*2.1. Biochemical Methane Potential (BMP) of Olive Mill Wastewater*

Experimental trials have been conducted at the laboratory scale to assess biochemical methane potential (BMP) of olive wastewater through anaerobic codigestion process.

#### 2.1.1. Anaerobic Codigestion Experiments

Olive mill wastewater (OMWW) was withdrawn during the 2020/2021 campaign from a private mill situated in the Province of Reggio Calabria (Southern Italy, 38◦23 28.70 N; 16◦04 31.10 E), which implements a three-phase extraction system. Digestate (Dig) was withdrawn from a biogas production plant situated in the same province and which already implements olive mill by-products among the feedstock.

Biochemical methane potential (BMP) tests were performed using 2000 mL DURAN® GL 45 laboratory glass bottles as reactors. These were later half-filled with mixtures containing 0% (blank), 20% and 30% (*v*/*v*) olive mill wastewater, the remaining content consisted in the digestate. The experimental design is reported in Table 2. Batch reactors were sealed and connected hermetically to the gasbags for biogas sampling. Each thesis was performed in triplicate. Once filled and before sealing, batch reactors were blown through with pure nitrogen (N2) to remove atmospheric air at the beginning of the fermentation and favor anaerobic conditions. Then, they were incubated for 30 days at 37 ◦C in a laboratory forced air oven (AgroLab, Italy, TCF 200) to guarantee mesophilic conditions, as shown in Figure 2.

**Table 2.** Experimental setup of biochemical methane potential (BMP) tests. Source: Our elaboration.


**Figure 2.** Biochemical methane potential (BMP) tests of olive mill wastewater under mesophilic conditions (37 ◦C). Source: Picture acquired in our own laboratory.

#### 2.1.2. Substrate and Matrix Characterization

Before each experiment, physical-chemical features of the employed matrices (olive mill wastewater and digestate) as well as the considered mixtures were characterized. This included pH using pH probe (Cryson, GLP 21+), dry content (DC) (%) at 105 ◦C using a moister analyzer (Ohaus, MB120) and volatile solids VS (%) after ignition at 550 ◦C using a muffle furnace (Fabber, FBL 70) [24]. Chemical oxygen demand (COD) (g.L<sup>−</sup>1) was measured using a bench photometer after reaction with Hanna high rate COD reagents. In addition, total polyphenols (PPs) were measured for OMWW according to Folin Ciocalteu method [25], total carbon (TC), total nitrogen (TN) and C/N ratio were determined with an elemental analyzer (Leco, CN628).
