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RATIONALE Studies of archaeal glycerol dibiphytanyl glycerol tetraethers (GDGTs) in the environment and cultures have exclusively focused on compounds with fully saturated alkyl moieties. Here we report a number of novel unsaturated GDGTs (uns-GDGTs) whose alkyl chains contain up to six double bonds and zero to two cyclopentyl moieties. METHODS The identification of these lipids was achieved via comparison of lipid distribution before and after hydrogenation, characteristic retention time patterns, and diagnostic ions using liquid chromatography/tandem mass spectrometry (LC/MS/MS), and ether cleavage products using gas chromatography/mass spectrometry (GC/MS). Isomerism resulting from different unsaturation patterns in the alkyl moieties was observed and specific positions of double bonds in the biphytene and biphytadiene moieties were tentatively assigned. RESULTS Uns-GDGTs were detected in sediment and microbial mat samples as both core lipids (CLs) and intact polar lipids (IPLs) associated with mono- or diglycosyl or phosphatidylglycerol headgroups. However, these lipids were overlooked in past investigations because conventional methods for archaeal lipid analysis are unsuitable for uns-GDGTs. Samples from distinct marine environments (Black Sea, Cariaco Basin, Discovery Basin, Eastern Mediterranean Sea, upwelling area off NW Africa, and seep sites off Crimea and Pakistan) were screened for uns-GDGTs using a new LC/MS protocol. The results show that uns-GDGTs contribute significantly to the archaeal lipid pool in anoxic methane-rich environments (Black Sea, Cariaco Basin, and both seep sites) but they were barely detected in the oxic or hypersaline settings. CONCLUSIONS The characteristic distribution of uns-GDGTs implies that they are attractive targets for future studies aiming at the chemotaxonomy of uncultivated archaea and regulation of uns-GDGT biosynthesis.

Archaea are ubiquitous and abundant microorganisms on Earth that mediate key global biogeochemical cycles. The headgroup attached to the sn-1 position of the glycerol backbone and the ether-linked isoprenoid lipids are among the diagnostic traits that distinguish Archaea from Bacteria and Eukarya. Over the last 30 years, numerous archaeal lipids have been purified and described in pure cultures. Coupled high-performance liquid chromatography (HPLC) ion-trap mass spectrometry (ITMS) now enables the detection and rapid identification of intact polar lipids in relatively small and complex samples, revealing a wide range of archaeal lipids in natural environments. Although major structural groups have been identified, the lack of a systematic evaluation of MS/MS fragmentation patterns has hindered the characterization of several atypical components that are therefore considered as unknowns. Here, we examined mass spectra resulting from lipid analysis of natural microbial communities using HPLC/electrospray ionization (ESI)-ITMS(n), and depicted the systematics in MS(2) fragmentation of intact archaeal lipids. This report will be particularly useful for environmental scientists interested in a rapid and straightforward characterization of intact archaeal membrane lipids.

Glycolipids are prominent constituents in the membranes of cells from all domains of life. For example, diglycosyl-glycerol dibiphytanyl glycerol tetraethers (2Gly-GDGTs) are associated with methanotrophic ANME-1 archaea and heterotrophic benthic archaea, two archaeal groups of global biogeochemical importance. The hydrophobic biphytane moieties of 2Gly-GDGTs from these two uncultivated archaeal groups exhibit distinct carbon isotopic compositions. To explore whether the isotopic compositions of the sugar headgroups provide additional information on the metabolism of their producers, we developed a procedure to analyze the δ 13 C values of glycosidic headgroups. Successful determination was achieved by (1) monitoring the contamination from free sugars during lipid extraction and preparation, (2) optimizing the hydrolytic conditions for glycolipids, and (3) derivatizing the resulting sugars into aldononitrile acetate derivatives, which are stable enough to withstand a subsequent column purification step. First results of δ 13 C values of sugars cleaved from 2Gly-GDGTs in two marine sediment samples, one containing predominantly ANME-1 archaea and the other benthic archaea, were obtained and compared with the δ 13 C values of the corresponding biphytanes. In both samples the dominant sugar headgroups were enriched in 13 C relative to the corresponding major biphytane. This 13 C enrichment was significantly larger in the putative major glycolipids from ANME-1 archaea (∼15‰) than in those from benthic archaea (