**3. Results and Discussion**

*Aspergillus oryzae* was selected as α-amylase-producer fungal strain for bioremediation pretreatment of starch from the paper mill wastewater. The wastewater used was derived from several processes of the pulp and paper industry. Thus, its characteristics depend on the type of process, type of wood materials, process technology involved, management practices, internal recirculation of the effluent for recovery, and the amount of water to be used in the specific process [21]. Mandal et al. reported the pH, TS, SS, BOD5, COD, and color characteristics of wastewater at various pulp and paper processes [22]. In this work, we aimed to exploit the amylase activity of *A. oryzae* for the pretreatment of starch-containing wastewater. The pretreatment of the wastewater (in this case proposed through a biotechnological process) is necessary as a preliminary step that precedes the generally employed "fluidized bed reactors" bioremediation [23,24]. In particular, the purpose of this work was to evaluate the feasibility of using *Aspergillus oryzae* to eliminate starch from paper mill wastewater, proposing a bioremediation approach as pre-treatment (elimination of starch) of the wastewater given its subsequent further remediation steps. In the experimental design, the stringent operational needs—typical of the industry were considered. To this end, attention was given to the amylase effect of the fungus in the wastewater, more than on the characteristics of the wastewater itself, or the fungal growth (which is, from an industrial point of view, assumed because of the observations of amylase activity). As mentioned, the composition of the wastewater undergoes great fluctuations in its composition due to various variables related to the paper processing processes. Our study, therefore, focused on the evaluation of the capacity of *Aspergillus* in the elimination of starch present in the wastewater. To do this, we decided to embrace the principles of the circular economy, using a production waste such as the rice hull, which, in this context, represented a raw material having a solid support role for the growth of *Aspergillus*, allowing us to obtain biomass through solid-state fermentation processes (SSF). Figure 1 reports the experimental strategy for the study of the pretreatment bioremediation feasibility by *A. oryzae*.

mediation feasibility by *A. oryzae*.

processes. Our study, therefore, focused on the evaluation of the capacity of *Aspergillus* in the elimination of starch present in the wastewater. To do this, we decided to embrace the principles of the circular economy, using a production waste such as the rice hull, which, in this context, represented a raw material having a solid support role for the growth of *Aspergillus*, allowing us to obtain biomass through solid-state fermentation processes (SSF). Figure 1 reports the experimental strategy for the study of the pretreatment biore-

Firstly, the amylase activity of *A. oryzae* was tested with a submerged liquid fermentation technology (SmF). The preliminary steps of *Aspergillus* growth in submerged culture allowed us to test the effect that starch could have on the growth of the fungus and, consequently, on the production of amylase, as evidence of the observed hydrolysis of the starch supplied as a standard substrate. In a controlled context such as that of SmF, we were able to study the effects of the presence or absence of starch. Furthermore, we tested the system starting from a growth medium represented by the paper mill wastewater itself. The observations obtained justified the subsequent experiments allowing us to develop the SSF process. Therefore, solid-state fermentation (SSF) tests were conducted changing the inoculum type. In this experimental design, the dried inoculated rice hull

will act as an additive for the bioremediation of starch in paper mill wastewater.

**Figure 1.** Draw chart of the experimental workflow of this study.

**Figure 1.** Draw chart of the experimental workflow of this study. Firstly, the amylase activity of *A. oryzae* was tested with a submerged liquid fermentation technology (SmF). The preliminary steps of *Aspergillus* growth in submerged culture allowed us to test the effect that starch could have on the growth of the fungus and, consequently, on the production of amylase, as evidence of the observed hydrolysis of the starch supplied as a standard substrate. In a controlled context such as that of SmF, we were able to study the effects of the presence or absence of starch. Furthermore, we tested the system starting from a growth medium represented by the paper mill wastewater itself. The observations obtained justified the subsequent experiments allowing us to develop the SSF process. Therefore, solid-state fermentation (SSF) tests were conducted changing the inoculum type. In this experimental design, the dried inoculated rice hull will act as an additive for the bioremediation of starch in paper mill wastewater.
