3.3. BMP Experiments

BMP experiments were carried out in duplicate at 35 °C in serum bottles of 160 mL, using a working volume of 100 mL. The experiments were performed according to the modified protocol of Owen and Chynoweth [33] using as inoculum sludge from the anaerobic digester of the Patras wastewater treatment plant, treating municipal sewage sludge and operating at steady state at an hydraulic retention time (HRT) of 15 d. The main characteristics of the sludge were: pH: 8.13, total chemical oxygen demand (T.COD): 25.4 g/L, dissolved COD (d.COD): 0.81 g/L, total suspended solids (TSS): 23.03 g/L and volatile suspended solids (VSS): 14.13 g/L.

BMP tests were performed either at the whole slurry or at the separated fractions, obtained after pretreatment. For the experiments with the whole slurry, 20 mL mixed anaerobic culture, 76 mL water and 4 mL of the whole slurry at a solid loading of 5% w/v, were used. For the experiments with the solids obtained after pretreatment, 20 mL mixed anaerobic culture, 80 mL water and appropriate amounts of samples were added, in order to acquire the desirable TS content of 2g TS/L. For the experiments with the hydolysates, 20 mL mixed anaerobic culture were seeded with water and appropriate volumes of hydrolysates, so as their final COD concentration, being 2 g/L. For all experiments, the microbial culture was supplemented with 10 mL/L of a (NH₄)₂HPO₄ (7.21 g/L) solution, 10 mL/L of a FeSO₄·7H₂O (0.7g/L) solution and 10 mL/L of a trace metals solution [34]. Control experiments for checking the methanogenic biomass activity using glucose and cellulose, as well as blank experiments in order to determine the background gas productivity of the inoculum, were also carried out. The content of the vials was gassed with a mixture of N₂/CO₂ (80/20) in order to secure anaerobic conditions. The vials were sealed with butyl rubber stoppers and aluminum crimps and methane production was monitored as a function of time according to Owen and Chynoweth [33].