Clumped isotope signatures of methane from mud volcanoes in Italy and Romania: implications for microbial activity

Natural gas seeps and mud volcanoes are widely distributed across terrestrial and shallow submarine sedimentary basins and contribute considerable amounts of fossil methane to the atmosphere. Methane emissions from these systems are commonly interpreted as dominantly thermogenic in origin; however, microbial activity may significantly contribute to, or overprint, these emissions through secondary methanogenesis or methane oxidation during gas migration and storage.

Conventional bulk isotope composition (δ¹³C and δD) and hydrocarbon concentration ratios are often insufficient to distinguish secondary microbial contributions from an initial thermogenic source. Independent of bulk isotopic signatures, methane clumped isotopes (Δ¹³CH₃D and Δ¹²CH₂D₂) provide direct constraints on methane formation pathways and post-generation alteration processes.　Recent studies have revealed low-temperature near-equilibrium clumped-isotope signatures in mud-volcano systems in Azerbaijan¹, indicative of strong microbial overprinting, whereas methane from Japanese mud volcanoes exhibits clumped isotope signatures spanning from far from equilibrium to near equilibrium values². For the latter, clumped isotope signatures of methane correlate with ¹³C-position-specific isotope composition of propane, suggesting the biodegradation of higher hydrocarbons is associated with progressive modification of methane clumped isotopes.

Here, we investigate methane emissions from mud volcanoes and gas seeps in central and southern Italy (n = 14) and Romania (n = 15). Methane bulk and clumped isotope composition (δ¹³C, δD, Δ¹³CH₃D and Δ¹²CH₂D₂) are analyzed using a quantum cascade laser absorption spectrometer (QCLAS) equipped with a customized gas-inlet system at Empa³. Propane concentrations span from below detection to 0.8%, indicating a wide range of potential microbial influence. Selected samples are further characterized by propane position-specific isotope analyses at Science Tokyo following established protocols by Gilbert et al. ⁴, providing constraints on the extent of secondary microbial processes affecting higher hydrocarbons.

Preliminary clumped-isotope results from Italian mud volcanoes indicate near-equilibrium signatures consistent with strong microbial influence, comparable to patterns reported from Azerbaijan mud-volcano systems. In contrast, Romanian samples exhibit pronounced variability in propane concentrations, providing a critical test case to explore whether methane clumped-isotope systematics transition toward more thermogenic-dominated patterns with secondary microbial influence, similar to those observed in Japanese systems. By integrating new datasets from Italy and Romania with published clumped-isotope and propane intramolecular isotope data, this study explores whether microbial influences on methane emissions follow consistent or system-specific patterns across mud-volcano and gas-seep systems globally.

 

[1] Liu et al., 2023 Geology

[2] Gilbert et al., 2025 EGU2025 Abstract

[3] Zhang et al., 2025 Anal. Chem.

[4] Gilbert et al. 2019 Proc. Natl. Acad. Sci.