In biogas production through a single-stage AD process, the CH4 formation takes away a significant
portion of the reactants, acetate and H2, which are produced by “H2-producing bacteria” and
simultaneously consumed by “H2-consuming bacteria”. In contrast, a two-stage AD produces H2 and
carbon dioxide (CO2) in the first stage, whereas the second stage produces CH4 and CO2. Nevertheless, to
increase the conservation potential in a two-stage digester, a pre-treatment to the feedstock and seed
should be applied. The pre-treatment should selectively inhibit methanogenic bacteria and increase the
production of acetic acid and acetate, thus achieve the highest possible H2 yield [4].