Abstract
Methane is an energy-dense fuel but is also a greenhouse gas 25 times more detrimental to the environment than CO2. Methane can be produced abiotically by serpentinization, chemically by Sabatier or Fisher-Tropsh chemistry, or biotically by microbes (Berndt et al., 1996; Horita and Berndt, 1999; Dry, 2002; Wolfe, 1982; Thauer, 1998; Metcalf et al., 2002). Methanogens are anaerobic archaea that grow by producing methane gas as a metabolic byproduct (Wolfe, 1982; Thauer, 1998). Our lab has developed and optimized three different gas chromatograph-utilizing assays to characterize methanogen metabolism (Catlett et al., 2015). Here we describe the end point and kinetic assays that can be used to measure methane production by methanogens or methane consumption by methanotrophic microbes. The protocols can be used for measuring methane production or consumption by microbial pure cultures or by enrichment cultures.
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 1. Crimper, gastight autosampler vials and gas-tight Hamilton syringes. A. Crimpers are used to seal autosampler vials using aluminum crimps and rubber stoppers. Hamilton syringes showing open (B) and closed (C) luer fittings. Figure 2. Dual-sided custom Coy anaerobic chamber showing Medlite clinical centrifuge (blue lid) Figure 3. Methane gas tank fitted with a septa Figure 4. Example standard curve Figure 5. Method for preparing gas standards and sampling gas headspace. A. An 18 G needle is pushed into the stopper about three-quarters of the way through. This acts as a guide for the Hamilton needle to push through the stopper and not bend. B. A Hamilton syringe is inserted into an 18 G needle and pushed through the rest of the stopper. Once the end of the Hamilton syringe needle is through the end of the stopper (arrow), headspace can be extracted. Do not push the Hamilton syringe so far into the stopper that the 18 G needle is pushed through the stopper, as this will allow headspace gas to quickly escape. Figure 6. Example kinetic assay results
Recipes
Notes:
Acknowledgments
This material is based upon work supported by the National Science Foundation under Grants IOS-1449525 and MCB-1449014, by the Water Environment Research Foundation grant NTRY6R14, and by the Nebraska Center for Energy Sciences Cycle 8 award to N. Buan. M. Smith was supported by an American Society for Microbiology Undergraduate Research Fellowship and a Pepsi UCARE Fellowship. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the funding sources. The authors declare no competing interests.
References
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