Tracing Microbial Methanogenesis and Methane Oxidation with Clumped Isotopologue Compositions of Methane

Author

• Jiawen Li, Dartmouth CollegeFollow

Author ORCID Identifier

https://orcid.org/0000-0002-2385-9446

Date of Award

Spring 2026

Document Type

Thesis (Ph.D.)

Department or Program

Earth Sciences

First Advisor

William D. Leavitt

Abstract

Methane plays a critical role in the global carbon cycle. It is also an energy resource, a potent greenhouse gas, and a potential biosignature for extraterrestrial life. Distinguishing between abiotic and biological methane sources and among microbial production pathways—remains a major challenge. While bulk carbon and hydrogen isotopes have long been used for this purpose, overlapping signatures and isotopic exchange processes with water and dissolved inorganic carbon limit their diagnostic power. Clumped isotopologues of methane (¹³CH₃D and ¹²CH₂D₂) offer additional constraints, as their abundances can indicate both formation/equilibration temperature and production/destruction pathways. However, microbial processes produce a wide range of clumped isotope signatures, necessitating a systematic investigation of their controls.

This thesis experimentally examines the thermodynamic and enzymatic factors governing clumped isotopic compositions in microbial methane production and oxidation. Chapters 2 and 3 focus on methanogenesis. Hydrogenotrophic methanogenesis yields distinct ¹²CH₂D₂ isotope signatures compared to other pathways using methylated organic compounds, which is driven by a “combinatorial effect” during the combination of methyl groups and intracellular hydrogen carriers. Additionally, isotopic signatures vary with Gibbs free energy yield, with clumped isotopes showing a reversal behavior around -35 kJ/mol. Chapters 4 and 5 investigate methane oxidation. Aerobic oxidation exhibits relatively consistent isotopic fractionation across enzymes (particulate and soluble methane monooxygenases) and temperatures (21-37 ºC), whereas anaerobic oxidation coupled to nitrate/nitrite or sulfate reduction shows strong variability, reflecting differences in enzymatic mechanisms and reaction reversibility. Finally, Chapter 6 broadens the scope and highlights enzymatic reactions and redox conditions as key factors controlling methane isotopic biosignatures, particularly in extraterrestrial contexts. Future studies focusing on the origin and evolution of metalloenzymes utilized by life, as well as the redox conditions on Mars and icy moons, are essential for defining the signals of life in these environments.

Overall, this work constrains the isotopic boundaries of biological methane and improves the application of isotopic tools for identifying methane sources and sinks in both terrestrial and extraterrestrial environments.

Original Citation

Li J, Ash JL, Cobban A, Kubik BC, Rizzo G, Thompson M, Guibourdenche L, Berger S, Morra K, Lin Y, Mueller EP, Masterson AL, Stein R, Fogel M, Torres MA, Feng X, Holden JF, Martini A, Welte CU, M. Jetten MS, Young ED, Leavitt WD. The Clumped Isotope Signatures of Multiple Methanogenic Metabolisms. Environ Sci Technol 2025;59:13798–810. https://doi.org/10.1021/acs.est.5c03255.

Li J, Chiu BK, Piasecki AM, Feng X, Landis JD, Marcum S, Young ED, Leavitt WD. The evolution of multiply substituted isotopologues of methane during microbial aerobic oxidation. Geochimica et Cosmochimica Acta 2024;381:223–38. https://doi.org/10.1016/j.gca.2024.06.032.

Recommended Citation

Li, Jiawen, "Tracing Microbial Methanogenesis and Methane Oxidation with Clumped Isotopologue Compositions of Methane" (2026). Dartmouth College Ph.D Dissertations. 491.
https://digitalcommons.dartmouth.edu/dissertations/491