*3.1. Preliminary Experiments: Amylase Activity Assays*

The amylase activity of *A. oryzae* was preliminarily tested in SmF. DNS assays were performed for the quantification of reducing sugars. The indirect estimation of the amylase activity of *A. oryzae*, growth on malt medium, paper mill wastewater, and PDB, was tested on standard starch and paper mill wastewater (with and without STD starch).

The first test aimed to assess the ability of *A. oryzae* of growing independently by the presence or absence of starch in the media, testing the flexibility of this microorganism for bioremediation purposes. Therefore, two standard-medium (malt medium and potato dextrose broth—PDB), as well as the paper mill wastewater, were tested for the *Aspergillus* growth. The exoenzymes were purified and the amylase activity was tested on known quantities of standard starch. The results are shown in Figure 2.

quantities of standard starch. The results are shown in Figure 2.

*3.1. Preliminary Experiments: Amylase Activity Assays* 

**Figure 2.** Effect of starch presence (paper mill wastewater and PDB) or absence (malt medium) on the concentration of reducing sugars. Assay of the reducing sugars after the hydrolysis of standard starch conducted with the purified enzymes from *A. oryzae* grown on PMW, PDB, and malt medium. **Figure 2.** Effect of starch presence (paper mill wastewater and PDB) or absence (malt medium) on the concentration of reducing sugars. Assay of the reducing sugars after the hydrolysis of standard starch conducted with the purified enzymes from *A. oryzae* grown on PMW, PDB, and malt medium.

The amylase activity of *A. oryzae* was preliminarily tested in SmF. DNS assays were performed for the quantification of reducing sugars. The indirect estimation of the amylase activity of *A. oryzae*, growth on malt medium, paper mill wastewater, and PDB, was tested on standard starch and paper mill wastewater (with and without STD starch).

The first test aimed to assess the ability of *A. oryzae* of growing independently by the presence or absence of starch in the media, testing the flexibility of this microorganism for bioremediation purposes. Therefore, two standard-medium (malt medium and potato dextrose broth—PDB), as well as the paper mill wastewater, were tested for the *Aspergillus* growth. The exoenzymes were purified and the amylase activity was tested on known

In our experimental design, we decided to exploit the parameter concerning the presence of reducing sugars, resulting from the hydrolytic activity of *Aspergillus* against starch. This parameter directed our experimental choices, allowing us to deduce the growth or not of *A. oryzae* as a function of the metabolic activity we found (specifically, its amylase activity). The industrial needs, which have guided our work, focused our attention on the feasibility of starch bioremediation by *Aspergillus*, justifying amylase activity as a key feature of the study of the process. As reported in Figure 2, the growth of *A. oryzae* seems to be not directly dependent on the presence of starch in the growth medium. Indeed, the quantity of reducing sugars, derived by the amylase activity of *A. oryzae* growth in presence of starch, or in its absence (malt medium), are comparable. However, in paper mill wastewater, the concentration of reducing sugars is higher in less time. In this context, probably, the presence of starch and additives in the paper mill wastewater could enhance the metabolic pathways of the expression of amylase. Moreover, essential for this work, In our experimental design, we decided to exploit the parameter concerning the presence of reducing sugars, resulting from the hydrolytic activity of *Aspergillus* against starch. This parameter directed our experimental choices, allowing us to deduce the growth or not of *A. oryzae* as a function of the metabolic activity we found (specifically, its amylase activity). The industrial needs, which have guided our work, focused our attention on the feasibility of starch bioremediation by *Aspergillus*, justifying amylase activity as a key feature of the study of the process. As reported in Figure 2, the growth of *A. oryzae* seems to be not directly dependent on the presence of starch in the growth medium. Indeed, the quantity of reducing sugars, derived by the amylase activity of *A. oryzae* growth in presence of starch, or in its absence (malt medium), are comparable. However, in paper mill wastewater, the concentration of reducing sugars is higher in less time. In this context, probably, the presence of starch and additives in the paper mill wastewater could enhance the metabolic pathways of the expression of amylase. Moreover, essential for this work, the growth of *Aspergillus* is not hindered by a complex growth medium such as the paper mill wastewater.

the growth of *Aspergillus* is not hindered by a complex growth medium such as the paper mill wastewater. Once the ability of the fungus to grow on paper mill wastewater was tested, as well as its production of amylase in this medium, the hydrolytic capacity of the enzymes pro-Once the ability of the fungus to grow on paper mill wastewater was tested, as well as its production of amylase in this medium, the hydrolytic capacity of the enzymes produced by the fungus was evaluated, not on starch standards, but against the starches present in the paper mill itself. The results are shown in Figure 3. *Fermentation* **2021**, *7*, x FOR PEER REVIEW 7 of 9

**Figure 3.** Reducing sugars from the amylase activity of *A. oryzae* in paper mill wastewater. **Figure 3.** Reducing sugars from the amylase activity of *A. oryzae* in paper mill wastewater.

As reported in Figure 3, *A. oryzae* has proven capable of biodegrading the starches present in the paper mill wastewater. In just 6 h, the amount of reducing sugars found in the medium amounted to 1.6 g/L. As reported in Figure 3, *A. oryzae* has proven capable of biodegrading the starches present in the paper mill wastewater. In just 6 h, the amount of reducing sugars found in the medium amounted to 1.6 g/L.
