Rumen bacterium R-25, which was isolated from the rumen of sheep and classified in the group U2 (Koike et al., 2010), was used in this study. Fibrobacter succinogenes S85, which is a fibrolytic bacterium, was purchased from American Type Culture Collection. Selenomonas ruminantium S137, which was isolated from sheep rumen (Sawanon et al., 2011), was used as a metabolite (lactate and succinate) utilizer. Basal medium was prepared anaerobically according to the method of Bryant (1972) to have the following composition (100 mL−1):
7.5 mL of mineral GSK2126458 mw solutions I and II (Bryant & Burkey, 1953), 0.1 mL of 0.1% resazurin, 40 mL of clarified rumen fluid, 39 mL of distilled water, 1 mL of 5% l-cysteine-HCl·H2O, and 5 mL of 8% Na2CO3. Rice straw was air-dried in an oven at 60 °C, ground to pass through a 1-mm sieve, and used as a substrate to measure fiber digestion. Strain R-25 and S. ruminantium S137 were grown to the end of log phase at 39 °C in basal medium containing cellobiose and glucose [0.5% (w/v) each] as carbon sources. After three passages, the cultures were centrifuged
(2300 g, 4 °C, 10 min) to pellet the bacteria. Anaerobic dilution solution (Bryant & Burkey, 1953) was added to the pellet to resuspend the bacteria to an optical density at 660 nm (OD660 nm) of 0.2. Fibrobacter succinogenes S85 was grown for 48 h in basal medium containing 1.0% (w/v) rice straw as the sole carbon source. After three passages, the culture was centrifuged (377 g, 4 °C, 1 min) to separate the rice straw particles and supernatant Sirolimus concentration containing bacterial cells (Minato & Suto, 1978). The supernatant was collected in another sterile tube and centrifuged (2300 g, 4 °C, 10 min) to pellet the bacteria. The pellet was resuspended as above, and these OD-adjusted cell suspensions were used as inocula. Each inoculum was added at 0.1 mL to 10 mL of basal medium containing 0.1 g of rice straw as the sole carbon source. Six test tubes were prepared for respective mono- and cocultures and incubated at 39 °C under anaerobic condition. On the basis of a previous
study (Shinkai et al., Obatoclax Mesylate (GX15-070) 2009), samples were collected at three time points after incubation; 0 h (corresponding to inocula), 48 h (middle of digestion) and 96 h (endpoint of digestion). Samples were collected from three of six test tubes at 48 h, and the rest of three test tubes were incubated until 96 h. Tubes with no inocula were prepared as a blank and treated in the same manner. After 96 h incubation, the cultures were cooled on ice for 30 min to detach bacterial cells from fiber particles (Minato & Suto, 1978) and centrifuged (377 g, 4 °C, 10 min), and the supernatant containing bacterial cells was collected. The residue was washed with 10 mL of 0.1 M potassium phosphate buffer and re-centrifuged (2300 g, 4 °C, 10 min). The washed residue was dried at 105 °C for 48 h and weighed to calculate dry matter (DM) digestion.