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, 2013). The 13C-labeling experiments conducted at both hot springs showed 13C-uptake into archaeal lipids with 13C-acetate for both batch-fed incubations and flow-through experiments (Figure ?(Figure4).4). From this it can be inferred that the SBCs can readily metabolize low molecular weight organic compounds such as selleck screening library acetate. Carbon uptake occurred particularly into archaeol and its ether cleavage product phytane (Tables ?(Tables33�C5), while biphytane 0 (which is derived from GDGT-0) only showed minimal 13C-incorporation at Octopus Spring or none at ��Bison Pool�� in the flow-through experiments and ��13C-values for biphytanes 1 or 2 remained unaltered. This result can best be understood when considering proposed biosynthetic routes of tetraether lipids, where GDGTs are proposed to form through condensation of two archaeols and subsequent coupling to form the membrane-spanning GDGTs, followed by their subsequent Selleck INCB028050 cyclization (Eguchi et al., 2003; Pearson, 2014). We therefore conclude that the biosynthesis of GDGTs was not fast enough to see 13C-label incorporation into tetraether lipids in the flow-through experiments. Formate assimilation was only observed at ��Bison Pool�� when hydrogen was added as supplementary source of reductant (Table ?(Table4,4, Figure ?Figure4),4), indicating that formate can be also utilized by the archaeal communities but only in the presence of additional energy sources. Uptake of 13C-glucose, representative of higher molecular weight organic compounds was not significant for archaeal lipids and thus does not seem to be a major carbon source on the timescales of our experiment. Labeling experiments with 13C-bicarbonate resulted only in negligible 13C-uptake rates into archaeal lipids at ��Bison Pool�� (Hamycin the potential for autotrophic growth of Archaea if additional energy donors are available. Summary This study used a stable isotope labeling approach combined with lipid biomarker analysis to explore modes of carbon assimilation by thermophilic SBCs found in outflow channels of alkaline hot springs. Uptake with 13C-acetate into almost all bacterial and archaeal lipids provided direct evidence for a heterotrophic lifestyle of both bacteria and archaea at the two hot springs investigated, ��Bison Pool�� and Octopus Spring. Heterotrophic carbon assimilation was furthermore confirmed by uptake of 13C-glucose into most fatty acids, but specifically iso and anteiso branched fatty acids, which are specific to the Thermus/Meiothermus and Firmicutes.