Mgso4 7h2o

The composition of the aqueous phase in all experiments was either luria broth or as described by Wubbolts et al30 (link). KH₂PO₄, 4 g/L; K₂HPO₄ (3H₂O), 15.9 g/L; Na₂HPO₄ (12H₂O), 7 g/L; (NH₄)₂SO₄, 1.2 g/L; NH₄Cl, 0.2 g/L (all from Sigma Aldrich); yeast extract (Oxoid), 5 g/L; L-leucine, 0.6 g/L; L-proline, 0.6 g/L; thiamine, 5 mg/L (All from Alfa Aesar). After autoclaving, MgSO₄ (7H₂O) (BDH), 1 g/L (BDH); 1 mL of trace minerals (composition below); 1 mL of 4% (w/v) CaCl₂(2H₂O) (Alfa Aesar) and 10 g/L D-glucose (Sigma Aldrich) were added having all been heat sterilized separately. 1 ml of 10 mg/ml filter sterilised tetracycline (Sigma Aldrich) was also added.
The solution of trace minerals contained per liter of 5 M HCL: FeSO₄ (7H₂O) (Sigma Aldrich), 40 g; MnSO₄ (H₂O) (Sigma Aldrich), 10 g; CoCl₂ (6H₂O) (Fluka), 4.75 g; ZnSO₄ (7H₂O) (VWR), 2 g; MoO₄Na₂ (2H₂O) (Sigma Aldrich), 2 g; CuCl₂, (2H₂O) (Riedel-de Haen), 1 g; H₃BO₃ (Sigma Aldrich), 0.50 g.

Bacterial strains used in this study are listed in Table 1, and in Petrovski et al. [29 (link)], and methods for their storage and cultivation were those described by Petrovski et al. [29 (link)]. These were all grown on R2A medium (0.5 g/L Yeast extract (Oxoid, Adelaide, Australia), 0.5 g/L Proteose peptone (Difco, North Ryde, Australia), 0.5 g/L Casamino acid (Difco, North Ryde, Australia), 0.5 g/L Glucose, 0.5 g/L soluble starch (Difco, North Ryde, Australia), 0.3 g/L K2HPO4, 0.005 g/L MgSO4.7H2O, 0.3 g/L sodium pyruvate (BDH, Murarrie, Australia)) broth and agar R2A + 14 g/L agar (Oxoid, Adelaide, Australia) at 25 C. All remaining chemicals were obtained from Sigma (Sydney, Australia), unless otherwise noted.

Anaerobic (N₂-sparged) batch fermentations were performed in triplicate using 10% fecal slurry (1% fecal inoculum) under controlled conditions [water-jacket vessels (Soham Scientific, Soham, United Kingdom), pH 6.8, temperature 37°C]. Basal medium contained per liter: 2 g peptone (Oxoid, Basingstoke, United Kingdom), 2 g yeast extract (Oxoid), 0.1 g NaCl (Fisher Scientific, Fair Lawn, NJ, United States), 0.04 g K₂HPO₄ (BDH, Toronto, ON, Canada), 0.04 g KH₂PO₄ (BDH), 0.01 g MgSO₄7H₂O (BDH), 0.01 g CaCl₂6H₂O (Honeywell, Morris Plains, NY, United States), 2 g NaHCO₃ (Oxoid), 2 mL Tween 80 (Sigma–Aldrich, Oakville, ON, Canada), 0.05 g Hemin (Sigma–Aldrich) dissolved in 1 mL of 4 M NaOH (Fisher Scientific), 10 μL Vitamin K (Sigma–Aldrich), 0.5 g l-Cysteine HCL (Sigma–Aldrich), 0.5 g Bile Salts (Oxoid), and 4 mL of Resazurin (Sigma–Aldrich) (0.025 g/100 mL). Vessels were dosed with the substrates (1% wt/vol) after simulated in vitro upper gastrointestinal digestion and dialysis (Mandalari et al., 2008 (link)) and inoculated with 10% fecal slurry. The final volume of each culture was 200 mL. Samples were harvested at time points 0 (immediately after incubation) and 24 h.