Sulfide ‘chimneys’ characteristic of seafloor hydrothermal venting are diverse microbial habitats. 13C/12C ratios of microbial lipids have rarely been used to assess carbon assimilation pathways on these structures, despite complementing gene- and culturebased approaches. Here, we integrate analyses of the diversity of intact polar lipids (IPL) and their sidechain ~d13C values (~d13Clipid) with 16S rRNA gene-based phylogeny to examine microbial carbon flow on active and inactive sulfide structures from the Manus Basin. Surficial crusts of active structures, dominated by Epsilonproteobacteria, yield bacterial ~d13Clipid values higher than biomass ~d13C (total organic carbon), implicating autotrophy via the reverse tricarboxylic acid cycle. Our data also suggest ~d13Clipid values vary on individual active structures without accompanying microbial diversity changes. Temperature and/or dissolved substrate effects – likely relating to variable advective–diffusive fluxes to chimney exteriors – may be responsible for differing 13C fractionation during assimilation. In an inactive structure, ~d13Clipid values lower than biomass ~d13C and a distinctive IPL and 16S rRNA gene diversity suggest a shift to a more diverse community and an alternate carbon assimilation pathway after venting ceases. We discuss here the potential of IPL and ~d13Clipid analyses to elucidate carbon flow in hydrothermal structures when combined with other molecular tools.
