Fecal Microbiome Cultivation in Anaerobic Bioreactors

Fecal samples (ca. 5 g) collected from three healthy donors (A: 6 year-old, male; B: 10 year-old, female; C: 8 year-old, male) receiving a fully diversified diet were transferred to a tube containing 25 mL of sterile, pre-reduced peptone water (0.1%, pH 7), placed in an anaerobic jar (Anaerojar, Oxoid, Hampshire, England) and immediately delivered to the laboratory. None of the children had been exposed to antibiotic treatment for three months prior to experimentation. Immobilization in 1–2 mm diameter gel beads composed of 2.5% gellan gum, 0.25% xanthan gum and 0.2% sodium citrate (w/v, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) was carried out as described previously [24] (link). The entire process was performed in an anaerobic chamber within 3 h after defecation and 60 ml fresh fecal gel beads from each donor were immediately transferred to inoculum reactors (IR, Sixfors, Infors, Bottmingen, Switzerland) of models A, B and C (IRA IRB and IRC) containing 140 ml nutritive medium (working volume: 200 ml). Beads were colonized for 48 h during batch cultures with conditions of the child proximal colon (T = 37°C; pH 5.7, control with addition of 2.5 N NaOH, continuous flow of pure CO₂ in the reactor headspace). The fermented medium was replaced every 12 h with fresh nutritive medium.