NITROGEN SOURCES AND SIGNATURES IN SULFURIC ACID CAVES

“Sulfidic” caves contain robust, chemosynthetic ecosystems where reduced, H₂S-rich groundwaters are exposed to oxygen at the cave water table. Cave streams host white mats created by sulfide-oxidizing microorganisms. Above the water table, viscous biofilms-forming sulfide oxidizers live on H₂S(g) vapors that degas from turbulent springs and streams. The chemoautotrophic nature of these cave ecosystems was recognized because d¹³C and d¹⁵N values of organic matter from the sulfidic water table are distinct from surface sources. In particular, d¹⁵N of organic material above the water table can be very low, with d¹⁵N values below -25‰, indicating that there are unusual sources or cycling of nitrogen in these areas. We are using molecular, metagenomic, and isotopic tools to evaluate different sources and key nitrogen cycling populations in this subaerial cave environment.

Previous authors have hypothesized that the extremely negative d¹⁵N values result from strong fractionation during NH₃(g) volatilization (Jones et al., 2008; Stern et al., 2003). By this hypothesis, trace NH₃(g) flux from circumneutral streams is trapped in acidic biofilms and water on the walls; however, the isotopic composition of NH₃/NH₄⁺ in the streams and air have not been measured, and other N sources have not been accounted for. We deployed passive NH₃(g) samplers for 2 and 9 months in the sulfidic Frasassi cave, in areas that are close to and far from the degassing streams. NH₄⁺ in the stream was consistently δ¹⁵N = -1‰, and preliminary results from passive samplers show a much lighter values between -13 and 27‰, consistent with a much lighter source in the cave atmosphere. We will validate these results with additional samplers, and are now testing an active sampling strategy.

Heavier organic d¹⁵N values further from the streams suggest that wall communities rely on N₂ fixation or another source. However, nifH amplification and high-throughput nifH amplicon sequencing shows that potential N₂-fixers are not abundant or diverse on cave walls, so these subaerial communities may rely on other N sources carried by air currents or waters. We will discuss N biogeochemistry in different cave zones, and compare microbial N cycling in Frasassi wall biofilms to those from other sulfidic caves with different groundwater geochemistry and surface connections.