*2.2. Fungal Pellet Obtention*

The white-rot fungus, *Phanerochaete chrysosporium* Burdsall 1974 was used. The freezedried strain (CECT 2798 from Spanish Type Culture Collection) was recovered in aseptic conditions by adding 100 μL of the resuspended fungus to 10 mL of malt extract (ME). Then, a Petri plate of 1.5% malt extract agar (MEA) was inoculated with 100 μL of this suspension and incubated at 26 ◦C for 6 days. Two subcultures of the fungus were necessary before use in the biological treatment. Fungal subcultures were routinely made every month to conserve the strain.

The methodology described by Díaz et al. [28] was followed to obtain the fungus pellets. To this aim, five cylinders of 1 cm diameter from the growing zone of inoculated plates were used to inoculate 500 mL Erlenmeyer flasks containing 150 mL of sterilised malt extract broth (VWR Chemicals BDH), with a pH between 4.5 and 5. The inoculated flasks were incubated at 26 ◦C and 135 rpm for 6 days. The fungal mycelial obtained after this process was separated with a sieve and homogenized with 0.8% NaCl (*w*/*v*) in a ratio of 1:3 (*w*/*v*). An amount of 600 μL of resulting suspension was used to inoculatea1L Erlenmeyer flask with 250 mL of sterilised ME. Finally, the inoculated ME was incubated at 26 ◦C and 135 rpm for 6 days. After that time, pellets were obtained, removed with a sieve, and preserved in 0.8% NaCl (*w*/*v*) solution at 4 ◦C until use.

#### *2.3. Fungal Treatment*

Several batch tests were carried out to treat diluted AL with *P. chrysosporium*. All the experiments were performed using 1 L Erlenmeyer flask with 250 mL of AL effluent.


The flasks were incubated at 26 ◦C and in an orbital shaking (150 rpm) for 10 days. During the treatment, the pH values were maintained within the range 5–7 by adding NaOH 0.5 M or HCl 0.5 M to ensure the optimal range for the fungus enzymatic system. Samples taken periodically were centrifuged at 15,000× *g* for 15 min and the supernatant

were conserved at 4 ◦C until analysed. The experiments were carried out in duplicate. Data shown in Results and Discussion section are the average values of both experiments. In all cases, standard deviations were lower than 15% with respect to average value.
