Signature lipids and stable carbon isotope analyses of Octopus Spring hyperthermophilic communities compared with those of Aquificales representatives

Abstract

The molecular and isotopic compositions of lipid biomarkers of cultured Aquificales genera have been used to study the community and trophic structure of the hyperthermophilic pink streamers and vent biofilm from Octopus Spring. Thermocrinis ruber, Thermocrinis sp. strain HI 11/12, Hydrogenobacter thermophilus TK-6, Aquifex pyrophilus, and Aquifex aeolicus all contained glycerol-ether phospholipids as well as acyl glycerides. The n-C(20:1) and cy-C(21) fatty acids dominated all of the Aquificales, while the alkyl glycerol ethers were mainly C(18:0). These Aquificales biomarkers were major constituents of the lipid extracts of two Octopus Spring samples, a biofilm associated with the siliceous vent walls, and the well-known pink streamer community (PSC). Both the biofilm and the PSC contained mono- and dialkyl glycerol ethers in which C(18) and C(20) alkyl groups were prevalent. Phospholipid fatty acids included both the Aquificales n-C(20:1) and cy-C(21), plus a series of iso-branched fatty acids (i-C(15:0) to i-C(21:0)), indicating an additional bacterial component. Biomass and lipids from the PSC were depleted in (13)C relative to source water CO(2) by 10.9 and 17.2 per thousand, respectively. The C(20-21) fatty acids of the PSC were less depleted than the iso-branched fatty acids, 18.4 and 22.6 per thousand, respectively. The biomass of T. ruber grown on CO(2) was depleted in (13)C by only 3.3 per thousand relative to C source. In contrast, biomass was depleted by 19.7 per thousand when formate was the C source. Independent of carbon source, T. ruber lipids were heavier than biomass (+1.3 per thousand). The depletion in the C(20-21) fatty acids from the PSC indicates that Thermocrinis biomass must be similarly depleted and too light to be explained by growth on CO(2). Accordingly, Thermocrinis in the PSC is likely to have utilized formate, presumably generated in the spring source region.

FIG. 1

Total ion chromatogram of biofilm-associated siliceous sinter from within Octopus Spring vent pool (92°C) showing predominance of C₁₈ mono- and dialkyl glycerol ethers. Structures are shown for 1,2-di-O-phytanylglycerol and C_18,18-diakylglycerol ether. Symbols designate FAME (n- or i-18), isoprenoid lipid (I-), monoalkyl glycerol (C₁₈), and dialkyl glycerol (C_18,18) with accompanying carbon chain lengths.

FIG. 2

16S rRNA gene-based phylogenetic tree of the Aquificales based on the results of a maximum-likelihood analysis showing C_20–22 signature lipids for this group. Reference sequences were chosen to represent the broadest diversity of Bacteria. Only sequence positions that have identical residues in 50% or more of all available bacterial 16S rRNA sequences were included for tree reconstruction. Accession numbers for the sequences are indicated. The scale bar represents 0.10 fixed mutations per nucleotide position. Of the sequences previously identified in the PSC (°), only EM17 is closely related to Aquificales from this study (*).

FIG. 3

Proposed carbon isotopic profile for PSC showing measured δ¹³C values for isolated PSC components on the left. In the center and to the right, an assumed value of ≈24‰ for Thermocrinis biomass is based on the PSC biomarker C₂₀ fatty acids and the established relationship between T. ruber biomass and fatty acids measured in laboratory cultures. On the right, suggested δ¹³C values for formate and CO₂ based on expressed discriminations measured in laboratory culture experiments. Isotopic value for formate in Octopus Spring vent (upper right) is unknown. In this scenario, mass balance between PSC organic C (hatched box) and Thermocrinis biomass requires a relatively heavy component (gray box) and leaves unresolved the relationship of iso-fatty acids and their associated biomass to the heavy organic carbon pool proposed.