USING HIGH-THROUGHPUT NIFH SEQUENCING TO CHARACTERIZE NITROGEN-FIXING MICROORGANISMS IN SULFURIC ACID CAVES: IMPLICATIONS FOR NITROGEN CYCLING IN SUBTERRANEAN CHEMOSYNTHETIC ECOSYSTEMS

Nitrogen is a key element for life on Earth, and nitrogen-fixing bacteria and archaea play a critical role in supplying bioavailable nitrogen to the ecosystem. The goal of this study is to characterize nitrogen-fixing microorganisms from the walls of the hydrogen sulfide-rich Frasassi cave system in Italy. At the cave water table, sulfidic springs and streams supply hydrogen sulfide to the cave atmosphere, which supports chemolithoautotrophic microbial communities on the walls and ceilings. However, the sources of nitrogen to these subaerial communities are not currently known. We hypothesized that nitrogen fixing microorganisms would be more abundant and diverse in regions of the cave that are farther from sulfidic streams, while communities closer to the water table rely on ammonia gas or other nitrogen compounds that volatilize from the sulfidic aquifer. We evaluated several primer sets to amplify nifH from the cave wall communities, and selected two, the Ueda19F/R6 and IGK3F/DVV primer pairs, that capture most known diazotrophs. We then modified and optimized these two primers with Nextera adaptors for high-throughput Illumina amplicon sequencing, and applied them to a set of cave wall samples collected from 0.5 to over 40 meters from the sulfidic water table. However, very few distinct nifH genes were recovered from the samples, and only half of the libraries were successful. The most abundant nifH sequences come from Methylovirgula spp., which was present in nearly all samples, as well as a close relative of Rhodopseudomonas palustris, which was present in more than half of the samples. Based on preliminary results, we did not observe a clear trend in diazotroph diversity, although overall we had more success amplifying nifH in samples collected at intermediate distances from the water table, but not in areas right above degassing streams. We will discuss the implications of this work for nitrogen cycling in the Frasassi cave system and other sulfidic caves, and for nitrogen fixation in caves and other subterranean habitats more broadly.