A Flexible System for Cultivation of Methanococcus and Other Formate-Utilizing Methanogens

Figure 5

Medium-scale cultivation system with culture bottle, sparging, and exhaust. The cultivation system comprises a 2 L cultivation bottle (A), water trap (B: 160 mL serum bottle with a blue butyl stopper and an aluminum seal), and a gas trap (C: 150 mL Erlenmeyer flask). The cultivation bottle is constructed from a Pyrex bottle (cat. number: 06-414-1E) with the top of a 28 mm aluminum seal tube attached as a side arm. The side arm is sealed with a blue butyl rubber stopper without the aluminum seal (Bellco Glass Inc., Vineland, NJ, cat. number: 2048-11800). Leaving off the aluminum seal allows the stopper to pop off and release any pressure that might form if the exhaust line becomes clogged. The gassing line (D, E, and F) is for sparging the medium after autoclaving and is assembled with F, a 2-cc interchangeable glass-filter syringe barrel (Micro-Mate™, cat. number: 14-825-1B) packed with cotton and sealed with a number 00 rubber stopper (Balch and Wolfe [35]), D: luer-to-tubing connector (Sigma-Aldrich, 1/16–3/32 in., cat. number: Z118028), and E: needle (BD, 22 G × 1 in., cat. number: 305155) with thin tubing (Zeus™ PTFE light wall tubing, P/N: TFT22-NT, AWG 22) connected. After sparging, the needle is removed prior to inoculation leaving the tubing in the culture. Other components include G: Nalgene™ tubing (80 PVC tubing-FDA/USPVI, 1/8 ID × 3/16 OD × 1/32 wall, Thermo Scientific cat. number: 8000-0010); H: Monoject™ needle (16 G × 1 1/2″, cat. number: 140394); and I: needle (BD, 15 G × 3 1/2 in., cat. number: 511108).