We describe a system for the cultivation of gaseous substrate utilizing microorganisms that overcomes some of the limitations of fixed volume culture vessels and the costs associated with sparging. Cali-5-Bond gas-sampling bag was used as the culture vessel. The bags contain approximately six times more mass of CO than the 40 mL vials at 1 atm of pressure and performed equally to the 40 mL vials in terms of their ability to maintain the composition of the gas over extended incubation times. Experiments using Clostridium ljungdahlii and CO as the sole carbon and energy source in both the gas sampling bag cultivation system and the traditional vial system demonstrated that this culture had a 15x increase in optical density in 24 h of incubation. The gas-sampling bags offer a viable alternative to gas sparging while overcoming the limitations of fixed volume culture vessels.