Biogeochemical and micropaleontological constraints on methane in the Amazon Fan gas hydrate province

Gas hydrate provinces along continental margins are major reservoirs of methane and play a key role in regulating carbon fluxes between the geosphere, hydrosphere, and biosphere. The Amazon Deep-Sea Fan, one of the largest sedimentary systems on Earth, hosts extensive gas-hydrate accumulations and widespread fluid-expulsion structures, yet the spatial and temporal dynamics of methane within this system remain poorly constrained. Here, we integrate organic geochemical biomarkers with micropaleontological and biostratigraphic data to assess methane occurrence and microbial processing within shallow sediments of the hydrate stability field. Seven piston cores recovered during the 2023 AMARYLLIS–AMAGAS expedition penetrated up to ~30 m below the seafloor across hydrate-rich areas of the Amazon Fan. Twenty sediment samples were selected based on total organic carbon content and stratigraphic position and were analyzed using solvent extraction, liquid chromatography, and GC–MS. In parallel, 367 samples from 17 piston and gravity cores were studied for planktonic foraminifera, supplemented by analyses of calcareous nannofossils and palynofacies, thereby providing a robust Quaternary stratigraphic framework. Biomarker distributions indicate a dominance of terrestrial organic matter, with long-chain odd-numbered n-alkanes (n-C27–n-C35) and immature hopane and sterane assemblages, reflecting rapid burial in a clay-rich, low-maturity depositional environment. Despite this strong terrigenous imprint, all analyzed samples contain 3-methylhopanoids, diagnostic lipids of aerobic methanotrophic or methylotrophic bacteria. Their ubiquitous occurrence demonstrates that methane is present and bioavailable throughout the shallow subsurface of the hydrate stability zone. Meanwhile, the absence of 2-methylhopanoids suggests that cyanobacterial or phototrophic inputs are negligible in this zone, emphasizing a subsurface microbial signal. In selected cores, pentamethylcosenes further indicate localized zones of elevated microbial lipid production, suggesting spatially heterogeneous methane oxidation associated with focused fluid flow. Micropaleontological data indicate that the upper tens of meters of sediment are entirely Quaternary but are strongly affected by sediment remobilization associated with mass-transport deposits and mud volcanism driven by gas hydrate dissociation. Biozonation based on the presence and absence of Globorotalia menardii reveals alternations between glacial and interglacial intervals, reflecting climatic control on sedimentation, productivity, and bottom-water properties. The frequent occurrence of reworked Cenozoic and even Cretaceous microfossils within Holocene and late Pleistocene strata provides independent evidence for upward sediment transport driven by methane-rich fluids. Together, these datasets reveal a tightly coupled system in which methane stored in hydrates is episodically mobilized, transported, and consumed by microbial communities within shallow Amazon Fan sediments. Biomarkers provide direct evidence for active methane cycling, while microfossils document the stratigraphic and depositional framework that modulates hydrate stability and fluid migration. This integrated approach highlights the Amazon Deep-Sea Fan as a dynamic methane system, sensitive to both climatic forcing and sedimentary processes, with implications for carbon cycling along tropical continental margins.