**3. Results and Discussion**

*3.1. Removal of Organic Matter*

The evolution of sCOD concentration during the fungal treatments is shown in Figure 1.

The initial sCOD value in the AL effluent was 4854 mg/L, which increased to 9243 mg/L after glucose addition. For C1 and C2 test, which were carried out without fungal inoculation, minor sCOD removals were observed. This degradation was carried out by the endogenous microbiota present in the effluent. With respect to test C1, no change was observed during the first two days and a 27% elimination of sCOD was achieved after four days of incubation. Afterwards, the sCOD value remained almost constant, reaching a final sCOD degradation of 30%. In the case of C2, where glucose where added, sCOD

degradation did not occur until the 4th day. The final percentage of sCOD removal was similar to that achieved with C1. However, it is necessary to point out that in C2 the final sCOD concentration was higher than the sCOD of AL before being supplemented with glucose. Therefore, the endogenous microorganisms were not able to assimilate even the sCOD provided by glucose added.

**Figure 1.** Changes in sCOD concentration during biological treatment. The dashed lines shown the non-inoculated tests C1 (-) and C2 (-), used as controls. The solid lines shown the inoculated tests E1 (•) and E2 (•). The standard deviation (SD) of the experimental data were in all cases less than 6.5% of mean value.

The addition of *P. chrysosporium* in E1 and E2, caused a fast decrease in sCOD from the beginning of the treatment, obtaining sCOD removals of 51% and 59%, respectively, after 10 days of treatment. In the experiment E1, carried out without the addition of glucose, 61% of the initial sCOD was degraded in only 4 days of treatment, which duplicate the degradation efficiency reached in C1 by endogenous microorganisms. Thus, the fungal inoculation gave an average rate of sCOD degradation of 0.51 mg/(L min) during the first 4 days, whereas the average rate in C1 was only 0.22 mg/(L min). Regarding test E2, again higher sCOD removals were obtained compared with the non-inoculated test C2. As in E1, the inoculation of the fungus duplicated the sCOD removal rate, which increased up to 0.61 mg/(L min) during first 8 days, whereas in C2 it was only of 0.27 mg/(L min).

To estimate the fungus growth, the TSS at the beginning of the experiments and after 10 days were measured. Data are shown in Table 2.


**Table 2.** TSS for control (C1 and C2) and inoculated (E1 and E2) tests at initial and final times of the fungal treatment.

As can be seen, in the control tests (C1 and C2), the supplementation with glucose increased the growth of the endogenous microbiota. Moreover, in the inoculated tests (E1 and E2), the TSS increases were higher than in the controls, which can be explained by the fungus growth. Comparing the increase in TSS observed in controls and inoculated tests, it can be estimated that fungus growth was similar in E1 and E2, around 0.6–0.7 g/L (dry matter), which is in agreement with the fact that final sCOD removals were also similar. Therefore, in this case, the addition of glucose was not effective for the AL treatment.

Results for sCOD removals here obtained were higher than have been previously reported. Aloui et al. [33], reported that a 44% of COD removal was achieved by a solid-state fermentation of AL using *P. chrysosporium* in a support of sugarcane bagasse. Ahmadi et al. [26] achieved a COD degradation around 50% using this fungus immobilized on loofah. Nogueira et al. [34] reported COD removals efficiencies lower than 44% for *P. chrysosporium* for an OMW pre-treat by photocatalytic oxidation.

The initial concentration of sBOD5 was 408 mg/L, with a biodegradability index of 0.08 (See Figure 2), which means that AL effluent has very low biodegradability. In the experiments C2 and E2, the initial biodegradability index (BI) was higher (0.13), as a consequence of the glucose addition.

**Figure 2.** Changes in sBOD5 concentration and biodegradability index for the different experiments at initial, intermediate, and final time of the treatment. Bars corresponds with sBOD5 concentration for non-inoculated tests C1 (-) and C2 (-), and inoculated tests E1 (-) and E2 (-). The dashed lines shown the biodegradability index (BI) for non-inoculated tests C1 (-) and C2 (-), used as controls, whereas the solid lines represent the inoculated tests E1 (•) and E2 (•).

In all cases, the sBOD5 concentration decreased throughout the fungal treatment, with final values lower than 100 mg/L. Moreover, the BI decreases with the treatment since biodegradable matter was consumed. The *P. chrysosporium* inoculated in E1 and E2 released enzymes able to break down recalcitrant organic matter into compounds more biodegradable. However, the fungus, as well as the endogenous microorganisms, consumed these compounds as they were produced, reducing the sCOD, the sBOD5, and the BI. Regardless, the addition of the fungus gave final BI higher than in the controls, even though it was low. If the enhancing of biodegradability were the objective, for example, as the previous step for the biomethanization process, an alternative could be to directly use the enzymes produced by the fungus instead of inoculating the fungus strain. In this way, the recalcitrant compounds present in the AL effluent could be broken down without the fungus using this organic matter as a nutrient source [35,36]. A sterilisation process may also be necessary to inactivate the endogenous microflora.

The evolution of reducing carbohydrates has been also measured, and results are shown in Figure 3. The reducing sugar concentration of the initial sample was 563 mg/L, and the ratio sBOD5/reducing sugars was 0.7, indicating that a great part of the sBOD5 measured is due to the reducing sugars. As expected, in the samples supplemented with 3 g/L of glucose, the initial concentration increased until 3662 mg/L. For the noninoculated sample C1, the amount of reducing sugars remained practically stable during the treatment. In contrast, the inoculated samples (E1 and E2) showed a significant decrease in the reducing sugars concentration, with final values of 176 and 140 mg/L, respectively. The initial reducing sugars concentration dropped abruptly in the experiments supplemented with glucose, especially in the one that had been inoculated with the fungus. In this sense, all the glucose that was practically added to test E2 was consumed during the first 24 h, whereas in the supplemented control (C2), the amount of reducing sugars dropped from

3663 mg/L to 1772 mg/L in 24 h, and afterwards remained almost constant, indicating that the endogenous microbiota was not able to degrade all the glucose added. Although the enzymatic activities of the fungus were not measured in this study, the literature has widely reported that the addition of glucose favours the synthesis of fungal enzymes, which in turn are related to the elimination of colour, COD and recalcitrant compounds [37–40]. This fact was reflected in E2, which showed a rapid degradation of reducing sugars, whereas sCOD removal was slower. Probably the fungus decomposed recalcitrant compounds that increased sCOD and, simultaneously, consumed them.

**Figure 3.** Changes in reducing sugars concentration during biological treatment. The dashed lines shown the non-inoculated tests C1 (-) and C2 (-), used as controls. The solid lines shown the inoculated tests E1 (•) and E2 (•). The standard deviation (SD) of the experimental data were in all cases less than 11% of mean value.
