Inoculum, nutrient medium, and enrichment setup

Duplicate glass bottles (325 mL total volume each) were used for enriching five different cultures. Activated sludge from a municipal wastewater treatment plant (1 mL) was added to the bottles as inoculum. The bottles were filled up to 250 mL with a nutrient medium as described by Marshall, et al.²⁶. The goal was to enrich hydrogenotrophic cultures performing methanogenesis, acetogenesis, sulfate reduction, nitrate reduction, as well as an acetate-oxidizing methanogenic culture. To accomplish this, the nutrient medium was amended with different electron acceptors and in some cases 2-bromoethanesulfonate to inhibit methanogens, as described in Table ^S1. The bottles were sealed with rubber caps and the head space (70 mL) was sparged with Ar/CO₂ gas (85%/15%) to remove oxygen. Then, the head space of the hydrogenotrophic bottles were filled with pure hydrogen gas at an overpressure of 160–180 kPa. During sampling, the amount of liquid that was extracted from the enrichment cultures was replaced by fresh medium. Every 3 weeks, fresh medium (20 mL) containing 20 mM NaSO₄, NaNO₃, and sodium acetate was added to the sulfate-reducing, denitrifiers and acetate-oxidizing enrichments. Sterile syringes (12 mL) and needles were used for collecting samples, sparging of gas, and addition of fresh medium for each enrichment.