An integrated study reveals diverse methanogens, Thaumarchaeota, and yet-uncultivated archaeal lineages in Armenian hot springs

Culture-independent and enrichment techniques, with an emphasis on members of the Archaea, were used to determine the composition and structure of microbial communities inhabiting microbial mats in the source pools of two geothermal springs near the towns of Arzakan and Jermuk in Armenia. Amplification of small-subunit rRNA genes using universal primers followed by pyrosequencing (pyrotags) revealed highly diverse microbial communities in both springs, with > 99 % of pyrosequences corresponding to members of the domain Bacteria. The spring in Arzakan was colonized by a photosynthetic mat dominated by Cyanobacteria, in addition to Proteobacteria, Bacteroidetes, Chloroflexi, Spirochaeta and a diversity of other Bacteria. The spring in Jermuk was colonized by phylotypes related to sulfur, iron, and hydrogen chemolithotrophs in the Betaproteobacteria and Epsilonproteobacteria, along with a diversity of other Bacteria. Analysis of near full-length small subunit rRNA genes amplified using Archaea-specific primers showed that both springs are inhabited by a diversity of methanogens, including Methanomicrobiales and Methanosarcinales and relatives of Methanomassiliicoccus luminyensis, close relatives of the ammonia-oxidizing archaeon (AOA) Candidatus Nitrososphaera gargensis, and the yet-uncultivated Miscellaneous Crenarchaeotal Group and Deep Hydrothermal Vent Crenarchaeota group 1. Methanogenic enrichments confirmed the predicted physiological diversity, revealing methylotrophic, acetoclastic, and hydrogenotrophic methanogenesis at 45 and 55 A degrees C, but not 65 A degrees C. This is one of only a few studies combining cultivation-independent and -dependent approaches to study archaea in moderate-temperature (37-73 A degrees C) terrestrial geothermal environments and suggests important roles for methanogenic archaea and AOA in the carbon and nitrogen biogeochemical cycles in these environments.