from the different experiments were used as substrate. Anaerobic
sludge collected at the wastewater treatment plant of Valladolid
(Spain) was used as inoculum. Anaerobic sludge presented total
solids (TS) concentration of 14.93 ± 1.25 g L1 and a ratio of volatile
solids (VS)/total solids (TS) of around 0.68. Inoculum and substrate
were mixed to achieve a CODt/VS ratio of 0.5 (g g1
). Bottles containing
only anaerobic sludge were run as blanks for quantification
of endogenous methane production. Controls using ethanol as
substrate were run to check the proper performance of the anaerobic
microorganisms. To keep anaerobic conditions, oxygen was
removed from the bottles by purging the headspace with helium.
Then, they were closed with butyl rubber seals and aluminium
caps. The bottles were incubated at 35 C with continuous agitation
at 130 rpm for 35 days. AD for the different conditions were run in
duplicates. The volume of biogas produced by the substrates was
calculated by periodically measuring the pressure of the bottle's
headspace. Methane yields were calculated under standard temperature
and pressure conditions (STP ¼ 0 C and 1 atm).