Enrichment, isolation and cultivation
A prereduced medium was used for enrichment cultures and cultivation of isolated strains. The medium contained (per liter deionized water): K2HPO4, 1.5 g; KH2PO4, 3 g; MgSO4 × 7 H2O, 0.3 g; CaCO3 × 2 H2O, 0.05 g; NH4Cl, 1.0 g; NaCl, 0.5 g; NaHCO3, 0.5 g; NiCl2 × 6 H2O, 2 mg; FeSO4 × 7 H2O, 1 mg; NH4Fe(III) citrate, 10 mg; MnSO4 × H2O, 5 mg; CoCl2 × 6 H2O, 1 mg; ZnSO4 × 7 H2O, 1 mg; CuSO4 × 5 H2O, 0.1 mg; H3BO4, 0.1 mg; Na2MoO4 × 2 H2O, 0.1 mg; Na2SeO3 × 5 H2O, 0.2 mg; Na2WoO4 × 2 H2O, 0.1 mg; nicotinic acid, 2 mg; cyanocobalamin, 0.25 mg; p-aminobenzoic acid, 0.25 mg; calcium pantothenate, 0.25 mg; thiamine-hydrochloride, 0.25 mg; riboflavin, 0.25 mg; lipoic acid, 0.25 mg; folic acid, 0.1 mg; biotin, 0.1 mg; pyridoxine-hydrochloride, 0.1 mg; yeast extract (Difco), 0.5 g; resazurin, 0.5 mg; Na2S × 9 H2O, 0.75 g.
The medium was prepared under anaerobic conditions under O2-free N2 and pH was adjusted to 7.2. The medium was dispensed into flasks and Hungate tubes under N2 and autoclaved.
Enrichment medium was inoculated with collected samples and incubated at 72°C in 50 ml flasks with 30 ml medium containing 4.3 g/l of cellulose (strips of filter paper Whatman No.1) and 20 g/l of untreated ground beech wood as substrate.
Cellulolytic strains were isolated by serial dilutions of single-cell colonies in Hungate roll tubes  with 30 g/l agar and 5 g/l acid-swollen amorphous cellulose. Non-cellulolytic strains were isolated by repeated serial dilutions in liquid medium with 5 g/l glucose.
Isolated strains were cultivated at 72°C in Hungate tubes or flasks with filter paper, microcrystalline cellulose (Avicel PH-101, Fluka), cellobiose, glucose, xylan from beech wood, xylose, washed or unwashed dilute sulfurous acid steam-explosion-pretreated lignocellulosic materials (poplar, spruce, miscanthus, wheat straw, whole corn plants, corn cobs, corn stalks, sugarcane bagasse, sweet sorghum, cotton stalks), dried distillers grains with solubles (DDGS), and waste paper as substrates. Hungate tubes were incubated without shaking. Flasks were incubated with shaking at 100 rpm.
Growth of bacteria was monitored by analysis of fermentation products and determination of optical density of the cultures (OD600). For the separation of cells from insoluble substrates, samples were centrifuged in 2 ml tubes for 20 s at 1.700 g.
In experiments on fermentation balance NaHCO3 was omitted from the media and insoluble substrates were separated from cells by centrifugation of the cultures in 50 ml tubes for 60 s at 1.700 g. Cells were washed with 0.9% NaCl and substrates were washed with distilled water. After centrifugation cells and substrates were dried at 90°C for 24 h.
Lignocellulosic substrates were pretreated by acidic steam explosion applying 2% (w/v) sulfurous acid at a temperature of 205°C for 5 min prior to sudden release of pressure. To obtain the insoluble carbohydrate fraction, pretreated substrates were washed three times with water at 72°C. Substrates were suspended in distilled water (100 g dry mass/5 liter) and incubated for 16 h at 72°C with stirring. After filtration under vacuum, substrates were washed two times (each time for 2 h with stirring) with equal volumes of water at 72°C. Washed substrates were removed by filtration, dried at 45°C for 66 h and used for growth experiments. Dry weight of washed and unwashed substrates was determined after drying at 105°C for 24 h.
Composition of raw untreated substrates and washed pretreated substrates was determined according to the laboratory analytical procedure "Determination of structural carbohydrates and lignin in biomass" from National Renewable Energy Laboratory (NREL) .
Fermentations were carried out in 2-liter stirred vessel fermentors (Biostat B-DCU, B.Braun / Sartorius AG) with a working volume of 1.2 liter. All vessels were equipped with double jackets for temperature control, two Rushton-type stirrer blades and pH-control loops. In order to maintain a constant pressure throughout the cultivation, vessels were additionally equipped with high-precision blow-off valves, controlling the pressure in the range of 1.3-1.5 bar. The medium as described above was supplemented with Avicel, pretreated poplar wood, xylan or glucose. The medium was set to pH 6.75 by automatic addition of NaOH solution and this value maintained throughout the fermentation run. In order to remove oxygen from the medium, the fermentor vessel was flushed with nitrogen for 1 h at a rate of 1 l/min; then Na2S × 9 H2O was added as described above while gas flushing was stopped. Each fermentor was inoculated with 100 ml of seed culture prepared as described above for cultivation of a single strain and with 50 ml of each seed culture for co-cultivation of two strains. Cellulolytic seed cultures were grown on 10 g/l Avicel. Non-cellulolytic seed cultures were grown on 5 g/l glucose. A temperature of 72°C was maintained during the entire fermentation run.