4 M ammonium chloride. The cultures were incubated http://www.selleckchem.com/products/cx-5461.html with sodium acetate (0.05 M) as the sole carbon and energy source. After depletion, new sodium acetate was added on two further occasions. Culture material was then transferred to fresh modified BM (20% v/v) supplemented with ammonium chloride and the cultures were again repeatedly fed with sodium acetate. After depletion of 0.15 M acetate in total, the cultures were again diluted in a new medium (10% v/v). Finally, after degradation of repeated additions of acetate, the cultures were serial-diluted

in agar (agar shakes). The agar used was washed four to five times in distilled water and added during the preparation of the modified BM to a final concentration of 20 g L−1. The agar medium was not supplemented with extra ammonium chloride, as this had a negative impact on the solidification properties of the agar. Four milliliters of agar solution were distributed into glass tubes (28 mL) in which anoxic conditions were maintained by flushing with N2/CO2 (80/20 v/v) and the tubes were sealed before autoclaving. After Panobinostat sterilization, the agar shakes were allowed to cool to 42 °C and the medium was

supplemented with solutions C1 and C2 and one of the following compounds as a substrate: formate (20 mM), ethylene glycol, fructose, glucose (10 mM), syringate or vanillate (3 mM). The tubes were then incubated until colonies appeared. Single colonies were withdrawn by a syringe and directly Digestive enzyme transferred and diluted in new agar shakes. The syntrophic acetate-oxidizing ability of strain Sp3T was determined by cocultivation with a hydrogen-utilizing

methanogen, Methanoculleus sp. strain MAB1 (Schnürer et al., 1994). Modified BM (225 mL) supplemented with ammonium chloride (0.2 M), sodium acetate (3 mM) and a plastic carrier (8% w/v, 10 mm Ø, AnoxKaldnes AB) was dispensed in vials (500 mL) under flushing with N2/CO2 (80/20 v/v). The vials were closed with butyl rubber stoppers and aluminum caps and autoclaved. After addition of the C1 and C2 solutions, the media were inoculated with the methanogen and finally H2/CO2 (80/20 v/v; 0.8 atm) was added as an electron and carbon source. After growth of the methanogen and depletion of added hydrogen, the medium was complemented with 0.05 M sodium acetate and the cultures were inoculated with the isolate Sp3T. Methane production was monitored using a Clarus 500 gas chromatograph equipped with a 7′ HayeSep N 60/80, 1/8″ SF column and an FID detector. Helium was used as the carrier gas, at a flow rate of 31 mL min−1. The column and the detector were operated at 60 and 250 °C, respectively, and injection was carried out at 40 °C. Acetate was quantified using HPLC analysis. The HPLC (Aligent 1100) was equipped with an ion exchange column (Rezex-ROA-Organic Acid H+) and a refractive index detector.