Anaerobic growth studies in shake flasks.

Anaerobic shake-flask based experiments were performed in a Lab Bactron 300 anaerobic workstation (Sheldon Manufacturing Inc., Cornelius, OR) containing an atmosphere of 85% N₂, 10% CO₂, and 5% H₂. Flat-bottom shake flasks of 50 ml were filled with 40 ml SMD-urea medium containing 50 g liter⁻¹ glucose as the carbon source to ensure depletion of the vitamin/growth factor of interest and 20 g liter⁻¹ glucose for the first transfer. Media were supplemented with vitamins, with and without pantothenic acid or nicotinic acid as indicated, and in all cases, supplemented with Tween 80 and ergosterol. Sterile medium was placed inside the anaerobic chamber 24 h prior to inoculation for removal of oxygen. Traces of oxygen were continuously removed with a regularly regenerated Pd catalyst for H₂-dependent oxygen removal placed inside the anaerobic chamber. Aerobic overnight shake-flask cultures on SMD-urea were used to inoculate the anaerobic shake flask without pantothenic acid or without nicotinic acid at an initial OD₆₀₀ of 0.2. Cultures were cultivated at 30°C with continuous stirring at 240 rpm on an IKA KS 260 Basic orbital shaker platform (Dijkstra Verenigde BV, Lelystad, the Netherlands). Periodic optical density measurements at a wavelength of 600 nm using an Ultrospec 10 cell density meter (Biochrom, Cambridge, United Kingdom) inside the anaerobic environment were used to follow the growth over time. After growth had ceased and the OD₆₀₀ no longer increased, the cultures were transferred to SMD-urea with 20 g liter⁻¹ glucose at an OD₆₀₀ of 0.2 (39).