Background

The vast majority of methane emitted worldwide is produced by a group of archaea known as methanogens. These microbes, which inhabit wetlands, ruminant guts, and hydrothermal vents, produce methane (CH₄) as a metabolic byproduct. In many of these environments, microbes known as methanotrophs coexist with methanogens, and convert the methane emitted into CO₂.

As part of an effort to better constrain the relationship between the cellular activity of these organisms and actual gas production and degradation rates, a need to culture these organisms arose in the lab. However, as methanotrophs require methane-rich environments, and methanogens require strictly anaerobic conditions, these organisms need to be grown under controlled atmospheres. This is accomplished by culturing in serum bottles (above), sealed with thick stoppers and crimp seals. However, to achieve the correct atmospheres within the serum bottles, a gas manifold is needed to quickly and accurately dispense and remove gases.

Microbe	Atmosphere	Pressure
Methanogens	80% H2, 20% CO2	20 psi
Methanotrophs	50% CH4, 50% Air	0 psi

Problem

This meant I needed to build a gas manifold, with the following constraints:

• Capable of dispensing N₂, H₂/CO₂, CH₄, and air
• Capable of pulling vacuum for changing headspace of cultures
• Can quickly switch between gas types and vacuum
• Can dispense sterile gases to avoid contamination
• Withstands pressures up to ~100 psi
• Included safeties to avoid over-pressurizing and prevent flammable gas leaks
• Scrubs anoxic gases to low ppm levels of O₂
• Able to monitor pressure of tanks, vacuum, and serum bottles
• Interfaces with multiple serum bottles simultaneously

Construction

End Product

The final product, termed The Methanifold, is capable of simultaneously filling, flushing, and/or replacing the headspace of six serum bottles. The vacuum and gas lines can be quickly switched between using a large, manually operated switching valve. The anoxic side of the manifold (on the right) can be sealed off from the oxic side (on the left), and includes a small, in-line oxygen scrubber. The lower pressure gauge displays the conditions created inside the serum bottles, and the upper two gauges show pressure coming from the connected tanks. Three 75 psi pressure relief valves (facing away from the front) are installed in case of regulator failure. Finally, two 30-minute timed switches, connected to solenoids on the CH₄ and H₂ tanks, act as dead man’s switches in case flammable gas supplies are accidently left on.