Isotope of Methane and Combined Metagenomic Elucidates Microbial Overprint to Methane Emissions from the Shallow-Water Hydrothermal System of Vulcano Island (Aeolian Archipelago, Italy)

The role of microorganisms in shaping Earth’s dynamics is becoming increasingly evident; therefore, understanding how they influence and control environmental processes is essential for deciphering the functioning of Earth’s systems and for the effective management of natural resources.

Hydrothermal systems offer natural laboratories for investigating the interplay between geological and microbial processes and in this context, Levante Bay on Vulcano Island (Aeolian Archipelago, Italy) represents an ideal setting to explore how these two components interact. This area is a typical hydrothermal system characterized by several CO₂-dominated fluid manifestations of varying intensity and temperature. These manifestations exhibit a pronounced H₂, CH₄ and fluid temperature gradient along a south–north direction, as consistently confirmed by long-term geochemical observations. A distinctive feature of this site is the unusually heavy δ¹³C values of CH₄ (up to -4.8‰ vs. V-PDB), which have led to the hypothesis of an abiotic origin for CH₄.

In particular, the geochemical observations indicate that elevated fluid temperatures co-occur with higher H₂ concentrations, whereas decreasing temperatures are accompanied by a marked increase in CH₄ concentrations. This evidence is consistent with cytofluorimetric detection of F₄₂₀+ autofluorescent cells, providing direct evidence for methanogenic archaea inhabiting the cooler points of the Bay. Our overarching hypothesis is that the observed CH₄ gradient is linked not only to geological dynamics but also to microbial activity, particularly methanogenic metabolism. 

To test this, we designed a microbial incubation experiment to assess whether, and to what extent, the microbial communities inhabiting five distinct points of the aquifer can influence gas and fluid chemistry, with a particular focus on elucidating their contribution to CH₄ production. In particular we set three treatments for each hydrothermal fluid sample: (i) BIO_H₂, unfiltered fluid incubated under an H₂:CO₂ (80:20) atmosphere to stimulate hydrogenotrophic methanogenesis; (ii) AB_H₂, filtered fluid under H₂:CO₂ (80:20) conditions serving as abiotic controls; and (iii) BIO_N₂, unfiltered fluid incubated under N₂:CO₂ (80:20) to maintain microbial communities while preventing H₂-driven methanogenesis. During the incubation period, we monitored both hydrothermal fluid and headspace gas composition, with particular focus on H₂ consumption, CH₄ production and stable carbon isotope composition of CH₄ (δ¹³C-CH₄). Microbiological characterization was conducted through 16S rRNA gene sequencing and shotgun metagenomics to detect shifts in taxonomic composition and functional potential, with a particular focus on metabolic pathways underpinning methanogenesis and other hydrogenotrophic processes. 

Preliminary results reveal that BIO_H₂ incubations showed increasing alkalinity, pH, and H₂S and CH₄ production compared to the other treatments. Surprisingly, the AB_H₂ condition also produced measurable CH₄, occasionally approaching biotic levels, pointing to the need to elucidate this phenomenon. δ¹³CH₄ signatures displayed strong site-specific variability, with high negative values under BIO_H₂ treatment and comparatively less negative signatures under BIO_N₂ conditions, indicating different CH₄ sources or pathways. Overall, these results highlight the complexity of CH₄ origin in Levante Bay and indicate that geological and biological controls on methane cycling remain insufficiently resolved.