*3.2. Solid-State Fermentation (SSF)*

in Table 1.

development.

*3.2. Solid-State Fermentation (SSF)*  The physiological and genetic properties of the microorganisms could make SSF advantageous against SmF biotechnology [13]. Thus, we tested the SSF of *A. oryzae* starting with the choice of the right inoculum strategy. Furthermore, the SSF is the best strategy of The physiological and genetic properties of the microorganisms could make SSF advantageous against SmF biotechnology [13]. Thus, we tested the SSF of *A. oryzae* starting with the choice of the right inoculum strategy. Furthermore, the SSF is the best strategy of bioremediation from an industrial point of view.

with the fungus growth on Petri dishes. To assay the effectiveness of the respective inocula, both suspensions, derived from SmF and Petri dishes, were collected for the CFU

**Table 1.** CFU/g of rice hull for several incubation times. Inoculum strategies tested: SmF and Petri

The two inocula were used in the SSF of the *A. oryzae* rice hull. Several incubation times, 3 to 10 days, were tested. The results concerning the CFU/g of rice hull are shown

> 3 8 × 105 ± 0.03 3 × 104 ± 0.04 5 3 × 106 ± 0.05 5 × 104 ± 0.05 7 4 × 107 ± 0.04 5 × 105 ± 0.03 10 4 × 108 ± 0.03 5 × 106 ± 0.02

As reported in Table 1, the most interesting inoculation method, at 10 days, is the inoculation from SmF. Although the SmF inoculum has fewer initial CFUs, it proved to be the best on analysis after 10 days. This is due to the presence of active fungi in the SmF inoculum, rather than the Petri dish inoculum. In fact, in the latter, only spores of the fungus are present, which typically need more time for the regeneration of the metabolically active fungus [25]. Furthermore, this is an advantage from the industrial point of view, where the management of a liquid inoculum does not determine obstacles in process

In the industrial management of the solid-state fermented product for bioremediation purposes, its use in dry form is interesting. Although the normal moisture content of the SSF is 80%, for the storage and use of *Aspergillus* on rice hulls in an industrial context the dry form is the most advantageous in terms of process management. Therefore, we tested the capacity of *Aspergillus oryzae* of producing amylase in paper mill wastewater

**SmF Petri Dishes** 

quantification. The CFU/mL were, respectively, 3 × 106 and 3 × 104.

dishes. The results are shown as average, and the standard deviation is shown.

**Days CFU/g rice hull**

bioremediation from an industrial point of view.

The solid rice hull was inoculated with the supernatant of *A. oryzae* growth in SmF (paper mill wastewater) compared to the inoculum of physiological solution in contact with the fungus growth on Petri dishes. To assay the effectiveness of the respective inocula, both suspensions, derived from SmF and Petri dishes, were collected for the CFU quantification. The CFU/mL were, respectively, 3 <sup>×</sup> <sup>10</sup><sup>6</sup> and 3 <sup>×</sup> <sup>10</sup><sup>4</sup> .

The two inocula were used in the SSF of the *A. oryzae* rice hull. Several incubation times, 3 to 10 days, were tested. The results concerning the CFU/g of rice hull are shown in Table 1.

**Table 1.** CFU/g of rice hull for several incubation times. Inoculum strategies tested: SmF and Petri dishes. The results are shown as average, and the standard deviation is shown.


As reported in Table 1, the most interesting inoculation method, at 10 days, is the inoculation from SmF. Although the SmF inoculum has fewer initial CFUs, it proved to be the best on analysis after 10 days. This is due to the presence of active fungi in the SmF inoculum, rather than the Petri dish inoculum. In fact, in the latter, only spores of the fungus are present, which typically need more time for the regeneration of the metabolically active fungus [25]. Furthermore, this is an advantage from the industrial point of view, where the management of a liquid inoculum does not determine obstacles in process development.

In the industrial management of the solid-state fermented product for bioremediation purposes, its use in dry form is interesting. Although the normal moisture content of the SSF is 80%, for the storage and use of *Aspergillus* on rice hulls in an industrial context the dry form is the most advantageous in terms of process management. Therefore, we tested the capacity of *Aspergillus oryzae* of producing amylase in paper mill wastewater also after a drying step. The test involved the quantification of reducing sugars as evidence of the amylase activity. In 6 h, 1.3 ± 0.2 g/L of reducing sugars were found as proof of maintained amylase activity. *Fermentation* **2021**, *7*, x FOR PEER REVIEW 8 of 9 also after a drying step. The test involved the quantification of reducing sugars as evidence of the amylase activity. In 6 h, 1.3 ± 0.2 g/L of reducing sugars were found as proof of maintained amylase activity.

> Finally, the SSF was tested in paper mill wastewater. The reducing sugars concentration was assessed after 72 h of treatment. The results are shown in Figure 4. Finally, the SSF was tested in paper mill wastewater. The reducing sugars concentration was assessed after 72 h of treatment. The results are shown in Figure 4.

strates.

**4. Conclusions** 

of the manuscript.

**Figure 4.** Effect of *A. oryzae* on the starch bioremediation of paper mill wastewater (72 h of incubation). C–, untreated PMW; C+, PMW treated with amylase STD. **Figure 4.** Effect of *A. oryzae* on the starch bioremediation of paper mill wastewater (72 h of incubation). C–, untreated PMW; C+, PMW treated with amylase STD.

sugars were found (except for those naturally present in the paper mill wastewater). On the contrary, reducing sugars were found when commercial amylase was added to the wastewater (as reported with positive control). This testifies that, in the PMW, starch is present and can be a substrate of the commercial amylase provided, validating the experimental design. If *A. oryzae* is supplied to the wastewater, it produces amylase which allows it to use the reducing sugars obtained from the hydrolysis of starch as a carbon source for its cellular metabolism. To evaluate this observation, the last column refers to the treatment of paper mill wastewater with *Aspergillus* and commercial amylase. The absence of reducing sugars supports that these are exploited by *Aspergillus* as metabolic sub-

In the results shown in Figure 4, the negative control refers to untreated paper mill

*Aspergillus oryzae* is a fungal strain widely exploited as an amylase producer. In this work, we aimed to study and test this fungus for the bioremediation of starch in industrial paper mill wastewater. For this purpose, submerged fermentation technologies (SmF) and solid-state fermentation (SSF) were studied. *A. oryzae* was found to grow on non-conventional media such as the paper mill wastewater. The SSF of *A. oryzae* was performed on rice hulls. In the bioremediation (as pretreatment) of paper mill wastewater, to remove starch, the fungus maintains its amylase activity and uses reducing sugars as metabolic substrates. This study opens new perspectives for the bioremediation of industrial efflu-

**Author Contributions:** Conceptualization, S.C. and E.T.; methodology, S.C.; validation, S.C., E.T. and R.B.; investigation, S.C, F.Z. and D.S.; writing—original draft preparation, F.Z. and E.T.; writing—review and editing, F.Z. and E.T. All authors have read and agreed to the published version

ents such as pulp-and-paper mill wastewater using *A. oryzae*.

**Funding:** This research article was funded by FLAG 2019. **Institutional Review Board Statement:** Not applicable.

In the results shown in Figure 4, the negative control refers to untreated paper mill wastewater. As expected, no reducing sugars were found. Indeed, in this context, the absence of *A. oryzae* and the following lack of amylase in the PMW suggest that no reducing sugars were found (except for those naturally present in the paper mill wastewater). On the contrary, reducing sugars were found when commercial amylase was added to the wastewater (as reported with positive control). This testifies that, in the PMW, starch is present and can be a substrate of the commercial amylase provided, validating the experimental design. If *A. oryzae* is supplied to the wastewater, it produces amylase which allows it to use the reducing sugars obtained from the hydrolysis of starch as a carbon source for its cellular metabolism. To evaluate this observation, the last column refers to the treatment of paper mill wastewater with *Aspergillus* and commercial amylase. The absence of reducing sugars supports that these are exploited by *Aspergillus* as metabolic substrates.
