{\text{C}(initial)} \times 100 \tag{2}$$

#### *2.5. Equilibrium Isotherms*

#### 2.5.1. Langmuir Isotherm

Langmuir isotherm is a mathematical deduction of kinetic equilibrium between the condensation and loss of adsorbed molecules.

$$\text{Ce/qe} = 1/\text{q}\_{\text{max}} \text{ b} + \text{Ce/q}\_{\text{max}} \tag{3}$$

where Ce is equilibrium concentration of Cd2<sup>+</sup> (mg/L), qe is amount of Cd2<sup>+</sup> adsorbed per gram of fungal biomass (mg/g), qmax is Langmuir constant, and b is the energy of adsorption. The values of b and qmax were evaluated by the intercept and slope of the graph, respectively.

#### 2.5.2. Freundlich Isotherm

This isotherm is the result of empirical consideration and expressed as

$$\text{Age} = \text{K}\_{\text{f}} \text{Ce}^{1/\text{n}} \tag{4}$$

where qe is the Cd2<sup>+</sup> adsorbed (mg/g), Ce is the equilibrium concentration of Cd2<sup>+</sup> (mg/L), K<sup>f</sup> is the adsorption coefficient (mg/g) and directly related to the standard free energy change, and n is the empirical constant.

The linear logarithmic form of the isotherm is:

$$\text{Log qe} = (1/\text{n}) \log \text{Ce} + \log \text{K}\_{\text{f}} \tag{5}$$

From the straight-line plot between log qe and log Ce, the values of of K<sup>f</sup> and 1/n were found.

#### *2.6. Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) Analysis*

The morphological changes in the fungi without and after incubating with Cd2<sup>+</sup> were visualized using Scanning Electron Microscopy (SEM; JSM-6100, JEOL, Tokyo, Japan). The changes in surface chemical profiling were analyzed and interpreted by Fourier transform infrared spectroscopy (FTIR) (Perkin Elmer Spectrum BXII, Waltham, MA, USA, in 4000–400 cm−<sup>1</sup> frequency range with a resolution of 1 cm−<sup>1</sup> ).

#### **3. Results and Discussion**

### *3.1. Isolation, Screening, and Identification of Cd2*+*-Resistant Fungal Biomass*

Twenty-five sludge, industrial effluents and sewage samples were collected from Panipat, Sonepat and Karnal districts of Haryana, India to isolate fungal isolates. All the fungi were maintained on PDA slants (pH 4.5) at 30 ◦C. Screening of these fungal isolates on PDA containing 25, 50, and 75 mg/L of Cd2<sup>+</sup> showed that 13 fungal isolates were tolerant to 25 mg/L Cd2+, 7 fungal isolates were tolerant to 50 mg/L, and 3 fungal isolates were tolerant to 75 mg/L (Table 1). Such phenomena indicate that some fungal isolates exhibited inhibition at higher metal ions concentrations. Out of three fungal isolates tolerant to 75 mg/L Cd2+, the highly Cd2+-tolerant fungal isolate FS7 and FS16 were identified as *Trichoderma fasciculatum* (ITCC 7547) and *Trichoderma longibrachiatum* (ITCC 7062) by the Indian Culture Collection, Indian Agricultural Research Institute. IARI, New Delhi, India.


**Table 1.** Growth profile of all the fungal isolates on potato dextrose agar (PDA) (pH 5.0, temperature 30 ◦C) containing 25, 50, and 75 mg/L of Cd2<sup>+</sup> after five days.

**Note**: Absent growth (−), least growth (+), less than normal growth (++), slightly less than normal growth (+++), and normal growth (++++).

#### *3.2. Optimization of Process Parameters*

Various surface and physiochemical properties—for instance, pH, incubation time, inoculum size, temperature, metal ion concentrations, quantity of biosorbents, etc. are affecting the process for the bioaccumulation of heavy metal ions in fungal biomass.

#### 3.2.1. pH Effect

In the process to remove the heavy metal ions from fungi, the pH plays an important and significant role. The observed results demonstrated that there was an enhancement in the Cd2<sup>+</sup> ion uptake and percentage removal up to pH 5.0 from the liquid medium by *T. fasciculatum* and *T. longibrachiatum.* After pH 5.0, the uptake and percentage removal was significantly decreased with the increasing pH (Figure 1a). The outcomes showed that the ideal pH for uptake and percentage removal of Cd2<sup>+</sup> + from the liquid medium by *T. fasciculatum* and *T. longibrachiatum* was 5.0.

**Figure 1.** Effect of operational parameters on the Cd2<sup>+</sup> removal efficiency by *T. fasciculatum* and *T. longibrachiatum* (**a**) pH (**b**) Incubation Time (**c**) Inoculum Size (**d**) Temperature (**e**) Concentration of Cd.

The observed uptake and percentage removal of Cd2<sup>+</sup> from the liquid medium by *T. fasciculatum* at pH 5.0 were 2.63 mg/g and 59.20%, respectively. In addition, the uptake and percentage removal of Cd2<sup>+</sup> by *T. longibrachiatum* were 3.59 mg/g and 81.60%, respectively. Interestingly, it has been reported that the optimum pH is important to get better results regarding the removal of heavy metal ions using fungal isolates [37,38]. In many cases, the optimal value of pH was 5.0, which shows a maximum removal of heavy metal ions (Pb(II), Cd(II), As(III), and Hg(II)) by fungus isolates such as *Penicillium purpurogenum* [37,38].

### 3.2.2. Incubation Time Effect

Figure 1b exhibits the incubation time effect on the Cd uptake and percentage removal from the liquid medium by *T. fasciculatum* and *T. longibrachiatum.* It was observed that at optimum pH 5.0, the uptake and percentage removal of Cd2<sup>+</sup> ions from the liquid medium by *T. fasciculatum* and *T. longibrachiatum* were increased with increasing the incubation time. Interestingly, it was seen the uptake and percentage removal were enhanced from 24 h to 120 h; however, they reduce after 120 h. The maximum uptake and percentage removal of Cd2<sup>+</sup> by *T. fasciculatum* at 120 h incubation time were 3.56 mg/g and 60.55%, respectively. Nevertheless, at 120 h incubation time, the uptake and percentage removal of Cd2<sup>+</sup> ion by *T. longibrachiatum* were 4.2 mg/g and 82.2%, respectively.

It is believed by most of the researchers that ion exchange or physical adsorption at the surface of the cell might relate with the initial rapid phase [39]. However, in the presence of salt, the metal ion transport into the cytoplasm across the cell membrane via an active metabolism-dependent transport is responsible for the slower phase [40]. Moreover, in the absence of salt, other biosorption mechanisms that consist of complexation, microprecipitation, etc. due to the diffusion of metal ions into the cell debris are responsible for the second slower phase [41].

#### 3.2.3. Inoculum Size Effect

Under the optimized conditions (pH 5.0, incubation time 120 h), with increasing the inoculum size from 0.5% to 3.0%, the Cd2<sup>+</sup> ion uptake and percentage removal was enhanced up to a particular size and then reduced. Figure 1c depicts the relation between the Cd2<sup>+</sup> ion uptake and percentage removal and inoculum size. The experimental observations confirmed that the uptake and percentage removal of the Cd2<sup>+</sup> ion was increased with increasing the inoculum size from 0.5% to 2.5% for *T. fasciculatum* and 2.0% for *T. longibrachiatum,* while it declined afterwards. The maximum uptake and percentage removal of Cd2<sup>+</sup> from the liquid medium by *T. fasciculatum* was approximately 2.15 mg/g and 62.60%, respectively. Moreover, under the same optimized experimental conditions, the Cd2<sup>+</sup> ion uptake and percentage removal in the liquid medium by *T. longibrachiatum* were 2.29 mg/g and 89.85%, respectively. Interestingly, after the inoculum size 2.5% for *T. fasciculatum* and 2.0% for *T. longibrachiatum*, the uptake and percentage removal of the Cd2<sup>+</sup> ions were decreased significantly. It has been reported that the percentage removal of heavy metal ions increases with enhancing the adsorbent dose due to the fact that it increases the adsorption sites, which leads to high adsorption and high removal efficiency [42–44].

#### 3.2.4. Temperature Effect

Interestingly, the adsorption medium temperature is also substantially important in the biosorption of the metal ions by microbial cells for energy-dependent mechanisms. As reported in the literature, mostly, the adsorption is considered as an exothermal process [45]. To understand the effect of temperature on the uptake and percentage removal of Cd2<sup>+</sup> ions, temperature-dependent experiments were performed by keeping all other optimized experimental conditions the same, i.e., pH 5, incubation time 120h, inoculum size 2% for *T. longibrachiatum*. Figure 1d exhibits the relation between the Cd2<sup>+</sup> ion uptake and percentage removal and adsorption temperature. The observed results revealed that under optimized conditions, the maximum Cd2+ion uptake and percentage removal from the liquid medium by both fungi were obtained at 30 ◦C. It was interesting to note that for *T. fasciculatum*, the maximum Cd2<sup>+</sup> uptake and percentage removal was 1.92 mg/g and 63.85%, respectively while for the *T. longibrachiatum*, the uptake and percentage removal was 2.40 mg/g and 84.25%, respectively.

#### 3.2.5. Initial Metal Concentration Effect

Initial metal ion concentrations also have a particular effect on the adsorption characteristics of heavy metal ions. Figure 1e shows the effect of initial metal ion concentration on the uptake and percentage removal of Cd2<sup>+</sup> ions using fungal isolates. To check the initial ion concentrations, several concentrations (5–50 mg/L) were tested, and all the experiments were performed at optimized experimental conditions, i.e., pH 5.0, incubation time (120 h), inoculum size (2.5% for *T. fasciculatum*, and 2.0% for *T. longibrachiatum*), and temperature 30 ◦C. The observed results confirmed that under optimized reaction conditions, for both fungal isolates, the Cd2<sup>+</sup> uptake and percentage removal increased with the increasing the Cd2<sup>+</sup> concentrations from 5 to 20 mg/L, and afterwards, a decline was seen. At an optimum concentration of 20 mg/L, the uptake and percentage removal of Cd2<sup>+</sup> from liquid medium was 2.27 mg/g and 67.10% for *T. fasciculatum* and 2.34 mg/g and 76.25% for *T. longibrachiatum,* respectively. This enhancement may be because of an increase in the electrostatic associations (comparative with covalent interactions), including sites of continuously lower affinity for metal ions [46].

#### *3.3. Adsorption Isotherms*

*longibrachiatum.*

*3.4. SEM and FTIR Analysis* 

Langmuir

Freundlich

(SEM), and the results are presented in Figure 3.

The availability of a finite number of binding sites can be expected in the Langmuir model, which equally distributed to the sorbent surface. It presents the same attraction for single-layer sorption with no interaction between sorbed species. The Freundlich equation provides the adsorption data for a limited concentration range and proposed an adsorption sites heterogeneity on the biomass [47]. By changing the pH from 3.5 to 6.0 for Cd2<sup>+</sup> ion removal from the liquid medium using *T. fasciculatum* and *T. longibrachiatum*, the obtained results were examined, and sorption isotherms were calculated. Figure 2 exhibits the calculated Langmuir and Freundlich isotherms for Cd2<sup>+</sup> removal by *T. fasciculatum* and *T. longibrachiatum*. As a result of the higher value of correlation coefficients R<sup>2</sup> > 0.90, the observed results revealed that the Cd2<sup>+</sup> removal using both the fungal isolates, i.e., *T. fasciculatum* and *T. longibrachiatum*, better fit with the Langmuir isotherm models compared with the Freundlich isotherm. Various parameters of Langmuir and Freundlich models for the Cd2<sup>+</sup> ion sorption on the live cells of *T. fasciculatum* and *T. longibrachiatum* are presented in Table 2. As observed, the maximum Cd2<sup>+</sup> ion adsorption capacities (qmax) for *T. fasciculatum* and *T. longibrachiatum* fungal isolates were 1.90 mg/g and 0.80 mg/g, while the calculated correlation coefficients (R<sup>2</sup> ) were 0.99 and 0.99 for both the fungal isolates, respectively. *Processes* **2020**, *8*, x FOR PEER REVIEW 8 of 14 *longibrachiatum* fungal isolates were 1.90 mg/g and 0.80 mg/g, while the calculated correlation coefficients (R2) were 0.99 and 0.99 for both the fungal isolates, respectively.

**Figure 2.** (**a**,**c**) Langmuir and (**b**,**d**) Fredundlich isotherms for Cd2+ removal by *T. fasciculatum* and *T. longibrachiatum*, respectively. **Figure 2.** (**a**,**c**) Langmuir and (**b**,**d**) Fredundlich isotherms for Cd2<sup>+</sup> removal by *T. fasciculatum* and *T. longibrachiatum*, respectively.

**Table 2.** Langmuir and Freundlich isotherm constants for removal of Cd2+ by *T. fasciculatum* and *T.* 

**Isotherms Parameters** *T. fasciculatum T. longibrachiatum*

R2 0.99 0.99

Kf (L/g) −0.15 0.02 n −3.11 −0.95 R2 0.84 0.84

The morphological changes before and after the Cd2+ ions accumulation in the fungal isolates, i.e., *T. fasciculatum* and *T. longibrachiatum*, were further analyzed by scanning electron microscopy

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+ 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+ 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].


**Table 2.** Langmuir and Freundlich isotherm constants for removal of Cd2<sup>+</sup> by *T. fasciculatum* and *T. longibrachiatum.*
