Acetate

To maintain
anaerobic conditions, inocula were seeded inside a glovebox (MBRAUN
MB200, Germany) with a N₂ atmosphere. After sieving to
remove large particles, inocula were diluted with BAm at 20:80 (v/v)
inside 118 mL serum glass bottles with 40 mL working volume and incubated
in the dark at 42 ± 1 °C. Subsequent re-inocula every 21
days were defined as cultivation generations. Each generation was
re-inoculated with stepwise increasing concentrations of ammonia,
in the form of NH₄Cl (Sigma-Aldrich, St. Louis, MO, U.S.A.).
Starting from the second generation, casein (from bovine milk, Sigma-Aldrich)
was used as the main carbon source (Table S1 of the Supporting Information) in the F/P2 reactors. Glucose (Carlo
Erba Reagents, Italy) and acetate (acetic acid glacial, Carlo Erba
Reagents) were also tested (Table S1 of
the Supporting Information). Yeast extract (YE, 26 Oxoid Limited,
London, U.K.) and vitamin solution were used to sustain microbial
growth. In the re-inoculation process, biogas production and pH were
measured and samples were collected and immediately frozen at −20
°C. Starting from the 10th generation, casein-fed cultures were
re-inoculated at two different NH₄Cl concentrations, with
2 g/L difference among them (Table S2 of
the Supporting Information), and grown independently. The adaptation
reached the 27th generation, corresponding to around 19.8 and 20.3
calculated g of NH₄⁺ N/L.

All chemicals were of analytical reagent grade, and deionized water was used for the preparation of all solutions throughout the experiment. A stock standard solution of iron (II) at 1000 mg L⁻¹ was prepared by dissolving 0.7022 g of ammonium ferrous sulfate hexahydrate (Sigma-Aldrich, England) in water containing 1.0% (v/v) concentrated sulfuric acid (BDH, England); then, the volume was adjusted to 100 mL. The stock standard solution of iron (III) at 1000 mg L⁻¹ was prepared in the same way with 0.1 g of ferric chloride (BDH, England). A series of working standard solutions were prepared daily by diluting the stock standard solution to the desired concentrations. Acetate buffer (0.2 M, 500 mL) pH 4.8 was prepared by dissolving 7.21 g of sodium Acetate trihydrate (Carlo Erba, Italy) in water containing 2.72 mL of acetic acid (Carlo Erba, Italy).