Intact Polar Membrane Lipids Research Articles

Dominance of mixed ether/ester, intact polar membrane lipids in five species of the order Rubrobacterales: Another group of bacteria not obeying the “lipid divide”

The composition of the core lipids and intact polar lipids (IPLs) of five Rubrobacter species was examined. Methylated (ω-4) fatty acids (FAs) characterized the core lipids of Rubrobacter radiotolerans, R. xylanophilus and R. bracarensis. In contrast, R. calidifluminis and R. naiadicus lacked ω-4 methyl FAs but instead contained abundant (i.e., 34–41 % of the core lipids) ω-cyclohexyl FAs not reported before in the order Rubrobacterales. Their genomes contained an almost complete operon encoding proteins enabling production of cyclohexane carboxylic acid CoA thioester, which acts as a building block for ω-cyclohexyl FAs in other bacteria. Hence, the most plausible explanation for the biosynthesis of these cyclic FAs in R. calidifluminis and R. naiadicus is a recent acquisition of this operon. All strains contained 1-O-alkyl glycerol ether lipids in abundance (up to 46 % of the core lipids), in line with the dominance (>90 %) of mixed ether/ester IPLs with a variety of polar headgroups. The IPL head group distribution of R. calidifluminis and R. naiadicus differed, e.g. they lacked a novel IPL tentatively assigned as phosphothreoninol. The genomes of all five Rubrobacter species contained a putative operon encoding the synthesis of the 1-O-alkyl glycerol phosphate, the presumed building block of mixed ether/ester IPLs, which shows some resemblance with an operon enabling ether lipid production in various other aerobic bacteria but requires more study. The uncommon dominance of mixed ether/ester IPLs in Rubrobacter species exemplifies our recent growing awareness that the lipid divide between archaea and bacteria/eukaryotes is not as clear cut as previously thought.

A new and improved protocol for extraction of intact polar membrane lipids from archaea

Knowledge of intact polar membrane lipids (IPLs) is central to investigations of the physiology of archaea in cultures and their prevalence in environmental samples. IPLs are typically extracted using modifications to the ‘Bligh and Dyer’ protocol developed decades before the chemical complexity of IPLs was fully appreciated. Recent studies have exposed both bias and a low extraction efficiency for archaeal lipids obtained by this technique. Here, we tested different solvent mixtures, mechanical rupture techniques and chemical detergents/enzymatic lysis solutions for their potential to increase the extraction efficiency of archaeal IPLs. Experiments were based on an iterative approach using Sulfolobus acidocaldarius biomass as a model organism and incrementally evaluating single-step modifications. Direct acid hydrolysis of biomass was used as a benchmark to assess the recovery of the archaeal lipids. After this initial phase, the resultant protocol was tested on various cultured archaea and environmental samples. Tests with different solvent mixtures revealed the highest S. acidocaldarius lipid yields when samples were extracted with a monophasic solvent system that included a trichloroacetic acid buffer. Freeze-thaw cycles before extraction enhanced the extraction efficiency by 1.8 times, while other mechanical rupture techniques showed no increase. Cetrimonium bromide, a quaternary ammonium surfactant, used in combination with freeze–thaw cycles resulted in up to 8 times higher lipid yields in S. acidocaldarius and other archaeal test cultures compared to experiments without this pretreatment. Extractions of environmental samples revealed contrasting results: while sediments showed little to no increase in archaeal lipid yields, suspended particulate organic matter yielded elevated abundances compared to the Bligh and Dyer extraction protocol.

Trimethylornithine Membrane Lipids: Discovered in Planctomycetes and Identified in Diverse Environments.

Intact polar membrane lipids (IPLs) are the building blocks of all cell membranes. There is a wide range of phosphorus-free IPL structures, including amino acid containing IPLs, that can be taxonomically specific. Trimethylornithine membrane lipids (TMOs) were discovered in northern wetland Planctomycete species that were isolated and described in the last decade. The trimethylated terminal nitrogen moiety of the ornithine amino acid in the TMO structure gives the lipid a charged polar head group, similar to certain phospholipids. Since their discovery, TMOs have been identified in various other recently described northern latitude Planctomycete species, and in diverse environments including tundra soil, a boreal eutrophic lake, meso-oligotrophic lakes, and hot springs. The majority of environments or enrichment cultures in which TMOs have been observed include predominately heterotrophic microbial communities involved in the degradation of recalcitrant material and/or low oxygen methanogenic conditions at primarily northern latitudes. Other ecosystems occupied with microbial communities that possess similar metabolic pathways, such as tropical peatlands or coastal salt marshes, may include TMO producing Planctomycetes as well, further allowing these lipids to potentially be used to understand microbial community responses to environmental change in a wide range of systems. The occurrence of TMOs in hot springs indicates that these unique lipids could have broad environmental distribution with different specialized functions. Opportunities also exist to investigate the application of TMOs in microbiome studies, including forensic necrobiomes. Further environmental and microbiome lipidomics research involving TMOs will help reveal the evolution, functions, and applications of these unique membrane lipids.

Shrinking majority of the deep biosphere

Living microorganisms were first discovered in sediment cores from scientific ocean drilling in the late 1980s when microbiologists observed and counted DNA-stained microbial cells under the microscope. During the following decade, new observations were made from drill sites in the Atlantic and Pacific Oceans and the Mediterranean Sea (1, 2). The combined data on cell numbers from these sites showed a systematic, log-log linear decrease with depth, from approximately 109 cells⋅cm−3 near the sediment surface to 106 cells⋅cm−3 at several hundred meters below seafloor, where the sediment had been deposited many million years ago (2) (Fig. 1). In their 1998 study, Whitman et al. (3) used these data to make a grand estimate of the total number of prokaryotic cells on Earth. Their astonishing conclusion was that 55% of all microbial cells occurred in the deep seabed whereas most of the rest were found in the deep terrestrial subsurface. Even more staggering, the combined deep biospheres appeared to harbor one third of the total living biomass on Earth. The publication of Whitman et al. (3) sparked a rapidly growing interest in the deep biosphere, and the data from this study were repeatedly cited in the literature in the following years. Their biomass estimate was later supported by data on intact polar membrane lipids as indicators of living cells (4). Now, these numbers are being challenged. In PNAS, Kallmeyer et al. (5) conclude that the total cell number for the deep subseafloor biosphere is probably 12-fold lower than the earlier estimate whereas the total biomass may be 74-fold lower. Is the deep biosphere losing significance?

Gradients in intact polar diacylglycerolipids across the Mediterranean Sea are related to phosphate availability

Abstract. Intact polar membrane lipids compose a significant fraction of cellular material in plankton and their synthesis imposes a substantial constraint on planktonic nutrient requirements. As a part of the Biogeochemistry from the Oligotrophic to the Ultraoligotrophic Mediterranean (BOUM) cruise we examined the distribution of several classes of intact polar diacylglycerolipids (IP-DAGs) across the Mediterranean, and found that phospholipid concentration as a percent of total lipids correlated with phosphate concentration. In addition, the ratios of non-phosphorus lipids to phospholipids – sulfoquinovosyldiacylglycerol (SQDG) to phosphatidylglycerol (PG), and betaine lipids to phosphatidylcholine (PC) – were also found to increase from west to east across the Mediterranean. Additionally, microcosm incubations from across the Mediterranean were amended with phosphate and ammonium, and in the course of several days nutrient amendments elicited a shift in the ratios of IP-DAGs. These experiments were used to assess the relative contribution of community shifts and physiological response to the observed change in IP-DAGs across the Mediterranean. The ratio of SQDG to chlorophyll-a was also explored as an indicator of phytoplankton response to nitrogen availability. This study is the first to demonstrate the dynamic response of membrane lipid composition to changes in nutrients in a natural, mixed planktonic community.

Symbiont–host relationships in chemosynthetic mussels: A comprehensive lipid biomarker study

Symbiosis with chemosynthetic microorganisms allows invertebrates from hydrothermal vents and cold seeps, such as mussels, snails and tubeworms, to gain nutrition independently of organic input from photosynthetic communities. Lipid biomarkers and their compound specific stable carbon isotopes ( δ 13C) have greatly aided the elucidation of chemosynthetic symbiosis. Due to recent methodological advances in liquid chromatography it is now possible to obtain a more holistic view of lipid biomarkers, including the analysis of intact polar membrane lipids (IPLs) and bacteriohopanepolyols (BHPs). This study provides an extensive examination of polar and apolar lipids in combination with stable carbon isotope analysis of three Bathymodiolus mussels ( Bathymodiolus childressi, Bathymodiolus cf. thermophilus, Bathymodiolus brooksi) hosting different types of bacterial symbiont (methane-oxidizing, sulfur-oxidizing and a dual symbiosis with methane- and sulfur-oxidizing symbionts, respectively). We propose that IPLs with C 16:1 acyl side chains, and phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE) head groups, which were only detected in the gill tissue, can be used as symbiont-characteristic biomarkers. These putative symbiont-specific IPLs provide the opportunity to detect and quantify the methanotrophic and thiotrophic symbionts within the gill tissue. Additional characteristic markers for methanotrophic symbionts were found in B. childressi and B. brooksi, including the BHP derivatives aminotriol and aminotetrol, 4-methyl sterols and diagnostic fatty acids (FAs), such as C 16:1ω9, C 16:1ω8, and C 18:1ω8. In general, the δ 13C values of FAs, alcohols and BHP-derived hopanols were in accordance with carbon assimilation pathways of the respective methanotrophic or thiotrophic symbionts in all three Bathymodiolus mussels. Differences in BHP distribution as well as δ 13C values in the two mussels hosting a methanotrophic symbiont may indicate the presence of different methanotrophic symbionts and/or changes in the nutritional status. In all three mussel species the δ 13C values of lipid biomarkers assigned to the symbionts were similar to those of the hosts, indicating the importance of the bacterial symbionts as the main carbon source for the mussel tissue.

Petroleum degradation and associated microbial signatures at the Chapopote asphalt volcano, Southern Gulf of Mexico

At the Chapopote Knoll in the Southern Gulf of Mexico, deposits of asphalt provide the substrate for a prolific cold seep ecosystem extensively colonized by chemosynthetic communities. This study investigates microbial life and associated biological processes within the asphalts and surrounding oil-impregnated sediments by analysis of intact polar membrane lipids (IPLs), petroleum hydrocarbons and stable carbon isotopic compositions (δ 13C) of hydrocarbon gases. Asphalt samples are lightly to heavily biodegraded suggesting that petroleum-derived hydrocarbons serve as substrates for the chemosynthetic communities. Accordingly, detection of bacterial diester and diether phospholipids in asphalt samples containing finely dispersed gas hydrate suggests the presence of hydrocarbon-degrading bacteria. Biological methanogenesis contributes a substantial fraction to the methane captured as hydrate in the shallow asphalt deposits evidenced by significant depletion in 13C relative to background thermogenic methane. In sediments, petroleum migrating from the subsurface stimulates both methanogenesis and methanotrophy at a sulfate–methane transition zone 6–7 m below the seafloor. In this zone, microbial IPLs are dominated by archaeal phosphohydroxyarchaeols and archaeal diglycosidic diethers and tetraethers. Bacterial IPLs dominate surface sediments that are impregnated by severely biodegraded oil. In the sulfate-reduction zone, diagnostic IPLs indicate that sulfate-reducing bacteria (SRB) play an important role in petroleum degradation. A diverse mixture of phosphohydroxyarchaeols and mixed phospho- and diglycosidic archaeal tetraethers in shallow oil-impregnated sediments point to the presence of anaerobic methane-oxidizing ANME-2 and ANME-1 archaea, respectively, or methanogens. Archaeal IPLs increase in relative abundance with increasing sediment depth and decreasing sulfate concentrations, accompanied by a shift of archaeol-based to tetraether-based archaeal IPLs. The latter shift is suggested to be indicative of a community shift from ANME-2 and/or methanogenic archaea in shallower sediments to ANME-1/methanogenic archaea and possibly benthic archaea in deeper sediments.

Stable carbon isotopic compositions of intact polar lipids reveal complex carbon flow patterns among hydrocarbon degrading microbial communities at the Chapopote asphalt volcano

Seepage of asphalt forms the basis of a cold seep system at 3000 m water depth at the Chapopote Knoll in the southern Gulf of Mexico. Anaerobic microbial communities are stimulated in the oil-impregnated sediments as evidenced by the presence of intact polar membrane lipids (IPLs) derived from archaea and Bacteria at depths up to 7 m below the seafloor. Detailed investigation of stable carbon isotope composition (δ 13C) of alkyl and acyl moieties derived from a range of IPL precursors with distinct polar head groups resolved the complexity of carbon metabolisms and utilization of diverse carbon sources by uncultured microbial communities. In surface sediments most of the polar lipid-derived fatty acids with phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) head groups could be tentatively assigned to autotrophic sulfate-reducing bacteria, with a relatively small proportion involved in the anaerobic oxidation of methane. Derivatives of phosphatidyl-( N)-methylethanolamine (PME) were abundant and could be predominantly assigned to heterotrophic oil-degrading bacteria. Archaeal IPLs with phosphate-based hydroxyarchaeols and diglycosidic glyceroldibiphytanylglyceroltetraethers (GDGTs) were assigned to methanotrophic archaea of the ANME-2 and ANME-1 cluster, respectively, whereas δ 13C values of phosphate-based archaeols and mixed phosphate-based and diglycosidic GDGTs point to methanogenic archaea. At a 7 m deep sulfate–methane transition zone that is linked to the upward movement of gas-laden petroleum, a distinct increase in abundance of archaeal IPLs such as phosphate-based hydroxyarchaeols and diglycosidic archaeol and GDGTs is observed; their δ 13C values are consistent with their origin from both methanotrophic and methanogenic archaea. This study reveals previously hidden, highly complex patterns in the carbon-flow of versatile microbial communities involved in the degradation of heavy oil including hydrocarbon gases that would not have been evident from classical compound-specific isotope analyses of either bulk IPL or apolar lipid derivatives.

Microbial sources of intact polar diacylglycerolipids in the Western North Atlantic Ocean

Intact polar membrane lipids are essential components of microbial membranes and recent work has uncovered a diversity of them occurring in the ocean. While it has long been understood that lipid composition varies across microbial groups, the microbial origins of the intact polar lipids in the surface ocean remain to be fully explained. This study focused on identifying the microbial sources of intact polar diacylglycerolipids (IP-DAGs) in the surface waters of the western North Atlantic Ocean. We used three approaches to define these microbial sources: (i) 13C tracing to identify photoautotrophic and heterotrophic production of the major classes of IP-DAGs, (ii) cell sorting flow cytometry of Prochlorococcus, Synechococcus and heterotrophic bacteria to determine IP-DAG composition and (iii) regrowth incubations targeting IP-DAG production by heterotrophic bacteria. Stable isotope tracing indicated that sulfoquinovosyldiacylglycerol (SQDG) and diacylglyceryl-trimethyl-homoserine (DGTS) were produced predominantly by photoautotrophs, while phosphatidylglycerol (PG) production was dominated by heterotrophic bacteria. Of the cells sorted with flow cytometry, Prochlorococcus and Synechococcus were found to have abundant glycolipids, while heterotrophic bacteria were dominated by phospholipids. The regrowth incubations showed that the growth of heterotrophic bacteria correlated with an increase in the concentration of PG, phosphatidylethanolamine (PE) and monoglycosyldiacylglycerol (MGDG). The finding of MGDG in heterotrophic bacteria differs from previous work, which had asserted that the membranes of heterotrophic bacteria in this environment were composed entirely of phospholipids. Overall, our findings indicate that phytoplankton are the primary source of SQDG and DGTS, while heterotrophic bacteria are the dominant source of PG, making these three compounds promising biomarkers for the study of microbes in the surface ocean.

Factors controlling the distribution of anaerobic methanotrophic communities in marine environments: Evidence from intact polar membrane lipids

Three distinct types of microbial consortia appear to mediate the anaerobic oxidation of methane with sulfate as electron acceptor in marine sediments and are distributed ubiquitously. These consortia consist of ANerobic MEthanotrophic (ANME) archaea of the ANME-1, ANME-2 and ANME-3 clades and their sulfate-reducing bacterial partners either of the Desulfosarcina– Desulfococcus (ANME-1/DSS and ANME-2/DSS) or Desulfobulbus spp. (ANME-3/DBB) branches. Frequently one consortium type dominates the community, but the selective factors are not well constrained. Here we analyzed patterns in the composition of intact polar lipids extracted from bacterial and archaeal communities of different marine seep environments. Further, we investigated if different environmental and geographical factors were responsible for the observed patterns, and hence could be important in the selection of seep communities. Intact polar lipids (IPLs) provide a more robust distinction of the composition of extant communities than their less polar derivatives. In ANME-1/DSS-dominated communities, glycosidic- and phospho-glyceroldialkylglyceroltetraethers were abundant, while ANME-2/DSS and ANME-3/DBB-dominated communities showed abundant archaeol-based IPLs, either with glycosidic and phospho-headgroups or only phospho-headgroups, respectively. The relative proportion of bacterial IPLs varied widely from 0% to 93% and was generally lower in samples of the ANME-1 type, suggesting lower bacterial biomasses in the respective communities. In addition to these lipid signatures, distinctive features were related to the habitat characteristics of these communities: lower amounts of phosphate-based IPLs were generally observed in communities from calcified microbial mats compared to sediments, which may reflect phosphate limitation. Based on statistical analyses of IPLs and environmental data this study constrained for the first time the occurrence of three environmental factors controlling the distribution of different ANME-associated communities in a wide range of hydrocarbon seep systems. Habitats dominated by ANME-1/DSS communities were characterized by high temperature and low oxygen content in bottom waters (or even anoxia), while ANME-2/DSS and ANME-3/DBB-dominated sediments were located in settings with lower temperatures and higher oxygen content in bottom waters. Furthermore, ANME-2/DSS communities were particularly prominent in environments in which a relatively high supply of sulfate was sustained.

Distribution of polar membrane lipids in permafrost soils and sediments of a small high Arctic catchment

The distribution of bacteriohopanepolyols (BHPs) and intact polar membrane lipids (IPLs) was studied in different soil profiles and in fluvial and marine sediments along the pathway of transport in a small catchment in the high Arctic at Ny-Ålesund, Svalbard (79°N, 12°E). Both groups of cellular membrane lipids have been used as biomarkers for determining major microbial groups and biogeochemical processes in natural environments. Little is known about their distribution and usefulness as environmental markers in the Arctic. In this study, we show that both membrane lipid groups are variably distributed in the drainage area of the Brøggerbreen Glacier, with highest abundance and diversity in the organic mat overlying the mineral soils, as well as in mossy surface soil. The BHP and IPL composition and abundance both indicate a larger community of phototrophic bacteria in the organic mats and fluvial sediments. The greater diversity and abundance of BHPs relative to IPLs in sub-soils, including probable markers for cyanobacteria, suggest a greater resistance to degradation. In contrast, IPL concentration decreased strongly with soil depth, pointing to their rapid degradation. Bacterial IPLs are thus thought to reflect more reliably the biomass of viable phototrophic bacteria. The high concentrations of proposed soil marker BHPs, adenosylhopane and related structures vs. published results and their relative increase with soil depth suggest better preservation and more effective accumulation of the compounds at low temperature.

Advances of Intact Polar Membrane Lipids as Chemical Biomarkers for Extant Microorganisms in Marine Sediments

Intact polar membrane lipids(IPLs)can serve as chemical biomarkers of living cells that can provide information on the extant microbial community structure and in situ microbial processes.IPLs analysis is a culture independent,rapid and non-selective procedure in contrast to biological methods.The compositions of cell membrane intact polar membrane lipids of bacteria and Achaea and their applications in the researches of biogeochemistry and microbial ecology are summarized in this paper.The recent progresses of IPLs applications are reviewed,with emphasis on those microorganisms that play a specific role in biogeochemical cycle,such as anaerobic ammonium-oxidizing bacteria,anaerobic methanotrophic Achaea,ammonia-oxidizing Achaea and marine Crenarcheota with tetraether lipids,or those special ecological systems,such as hydrothermal vent,cold seep and marine deep biosphere.The advances of analytical method for microbial IPLs in marine sediments are briefly introduced,and their application prospects are proposed.

Fossilization and degradation of intact polar lipids in deep subsurface sediments: A theoretical approach

Intact polar membrane lipids (IPLs) are frequently used as markers for living microbial cells in sedimentary environments. The assumption with these studies is that IPLs are rapidly degraded upon cell lysis and therefore IPLs present in sediments are derived from in situ microbial production. We used a theoretical approach to assess whether IPLs in surface sediments can potentially represent fossilized IPLs derived from the upper part of the water column and whether IPLs can be preserved during sediment burial. Previous studies which examined the degradation kinetics of IPLs show that phospholipids, i.e. ester-linked lipids with a phosphor-containing head group, degrade more rapidly than glycosidic ether lipids, i.e. ether-linked lipids with a glycosidically bound sugar moiety. Based on these studies, we calculate that only a minor fraction of phospholipids but a major fraction of glycosidic ether lipids biosynthesized in the upper part of the water column can potentially reach deep-sea surface sediments. Using a simple model and power law kinetic degradation parameters reported in the literature, we also evaluated the degradation of IPLs during sediment burial. Our model predicts a log–log relationship between IPL concentrations and depth, consistent with what has been observed in studies of IPLs in subsurface sediments. Although our results do not exclude production of IPLs in subsurface sediment, they do suggest that IPLs present in the deep biosphere may contain a substantial fossil component potentially masking in situ IPL production.

Detection of microbial biomass by intact polar membrane lipid analysis in the water column and surface sediments of the Black Sea

The stratified water column of the Black Sea produces a vertical succession of redox zones, stimulating microbial activity at the interfaces. Our study of intact polar membrane lipids (IPLs) in suspended particulate matter and sediments highlights their potential as biomarkers for assessing the taxonomic composition of live microbial biomass. Intact polar membrane lipids in oxic waters above the chemocline represent contributions of bacterial and eukaryotic photosynthetic algae, while anoxygenic phototrophic bacteria and sulfate-reducing bacteria comprise a substantial amount of microbial biomass in deeper suboxic and anoxic layers. Intact polar membrane lipids such as betaine lipids and glycosidic ceramides suggest unspecified anaerobic bacteria in the anoxic zone. Distributions of polar head groups and core lipids show planktonic archaea below the oxic zone; methanotrophic archaea are only a minor fraction of archaeal biomass in the anoxic zone, contrasting previous observations based on the apolar derivatives of archaeal lipids. Sediments contain algal and bacterial IPLs from the water column, but transport to the sediment is selective; bacterial and archaeal IPLs are also produced within the sediments. Intact polar membrane lipid distributions in the Black Sea are stratified in accordance with geochemical profiles and provide information on vertical successions of major microbial groups contributing to suspended biomass. This study vastly extends our knowledge of the distribution of complex microbial lipids in the ocean.

Structural diversity and fate of intact polar lipids in marine sediments

Marine sediments harbor an enormous quantity of microorganisms, including a multitude of novel species. The habitable zone of the marine sediment column begins at the sediment–water interface and probably extends to depths of several thousands of meters. Studies of the microbial diversity in this ecosystem have mostly relied on molecular biological techniques. We used a complementary method – analysis of intact polar membrane lipids – to characterize the in-situ microbial community in sediments covering a wide range of environmental conditions from Peru Margin, Equatorial Pacific, Hydrate Ridge, and Juan de Fuca Ridge. Bacterial and eukaryotic phospholipids were only detected in surface sediments from the Peru Margin. In contrast, deeply buried sediments, independent of their geographic location, were dominated by archaeal diether and tetraether lipids with various polar head groups and core lipids. We compared ring distributions of archaeal tetraether lipids derived from polar glycosidic precursors with those that are present as core lipids. The distributions of these related compound pools were distinct, suggestive of different archaeal sources, i.e., the polar compounds derive from sedimentary communities and the core lipids are fossil remnants from planktonic communities with possible admixtures of decayed sedimentary archaea. This in-situ production of distinct archaeal lipid populations potentially affects applications of the TEX86 paleotemperature proxy as demonstrated by offsets in reconstructed temperatures between both pools. We evaluated how varying cell and lipid stabilities will influence the sedimentary pool by using a box-model. The results are consistent with (i) a requirement of continuous inputs of freshly synthesized lipids in subsurface sediments for explaining the observed distribution of intact polar lipids, and (ii) decreasing lipid inputs with increasing burial depth.

Methods for Recovery of Microorganisms and Intact Microbial Polar Lipids from Oil−Water Mixtures: Laboratory Experiments and Natural Well-Head Fluids

Most of the world's remaining petroleum resource has been altered by in-reservoir biodegradation which adversely impacts oil quality and production, ultimately making heavy oil. Analysis of the microorganisms in produced reservoir fluid samples is a route to characterization of subsurface biomes and a better understanding of the resident and living microorganisms in petroleum reservoirs. The major challenges of sample contamination with surface biota, low abundances of microorganisms in subsurface samples, and viscous emulsions produced from biodegraded heavy oil reservoirs are addressed here in a new analytical method for intact polar lipids (IPL) as taxonomic indicators in petroleum reservoirs. We have evaluated the extent to which microbial cells are removed from the free water phase during reservoir fluid phase separation by analysis of model reservoir fluids spiked with microbial cells and have used the resultant methodologies to analyze natural well-head fluids from the Western Canada Sedimentary Basin (WCSB). Analysis of intact polar membrane lipids of microorganisms using liquid chromatography-mass spectrometry (LC-MS) techniques revealed that more than half of the total number of microorganisms can be recovered from oil-water mixtures. A newly developed oil/water separator allowed for filtering of large volumes of water quickly while in the field, which reduced the chances of contamination and alterations to the composition of the subsurface microbial community after sample collection. This method makes the analysis of IPLs (or indirectly microorganisms) from well-head fluids collected in remote field settings possible and reliable. To the best of our knowledge this is the first time that IPLs have been detected in well-head oil-water mixtures.

Significant contribution of Archaea to extant biomass in marine subsurface sediments

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells. 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 ecosystem. 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 cells, 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.

Methane-Producing Microbial Community in a Coal Bed of the Illinois Basin

A series of molecular and geochemical studies were performed to study microbial, coal bed methane formation in the eastern Illinois Basin. Results suggest that organic matter is biodegraded to simple molecules, such as H(2) and CO(2), which fuel methanogenesis and the generation of large coal bed methane reserves. Small-subunit rRNA analysis of both the in situ microbial community and highly purified, methanogenic enrichments indicated that Methanocorpusculum is the dominant genus. Additionally, we characterized this methanogenic microorganism using scanning electron microscopy and distribution of intact polar cell membrane lipids. Phylogenetic studies of coal water samples helped us develop a model of methanogenic biodegradation of macromolecular coal and coal-derived oil by a complex microbial community. Based on enrichments, phylogenetic analyses, and calculated free energies at in situ subsurface conditions for relevant metabolisms (H(2)-utilizing methanogenesis, acetoclastic methanogenesis, and homoacetogenesis), H(2)-utilizing methanogenesis appears to be the dominant terminal process of biodegradation of coal organic matter at this location.

Intact polar membrane lipids in prokaryotes and sediments deciphered by high-performance liquid chromatography/electrospray ionization multistage mass spectrometry--new biomarkers for biogeochemistry and microbial ecology.

Lipids from prokaryotic cell membranes can serve as sources of information on the biogeochemistry and microbial ecology of natural ecosystems. Traditionally, apolar derivatives of the intact polar membrane molecules, e.g., fatty acids, have been the major target of lipid-based biogeochemical studies. However, when still intact, i.e., as glycerol esters and ethers with attached polar headgroups, membrane lipids are diagnostic for living prokaryotes, which makes them excellent biomarkers for the study of in situ microbial processes in geological systems such as sediments or soils. Intact polar lipids (IPLs) are attractive analytical targets because they are taxonomically more specific than their apolar derivatives and avoid exclusion of signals from prokaryotes that primarily build their membranes with ether-bound lipids such as archaea and some bacteria. Here we report results from analyses of IPLs in pure cultures of biogeochemically relevant prokaryotes and marine sediments by high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry (HPLC/ESI-IT-MSn). This technique is suitable for screening of biomass and environmental samples for distinctive taxonomic structural features such as distribution of polar lipid headgroups, types of bonds between alkyl moiety and glycerol backbone, and the chain length and degree of unsaturation in the alkyl moieties. We present analytical protocols to decipher structural information from mass spectral data. The IPL contents in selected archaeal and bacterial species are diverse and qualify as molecular fingerprints. Applied to marine sediments, the approach provided detailed information on the dominant microbial groups. The IPLs from bacterial members of anaerobic methanotrophic communities in surface sediments at Hydrate Ridge resemble those found in Desulfosarcina variabilis. The presence of dietherglycerophospholipids, however, suggests the presence of other bacteria possibly affiliated with the deepest phylogenetic branches in the tree of life. Sediments from approximately 90 m below the seafloor on the Peruvian continental margin are dominated by intact archaeal tetraethers with glycosidically bound hexoses as headgroups, consistent with a significant fraction of the community being archaea. Additional calditol-based tetraethers imply that the sedimentary archaea are taxonomically linked to the crenarchaeal Sulfolobales.