Unless specified otherwise, all anaerobic media and additives in this study were prepared using strict anaerobic techniques described in McSweeney et al., 2005. Final media preparations were maintained under anaerobic condition in either 16 × 125 mm Hungate tubes (Bellco) sealed with open top screw caps and rubber stoppers (Bellco) or 50 ml glass serum vials (Supelco) plugged with butyl rubber stoppers and aluminium seals (Supelco). Liquid medium was sterilized by autoclaving at 121℃ for 20 min. Any additions of components into sterilized media were carried out by inserting N2-flushed syringes and needles through the stoppers or inside a UV-sterilized anaerobic chamber (COY Laboratory Products Inc. Michigan).
2.1.2 Basal nutrient (BN) medium
Basal nutrient medium was used as an enrichment medium for pectin-utilizing bacteria from human faeces. Per litre of basal nutrient medium contained 2 g Difco™ peptone water; 2 g Bacto™ yeast extract; 0.5 g bile salts (Oxoid); 10 ml Mineral A stock solution (0.4 g K2HPO4 dissolved in 100 ml distilled water); 10 ml Mineral B stock solution (0.4 g KH2PO4; 0.1 g MgSO4.7H2O; 0.1 g
CaCl2.6H2O; and 1.0 g NaCl dissolved in 100 ml distilled water); 2 ml Tween 80 (Fischer Scientific); 0.05 g hemin (Sigma); 10 µl vitamin K (Sigma); 0.5 g L-cysteine-HCl; 0.02 g resazurin; and 4.2 g NaHCO3. All components except hemin, vitamin K, L-cysteine-HCl, and NaHCO3 were dissolved in distilled water and boiled, and then were transferred on ice while hot. The medium was bubbled with N2 while being cooled on ice. Hemin, vitamin K, L-cysteine-HCl, and NaHCO3 were added into the cooled medium. Required volumes of the medium were dispensed into Hungate tubes or serum vials under N2, resulting in the headspace of the tubes to be filled with 100 % N2. Vessels sealed with caps and stoppers were sterilized by autoclaving. Pectin substrates and 5 % (v/v) clarified rumen fluid were added immediately prior to inoculation.
2.1.3 Basal nutrient (BN) agar medium prepared in roll-tubes
9 ml of basal nutrient medium was dispensed into Hungate tubes containing 150 mg of agar. Medium was sterilized by autoclaving and cooled to 50 ℃ in a water bath. Additives were injected through rubber stoppers to make up the final volume of the medium to 10 ml. Hot medium was spread over the inner surface of stoppered tubes under cold tap water as described in Hungate (1969).
2.1.4 Mineral medium
Routine cultivation and characterization of strain 14T was carried out using mineral medium. Per litre of the medium contained 1.4 g KH2PO4; 0.6 g (NH4)2SO4; 1.5 g KCl; 1 g yeast extract; 0.02 g resazurin; 4.2 g NaHCO3; and 0.5 g L-cysteine-HCl. NaHCO3 and L-cysteine-HCl were added after boiling and cooling the medium on ice under 100 % CO2. Required volumes of the medium were dispensed into Hungate tubes or serum vials under CO2. Sealed vessels were sterilized by autoclaving. Substrates and 5 % (v/v) clarified rumen fluid were added to the medium prior to inoculation.
2.1.5 Reinforced clostridial (RC) medium
Genomic DNA was extracted from strain 14T grown on RC medium (CM0149) purchased from Oxoid. RCM contains 13 g/L yeast extract; 10 g/L peptone; 5 g/L glucose; 1 g/L soluble starch; 5 g/L NaCl; 3 g/L sodium acetate; 0.5 g/L; cysteine hydrochloride; and 0.5 g/L agar. Following the
manufacturer’s instruction, 38 g of RCM was dissolved in 950 ml of distilled water in 1 litre Duran® glass bottles. The pH of the medium was adjusted to 8.0 using 2M NaOH. Medium was boiled and cooled on ice while bubbling with N2. Medium was autoclaved and kept inside a UV-sterilized anaerobic chamber overnight to equilibrate with the anaerobic ambient. Additives (2 % (v/v) clarified rumen fluid and 50 ml of 10 % (w/v) D-fructose solution, filter-sterilized) were aseptically added to the medium prior to inoculation.
2.1.6 Luria-Bertani (LB) broth and agar medium
20 g of LB broth powder (Sigma) was dissolved in 950 ml of distilled water. Medium was sterilized by autoclaving. Autoclaved medium was allowed to cool to 50℃ in a water bath before adding antibiotics if required. LB agar medium was prepared by adding 15 g of agar per litre of the medium before autoclaving. Autoclaved agar medium containing antibiotics was poured into Petri dishes.
2.1.7 Clarified rumen fluid
Rumen fluid collected from hay-fed fistulated cows was provided by AgResearch Ltd (Grasslands Research Centre, Palmerston North). Rumen fluids were bubbled with N2 for 20 min and dispensed into 50 ml serum vials sealed with rubber stoppers and aluminium caps. Rumen fluid was autoclaved. Autoclaved rumen fluid was poured into a beaker and mixed thoroughly with MgCl2.6H2O (1.63 g per 100 ml) and CaCl2.2H2O (1.18 g per 100 ml) by stirring for 30 min. Precipitates were removed by centrifuging at 6,000 g for 20 min at 4℃. Supernatant was collected in a clean beaker and bubbled with N2 for 20 min. While under N2, supernatant was filter-sterilized through 0.22 µm filter (Merck Millipore) into sterile and N2-washed serum vials sealed with rubber stoppers and aluminium caps. 2 % (v/v) anaerobically prepared vitamin solution was added by using N2-washed syringes and needles. The resulting mixture was termed ‘Clarified rumen fluid’ in this study.
2.1.8 Vitamin solution
RPMI-1640 Vitamin Solution (R 7256) was purchased from Sigma-Aldrich. RPMI-1640 Vitamin Solution contains 0.02 g/L D-biotin; 0.3 g/L Choline Chloride; 0.1 g/L Folic Acid; 3.5 g/L myo- Inositol; 0.1 g/L Niacinamide; 0.1 g/L p-Amino Benzoic Acid; 0.025 g/L D-Pantothenic Acid· 1 2⁄ Ca; 0.1 g/L Pyridoxine·HCl; 0.02 g/L riboflavin; 0.1 g/L Thiamine·HCl; 0.0005 g/L Vitamin B12; 0.2 g/L KCl; 0.2 g/L KH2PO4 (anhyd); 8 g/L NaCl; and 1.15 g/L Na2HPO4 (anhyd). The solution was poured into a beaker and bubbled with N2 for 10 min. While bubbling with N2, the solution was filter- sterilized (0.22 µm) into sterile and N2-washed serum vials. Vitamin Solution was stored frozen at - 20℃ until use.
2.1.9 Anaerobic water
1 litre of distilled water was boiled and cooled on ice under a continuous flow of N2. 0.5 g of L- cysteine HCl was added. Required volumes of water were dispensed into Hungate tubes or serum vials and sealed using caps and rubber stoppers under N2. Anaerobically prepared water was sterilized by autoclaving.
2.1.10 Anaerobic phosphate buffered saline (PBS)
1 litre of 10 mM PBS (MP Biomedicals) was boiled and cooled on ice under a continuous flow of N2. 0.5 g of L-cysteine HCl was added to the cooled solution. Required volumes of PBS were dispensed into anaerobic vessels and sealed under N2. PBS was sterilized by autoclaving.
2.1.11 Anaerobic glycerol solution for a long-term preservation of bacteria
50 % (v/v) glycerol was mixed with distilled water and boiled and cooled on ice under N2. 0.5 g of L- cysteine HCl was added per litre of glycerol solution. Solution was dispensed into anaerobic glass vessels under N2 and sealed using caps and rubber stoppers. Solution was sterilized by autoclaving. 50 % glycerol solution was mixed vigorously with exponentially growing bacterial culture at 1:1 ratio. The mixture was stored at -80℃.