Methanogen Growth and Culturing Protocols

Methanococcus maripaludis strain S2 was obtained from our laboratory collection (Whitman et al.) [27 (link)] and cultured at 37°C. Methanothermococcus okinawensis strain IH1 was obtained from Takai et al. and cultured at 62°C [28 (link)].
Cultures were grown in H₂/CO₂ medium (McNA, a minimal medium with 10 mM sodium acetate) or formate medium (McF) reduced with 3 mM cysteine hydrochloride. The 5 mL cultures were grown in 28 mL aluminum-sealed tubes. For McNA, the tubes were pressurized to 276 kPa with H₂/CO₂ (4 : 1, v/v) and refilled with the same gas every 24 hours after inoculation. Detailed protocols for growth on formate are given in Appendix A. Briefly, McF medium contained 0.4 M sodium formate and was buffered with 0.2 M glycylglycine (pH = 8.0). The medium was first sparged with N₂ to remove most of the O₂, and 3 mM cysteine chloride was then added. Tubes were pressurized to 103 kPa with N₂/CO₂ (4 : 1, v/v) before autoclaving. Prior to inoculation, 3 mM sodium sulfide was added as the sulfur source.
The buffers tested were obtained from Sigma Chemical Co. and included (with the counter ion) Tricine/NaOH (N-[Tris(hydroxymethyl)methyl]glycine), Bicine/NaOH (N,N-bis(2-hydroxyethyl)glycine), Tris/HCl (2-amino-2-hydroxymethyl-propane-1,3-diol), glycine/NaOH, and glycylglycine/NaOH. During formate medium preparation, ingredients were added as listed in the appendices, and the organic buffers were added from stock solutions at pH 7. The concentration of NaCl was adjusted depending upon the amount of sodium formate and sodium in the buffer used so that the final concentration of sodium ion was 0.4 M.
The final medium was also tested for plating (Appendix B) and growth of 1.5 L cultures (Appendix C).