Marine microorganisms adapt to their habitat by structural modification of their membrane lipids. This concept is the basis of numerous molecular proxies used for paleoenvironmental reconstruction. Archaeal tetraether lipids from ubiquitous marine planktonic archaea are particularly abundant, well preserved in the sedimentary record and used in several molecular proxies. We here introduce the direct, extraction-free analysis of these compounds in intact sediment core sections using laser desorption ionization (LDI) coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). LDI FTICR-MS can detect the target lipids in single submillimeter-sized spots on sediment sections, equivalent to a sample mass in the nanogram range, and could thus pave the way for biomarker-based reconstruction of past environments and ecosystems at subannual to decadal resolution. We demonstrate that ratios of selected archaeal tetraethers acquired by LDI FTICR-MS are highly correlated with values obtained by conventional liquid chromatography/MS protocols. The ratio of the major archaeal lipids, caldarchaeol and crenarchaeol, analyzed in a 6.2-cm intact section of Mediterranean sapropel S1 at 250-µm resolution (∼4-y temporal resolution), provides an unprecedented view of the fine-scale patchiness of sedimentary biomarker distributions and the processes involved in proxy signal formation. Temporal variations of this lipid ratio indicate a strong influence of the ∼200-y de Vries solar cycle on reconstructed sea surface temperatures with possible amplitudes of several degrees, and suggest signal amplification by a complex interplay of ecological and environmental factors. Laser-based biomarker analysis of geological samples has the potential to revolutionize molecular stratigraphic studies of paleoenvironments.

Deep subseafloor sediments host a microbial biosphere with unknown impact on global biogeochemical cycles. This study tests previous evidence based on microbial intact polar lipids (IPLs) as proxies of live biomass, suggesting that Archaea dominate the marine sedimentary biosphere. We devised a sensitive radiotracer assay to measure the decay rate of ([14C]glucosyl)-diphytanylglyceroldiether (GlcDGD) as an analog of archaeal IPLs in continental margin sediments. The degradation kinetics were incorporated in model simulations that constrained the fossil fraction of subseafloor IPLs and rates of archaeal turnover. Simulating the top 1 km in a generic continental margin sediment column, we estimated degradation rate constants of GlcDGD being one to two orders of magnitude lower than those of bacterial IPLs, with half-lives of GlcDGD increasing with depth to 310 ky. Given estimated microbial community turnover times of 1.6–73 ky in sediments deeper than 1 m, 50–96% of archaeal IPLs represent fossil signals. Consequently, previous lipid-based estimates of global subseafloor biomass probably are too high, and the widely observed dominance of archaeal IPLs does not rule out a deep biosphere dominated by Bacteria. Reverse modeling of existing concentration profiles suggest that archaeal IPL synthesis rates decline from around 1,000 pg⋅mL−1 sediment⋅y−1 at the surface to 0.2 pg⋅mL−1⋅y−1 at 1 km depth, equivalent to production of 7 × 105 to 140 archaeal cells⋅mL−1 sediment⋅y−1, respectively. These constraints on microbial growth are an important step toward understanding the relationship between the deep biosphere and the carbon cycle.

We conducted a survey of archaeal GDGT (glycerol dibiphytanyl glycerol tetraether) distributions in marine sediments deposited in a range of depositional settings. The focus was comparison of two pools presumed to have distinct geobiological significance, i.e. intact polar GDGTs (IP GDGTs) and core GDGTs (C GDGTs). The former pool has been suggested to be related to living communities of benthic archaea in marine sediments, while the latter is commonly interpreted to consist of molecular fossils from past planktonic archaeal communities that inhabited the surface ocean. Understanding the link between these two pools is important for assessment of the validity of current molecular proxies for sedimentary archaeal biomass and past sea surface temperatures. The relative distributions of GDGTs in the two pools in a core at a CH4 rich site in the Black Sea provide evidence for in situ production of glycosidic IP GDGTs and their subsequent degradation to corresponding C GDGTs on timescales that are short in geological terms. In addition, we monitored the relationship between the IP GDGT and C GDGT pools in a sample set from various ocean basins with subseafloor depth from a few cm to 320 m and 0 to 4 Myr in age. Notable differences between the two pools can be summarized as follows: the GDGT with acyclic biphytanes, GDGT-0, and its analogues with two and three cyclopentane moieties (GDGT-2 and -3) are generally more abundant in the pool of IP GDGTs, while crenarchaeol tends to be more abundant in the C GDGT pool. Consequently, the ring index is generally higher for the C GDGTs while TEX86, a molecular proxy ratio not considering the two major GDGTs, tends to be higher in the IP GDGT pool. These differences in the proportion of individual GDGTs in the two pools are probably due to in situ production of IP GDGTs with distributions differing from those of C GDGTs. Despite these differences, we observed significant correlation of these two ratios between the two pools. Specifically, in both pools TEX86 is high in sediments from warm oceanic regimes and low in cold regimes. We discuss these relationships and suggest that recycling of core GDGTs by benthic archaea is an important mechanism linking both molecular pools.

Two types of intact branched glycerol dialkyl glycerol tetraethers (GDGTs) were detected in peat bog samples from Bullenmoor, Northern Germany. Glucuronosyl and glucosyl branched GDGTs comprise on average ca. 4% of the microbial intact polar lipids in the anoxic, acidic peat layer ca. 20 cm below the surface of the bog, suggesting an important ecological role for the source microorganisms. No corresponding phospholipids were detected. Notably, glycosidic branched GDGTs are 5–10 times less abundant than their intact isoprenoid counterparts derived from Archaea, while branched GDGT core lipids exceed their isoprenoid analogues by about an order of magnitude. These contrasting relationships may reflect lower standing stocks of the biomass of producers of branched GDGTs, combined with higher population growth rates relative to soil Archaea. Search strategies for the microbial producers of these conspicuous orphan lipids should benefit from the discovery of their intact polar precursors.

Previous biomarker studies of microbes involved in anaerobic oxidation of methane (AOM) have targeted non-polar lipids. We have extended the biomarker approach to include intact polar lipids (IPLs) and show here that the major community types involved in AOM at marine methane seeps can be clearly distinguished by these compounds. The lipid profile of methanotrophic communities with dominant ANME-1 archaea mainly comprises diglycosidic GDGT derivatives. IPL distributions of microbial communities dominated by ANME-2 or ANME-3 are consistent with their phylogenetic affiliation with the euryarchaeal order Methanosarcinales, i.e., the lipids are dominated by phosphate-based polar derivatives of archaeol and hydroxyarchaeol. IPLs of associated bacteria strongly differed among the three community types analyzed here; these differences testify to the diversity of bacteria in AOM environments. Generally, the bacterial members of methanotrophic communities are dominated by phosphatidylethanolamine and phosphatidyl-(N,N)-dimethylethanolamine species; polar dialkylglycerolethers are dominant in the ANME-1 community while in ANME-2 and ANME-3 communities mixed acyl/ether glycerol derivatives are most abundant. The relative concentration of bacterial lipids associated with ANME-1 dominated communities appears significantly lower than in ANME-2 and ANME-3 dominated communities. Our results demonstrate that IPL analysis provides valuable molecular fingerprints of biomass composition in natural microbial communities and enables taxonomic differentiation at the rank of families to orders.

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells1, 2. Extrapolation of direct counts of stained microbial cells to the total volume of habitable marine subsurface sediments suggests that between 56 Pg (ref. 1) and 303 Pg (ref. 3) of cellular carbon could be stored in this largely unexplored habitat. From recent studies using various culture-independent techniques, no clear picture has yet emerged as to whether Archaea or Bacteria are more abundant in this extensive ecosystem4, 5, 6, 7. Here we show that in subsurface sediments buried deeper than 1 m in a wide range of oceanographic settings at least 87% of intact polar membrane lipids, biomarkers for the presence of live cells7, 8, are attributable to archaeal membranes, suggesting that Archaea constitute a major fraction of the biomass. Results obtained from modified quantitative polymerase chain reaction and slot-blot hybridization protocols support the lipid-based evidence and indicate that these techniques have previously underestimated archaeal biomass. The lipid concentrations are proportional to those of total organic carbon. On the basis of this relationship, we derived an independent estimate of amounts of cellular carbon in the global marine subsurface biosphere. Our estimate of 90 Pg of cellular carbon is consistent, within an order of magnitude, with previous estimates, and underscores the importance of marine subsurface habitats for global biomass budgets.

Studies of deeply buried, sedimentary microbial communities and associated biogeochemical processes during Ocean Drilling Program Leg 201 showed elevated prokaryotic cell numbers in sediment layers where methane is consumed anaerobically at the expense of sulfate. Here, we show that extractable archaeal rRNA, selecting only for active community members in these ecosystems, is dominated by sequences of uncultivated Archaea affiliated with the Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group, whereas known methanotrophic Archaea are not detectable. Carbon flow reconstructions based on stable isotopic compositions of whole archaeal cells, intact archaeal membrane lipids, and other sedimentary carbon pools indicate that these Archaea assimilate sedimentary organic compounds other than methane even though methanotrophy accounts for a major fraction of carbon cycled in these ecosystems. Oxidation of methane by members of Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group without assimilation of methane–carbon provides a plausible explanation. Maintenance energies of these subsurface communities appear to be orders of magnitude lower than minimum values known from laboratory observations, and ecosystem-level carbon budgets suggest that community turnover times are on the order of 100–2,000 years. Our study provides clues about the metabolic functionality of two cosmopolitan groups of uncultured Archaea.