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Understanding how microbial lipidomes adapt to environmental and nutrient stress is crucial for comprehending microbial survival and functionality. Certain anaerobic bacteria can synthesize glycerolipids with ether/ester bonds, yet the complexities of their lipidome remodeling under varying physicochemical and nutritional conditions remain largely unexplored. In this study, we thoroughly examined the lipidome adaptations of Desulfatibacillum alkenivorans strain PF2803 T , a mesophilic anaerobic sulfate-reducing bacterium known for its high proportions of alkylglycerol ether lipids in its membrane, under various cultivation conditions including temperature, pH, salinity, and ammonium and phosphorous concentrations. Employing an extensive analytical and computational lipidomic methodology, we identified an assemblage of nearly 400 distinct lipids, including a range of glycerol ether/ester lipids with various polar head groups. Information theory-based analysis revealed that temperature fluctuations and phosphate scarcity profoundly influenced the lipidome's composition, leading to an enhanced diversity and specificity of novel lipids. Notably, phosphorous limitation led to the biosynthesis of novel glucuronosylglycerols and sulfur-containing aminolipids, termed butyramide cysteine glycerols, featuring various ether/ester bonds. This suggests a novel adaptive strategy for anaerobic heterotrophs to thrive under phosphorus-depleted conditions, characterized by a diverse array of nitrogen- and sulfur-containing polar head groups, moving beyond a reliance on conventional nonphospholipid types., Competing Interests: Competing interests statement:The authors declare no competing interest.

The composition of membrane lipids varies in a number of ways as adjustment to growth conditions. Variations in head group composition and carbon skeleton and degree of unsaturation of glycerol-bound acyl or alkyl chains results in a high structural complexity of the lipidome of bacterial cells. We studied the lipidome of the mesophilic, sulfate-reducing bacterium, Desulfatibacillum alkenivorans strain PF2803 T by ultra-high-pressure liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS n ). This anaerobic bacterium has been previously shown to produce high amounts of mono-and di-alkyl glycerol ethers as core membrane lipids. Our analyses revealed that these core lipids occur with phosphatidylethanomamine (PE) and phosphatidylglycerol (PG) head groups, representing each approximately one third of the phospholipids. The third class was a novel group of phospholipids, i.e., cardiolipins (CDLs) containing one (monoether/triester) to four (tetraether) ether-linked saturated straight-chain or methyl-branched alkyl chains. Tetraether CDLs have been shown to occur in archaea (with isoprenoid alkyl chains) but have not been previously reported in the bacterial Domain. Structurally related CDLs with one or two alkyl/acyl chains missing, so-called monolyso-and dilyso-CDLs, were also observed. The potential biosynthetic pathway of these novel CDLs was investigated by examining the genome of D. alkenivorans . Three CDL synthases were identified; one catalyzes the condensation of two PGs, the other two are probably involved in the condensation of a PE with a PG. A heterologous gene expression experiment showed the in vivo production of dialkylglycerols upon anaerobic expression of the glycerol ester reductase enzyme of D. alkenivorans in E. coli . Reduction of the ester bonds probably occurs first at the sn -1 and subsequently at the sn -2 position after the formation of PEs and PGs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hopmans, Grossi, Sahonero-Canavesi, Bale, Cravo-Laureau and Sinninghe Damsté.)

Sulfurimonas species are among the most abundant sulfur-oxidizing bacteria in the marine environment. They are capable of using different electron acceptors, this metabolic flexibility is favorable for their niche adaptation in redoxclines. When oxygen is depleted, most Sulfurimonas spp. (e.g., Sulfurimonas gotlandica) use nitrate ([Formula: see text]) as an electron acceptor to oxidize sulfur, including sulfide (HS - ), S 0 and thiosulfate, for energy production. Candidatus Sulfurimonas marisnigri SoZ1 and Candidatus Sulfurimonas baltica GD2, recently isolated from the redoxclines of the Black Sea and Baltic Sea respectively, have been shown to use manganese dioxide (MnO 2 ) rather than [Formula: see text] for sulfur oxidation. The use of different electron acceptors is also dependent on differences in the electron transport chains embedded in the cellular membrane, therefore changes in the membrane, including its lipid composition, are expected but are so far unexplored. Here, we used untargeted lipidomic analysis to reveal changes in the composition of the lipidomes of three representative Sulfurimonas species grown using either [Formula: see text] and MnO 2 . We found that all Sulfurimonas spp. produce a series of novel phosphatidyldiazoalkyl-diacylglycerol lipids. Ca. Sulfurimonas baltica GD2 adapts its membrane lipid composition depending on the electron acceptors it utilizes for growth and survival. When carrying out MnO 2 -dependent sulfur oxidation, the novel phosphatidyldiazoalkyl-diacylglycerol headgroup comprises shorter alkyl moieties than when sulfur oxidation is [Formula: see text]-dependent. This is the first report of membrane lipid adaptation when an organism is grown with different electron acceptors. We suggest novel diazoalkyl lipids have the potential to be used as a biomarker for different conditions in redox-stratified systems., (© 2022. The Author(s).)

Metabolites exuded by primary producers comprise a significant fraction of marine dissolved organic matter, a poorly characterized, heterogenous mixture that dictates microbial metabolism and biogeochemical cycling. We present a foundational untargeted molecular analysis of exudates released by coral reef primary producers using liquid chromatography-tandem mass spectrometry to examine compounds produced by two coral species and three types of algae (macroalgae, turfing microalgae, and crustose coralline algae [CCA]) from Mo'orea, French Polynesia. Of 10,568 distinct ion features recovered from reef and mesocosm waters, 1,667 were exuded by producers; the majority (86%) were organism specific, reflecting a clear divide between coral and algal exometabolomes. These data allowed us to examine two tenets of coral reef ecology at the molecular level. First, stoichiometric analyses show a significantly reduced nominal carbon oxidation state of algal exometabolites than coral exometabolites, illustrating one ecological mechanism by which algal phase shifts engender fundamental changes in the biogeochemistry of reef biomes. Second, coral and algal exometabolomes were differentially enriched in organic macronutrients, revealing a mechanism for reef nutrient-recycling. Coral exometabolomes were enriched in diverse sources of nitrogen and phosphorus, including tyrosine derivatives, oleoyl-taurines, and acyl carnitines. Exometabolites of CCA and turf algae were significantly enriched in nitrogen with distinct signals from polyketide macrolactams and alkaloids, respectively. Macroalgal exometabolomes were dominated by nonnitrogenous compounds, including diverse prenol lipids and steroids. This study provides molecular-level insights into biogeochemical cycling on coral reefs and illustrates how changing benthic cover on reefs influences reef water chemistry with implications for microbial metabolism., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Membrane-spanning lipids are present in a wide variety of archaea, but they are rarely in bacteria. Nevertheless, the (hyper)thermophilic members of the order Thermotogales harbor tetraester, tetraether, and mixed ether/ester membrane-spanning lipids mostly composed of core lipids derived from diabolic acids, C 30 , C 32 , and C 34 dicarboxylic acids with two adjacent mid-chain methyl substituents. Lipid analysis of Thermotoga maritima across growth phases revealed a decrease of the relative abundance of fatty acids together with an increase of diabolic acids with independence of growth temperature. We also identified isomers of C 30 and C 32 diabolic acids, i.e., dicarboxylic acids with only one methyl group at C-15. Their distribution suggests they are products of the condensation reaction but are preferably produced when the length of the acyl chains is not optimal. Compared with growth at the optimal temperature of 80°C, an increase of glycerol ether-derived lipids was observed at 55°C. Our analysis only detected diabolic acid-containing intact polar lipids with phosphoglycerol (PG) head groups. Considering these findings, we hypothesize a biosynthetic pathway for the synthesis of membrane-spanning lipids based on PG polar lipid formation, suggesting that the protein catalyzing this process is a membrane protein. We also identified, by genomic and protein domain analyses, a gene coding for a putative plasmalogen synthase homologue in T. maritima that is also present in other bacteria producing sn -1-alkyl ether lipids but not plasmalogens, suggesting it is involved in the conversion of the ester-to-ether bond in the diabolic acids bound in membrane-spanning lipids. IMPORTANCE Membrane-spanning lipids are unique compounds found in most archaeal membranes, but they are also present in specific bacterial groups like the Thermotogales . The synthesis and physiological role of membrane-spanning lipids in bacteria represent an evolutionary and biochemical open question that points to the differentiation of the membrane lipid composition. Understanding the formation of membrane-spanning lipids is crucial to solving this question and identifying the enzymatic and biochemical mechanism performing this procedure. In the present work, we found changes at the core lipid level, and we propose that the growth phase drives the biosynthesis of these lipids rather than temperature. Our results identified physiological conditions influencing the membrane-spanning lipid biosynthetic process, which can further clarify the pathway leading to the biosynthesis of these compounds.

Particulate pyrogenic carbon (PyC) transported by rivers and aerosols, and deposited in marine sediments, is an important part of the carbon cycle. The chemical composition of PyC is temperature dependent and levoglucosan is a source-specific burning marker used to trace low-temperature PyC. Levoglucosan associated to particulate material has been shown to be preserved during riverine transport and marine deposition in high- and mid-latitudes, but it is yet unknown if this is also the case for (sub)tropical areas, where 90% of global PyC is produced. Here, we investigate transport and deposition of levoglucosan in suspended and riverbed sediments from the Amazon River system and adjacent marine deposition areas. We show that the Amazon River exports negligible amounts of levoglucosan and that concentrations in sediments from the main Amazon tributaries are not related to long-term mean catchment-wide fire activity. Levoglucosan concentrations in marine sediments offshore the Amazon Estuary are positively correlated to total organic content regardless of terrestrial or marine origin, supporting the notion that association of suspended or dissolved PyC to biogenic particles is critical in the preservation of PyC. We estimate that 0.5-10 × 10 6  g yr -1 of levoglucosan is exported by the Amazon River. This represents only 0.5-10 ppm of the total exported PyC and thereby an insignificant fraction, indicating that riverine derived levoglucosan and low-temperature PyC in the tropics are almost completely degraded before deposition. Hence, we suggest caution in using levoglucosan as tracer for past fire activity in tropical settings near rivers., (© 2021. The Authors.)

Lipids, as one of the main building blocks of cells, can provide valuable information on microorganisms in the environment. Traditionally, gas or liquid chromatography coupled to mass spectrometry (MS) has been used to analyze environmental lipids. The resulting spectra were then processed through individual peak identification and comparison with previously published mass spectra. Here, we present an untargeted analysis of MS 1 spectral data generated by ultra-high-pressure liquid chromatography coupled with high-resolution mass spectrometry of environmental microbial communities. Rather than attempting to relate each mass spectrum to a specific compound, we have treated each mass spectrum as a component, which can be clustered together with other components based on similarity in their abundance depth profiles through the water column. We present this untargeted data visualization method on lipids of suspended particles from the water column of the Black Sea, which included >14,000 components. These components form clusters that correspond with distinct microbial communities driven by the highly stratified water column. The clusters include both known and unknown compounds, predominantly lipids, demonstrating the value of this rapid approach to visualize component distributions and identify novel lipid biomarkers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bale, Ding, Hopmans, Arts, Villanueva, Boschman, Haas, Schouten and Sinninghe Damsté.)

Structurally diverse, specialized lipids are crucial components of microbial membranes and other organelles and play essential roles in ecological functioning. The detection of such lipids in the environment can reveal not only the occurrence of specific microbes but also the physicochemical conditions to which they are adapted to. Traditionally, liquid chromatography coupled with mass spectrometry allowed for the detection of lipids based on chromatographic separation and individual peak identification, resulting in a limited data acquisition and targeting of certain lipid groups. Here, we explored a comprehensive profiling of microbial lipids throughout the water column of a marine euxinic basin (Black Sea) using ultra high-pressure liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS). An information theory framework combined with molecular networking based on the similarity of the mass spectra of lipids enabled us to capture lipidomic diversity and specificity in the environment, identify novel lipids, differentiate microbial sources within a lipid group, and discover potential biomarkers for biogeochemical processes. The workflow presented here allows microbial ecologists and biogeochemists to process quickly and efficiently vast amounts of lipidome data to understand microbial lipids characteristics in ecosystems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ding, Bale, Hopmans, Villanueva, Arts, Schouten and Sinninghe Damsté.)

Archaea synthesize membranes of isoprenoid lipids that are ether-linked to glycerol-1-phosphate (G1P), while Bacteria/Eukarya produce membranes consisting of fatty acids ester-bound to glycerol-3-phosphate (G3P). This dichotomy in membrane lipid composition (i.e., the 'lipid divide') is believed to have arisen after the Last Universal Common Ancestor (LUCA). A leading hypothesis is that LUCA possessed a heterochiral 'mixed archaeal/bacterial membrane'. However, no natural microbial representatives supporting this scenario have been shown to exist today. Here, we demonstrate that bacteria of the Fibrobacteres-Chlorobi-Bacteroidetes (FCB) group superphylum encode a putative archaeal pathway for ether-bound isoprenoid membrane lipids in addition to the bacterial fatty acid membrane pathway. Key genes were expressed in the environment and their recombinant expression in Escherichia coli resulted in the formation of a 'mixed archaeal/bacterial membrane'. Genomic evidence and biochemical assays suggest that the archaeal-like lipids of members of the FCB group could possess either a G1P or G3P stereochemistry. Our results support the existence of 'mixed membranes' in natural environments and their stability over a long period in evolutionary history, thereby bridging a once-thought fundamental divide in biology.

Diversity analyses of microbial enrichments obtained from deep sulfidic water (2000 m) collected from the Black Sea indicated the presence of eleven novel putative lineages of bacteria affiliated to the family Marinifilaceae of the phylum Bacteroidetes. Pure cultures were obtained for four strains (i.e. M1P T , M3P, A4 T and 44) of this family, which could be grouped into two different clades based on their 16S rRNA gene sequences. All four strains were Gram-negative, rod-shaped and facultative anaerobic bacteria. The genomes of all strains were sequenced and physiological analyses were performed. All strains utilized a wide range of carbon sources, which was supported by the presence of the pathways involved in carbon utilization encoded by their genomes. The strains were able to grow at elevated hydrostatic pressure (up to 50 MPa), which coincided with increased production of unsaturated and branched fatty acids, and a decrease in hydroxy fatty acids. Intact polar lipid analysis of all four strains showed the production of ornithine lipids, phosphatidylethanolamines and capnine lipids as major intact polar lipids (IPLs). Genes involved in hopanoid biosynthesis were also identified. However, bacteriohopanepolyols (BHPs) were not detected in the strains. Based on distinct physiological, chemotaxonomic, genotypic and phylogenetic differences compared to other members of the genera Ancylomarina and Labilibaculum, it was concluded that strains M1P T and A4 T represented two novel species for which the names Ancylomarina euxinus sp. nov. and Labilibaculum euxinus sp. nov., respectively, are proposed., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The conventional perception that the zone of sulfate reduction and methanogenesis are separated in high- and low-sulfate-containing marine sediments has recently been changed by studies demonstrating their co-occurrence in sediments. The presence of methanogens was linked to the presence of substrates that are not used by sulfate reducers. In the current study, we hypothesized that both groups can co-exist, consuming common substrates (H 2 and/or acetate) in sediments. We enriched butyrate-degrading communities in sediment slurries originating from the sulfate, sulfate-methane transition, and methane zone of Aarhus Bay, Denmark. Sulfate was added at different concentrations (0, 3, 20 mM), and the slurries were incubated at 10 °C and 25 °C. During butyrate conversion, sulfate reduction and methanogenesis occurred simultaneously. The syntrophic butyrate degrader Syntrophomonas was enriched both in sulfate-amended and in sulfate-free slurries, indicating the occurrence of syntrophic conversions at both conditions. Archaeal community analysis revealed a dominance of Methanomicrobiaceae . The acetoclastic Methanosaetaceae reached high relative abundance in the absence of sulfate, while presence of acetoclastic Methanosarcinaceae was independent of the sulfate concentration, temperature, and the initial zone of the sediment. This study shows that there is no vertical separation of sulfate reducers, syntrophs, and methanogens in the sediment and that they all participate in the conversion of butyrate.

The marine pelagic archaeal community is dominated by three major groups, the marine group I (MGI) Thaumarchaeota, and the marine groups II and III (MGII and MGIII) Euryarchaeota. Studies of both MGI cultures and the environment have shown that the MGI core membrane lipids are predominantly composed of glycerol dibiphytanyl glycerol tetraether (GDGT) lipids and the diether lipid archaeol. However, there are no cultured representatives of MGII and III archaea and, therefore, both their membrane lipid composition and potential contribution to the marine archaeal lipid pool remain unknown. Here, we show that GDGTs present in suspended particulate matter of the (sub)surface waters of the North Atlantic Ocean and the coastal North Sea are derived from MGI archaea, and that MGII archaea do not significantly contribute to the pool of GDGTs and archaeol. This implies, in contrast to previous suggestions, that their lipids do not affect the widely used sea surface temperature proxy TEX 86 . These findings also indicate that MGII archaea are not able to produce any known archaeal lipids, implying that our understanding of the evolution of membrane lipid biosynthesis in Archaea is far from complete.

Fire regime shifts are driven by climate and natural vegetation changes, but can be strongly affected by human land management. Yet, it is poorly known how humans have influenced fire regimes prior to active wildfire suppression. Among the last 250 years, the human contribution to the global increase in fire occurrence during the mid-19th century is especially unclear, as data sources are limited. Here, we test the extent to which forest management has driven fire regime shifts in a temperate forest landscape. We combine multiple fire proxies (macroscopic charcoal and fire-related biomarkers) derived from highly resolved lake sediments (i.e., 3-5 years per sample), and apply a new statistical approach to classify source area- and temperature-specific fire regimes (biomass burnt, fire episodes). We compare these records with independent climate and vegetation reconstructions. We find two prominent fire regime shifts during the 19th and 20th centuries, driven by an adaptive socio-ecological cycle in human forest management. Although individual fire episodes were triggered mainly by arson (as described in historical documents) during dry summers, the biomass burnt increased unintentionally during the mid-19th century due to the plantation of flammable, fast-growing pine tree monocultures needed for industrialization. State forest management reacted with active fire management and suppression during the 20th century. However, pine cover has been increasing since the 1990s and climate projections predict increasingly dry conditions, suggesting a renewed need for adaptations to reduce the increasing fire risk., Competing Interests: The authors have declared that no competing interests exist.

Archaea are ubiquitous in the modern ocean where they are involved in the carbon and nitrogen biogeochemical cycles. However, the majority of Archaea remain uncultured. Archaeal specific membrane intact polar lipids (IPLs) are biomarkers of the presence and abundance of living cells. They comprise archaeol and glycerol dibiphytanyl glycerol tetraethers (GDGTs) attached to various polar headgroups. However, little is known of the IPLs of uncultured marine Archaea, complicating their use as biomarkers. Here, we analyzed suspended particulate matter (SPM) obtained in high depth resolution from a coastal and open ocean site in the eastern tropical South Pacific (ETSP) oxygen deficient zone (ODZ) with the aim of determining possible biological sources of archaeal IPL by comparing their composition by Ultra High Pressure Liquid Chromatography coupled to high resolution mass spectrometry with the archaeal diversity by 16S rRNA gene amplicon sequencing and their abundance by quantitative PCR. Thaumarchaeotal Marine Group I (MGI) closely related to Ca . Nitrosopelagicus and Nitrosopumilus dominated the oxic surface and upper ODZ water together with Marine Euryarchaeota Group II (MGII). High relative abundance of hexose phosphohexose- (HPH) crenarchaeol, the specific biomarker for living Thaumarchaeota, and HPH-GDGT-0, dihexose- (DH) GDGT-3 and -4 were detected in these water masses. Within the ODZ, DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaea) of the Woesearchaeota DHVE-6 group and Marine Euryarchaeota Group III (MGIII) were present together with a higher proportion of archaeol-based IPLs, which were likely made by MGIII, since DPANN archaea are supposedly unable to synthesize their own IPLs and possibly have a symbiotic or parasitic partnership with MGIII. Finally, in deep suboxic/oxic waters a different MGI population occurred with HPH-GDGT-1, -2 and DH-GDGT-0 and -crenarchaeol, indicating that here MGI synthesize membranes with IPLs in a different relative abundance which could be attributed to the different detected population or to an environmental adaptation. Our study sheds light on the complex archaeal community of one of the most prominent ODZs and on the IPL biomarkers they potentially synthesize.

We analyzed the polar membrane lipids of 13 strains of halo(alkali)philic euryarchaea from hypersaline lakes. Nine belong to the class Halobacteria , representing two functional groups: aerobic polysaccharide utilizers and sulfur-respiring anaerobes. The other four strains represent halo(alkali)philic methanogens from the class Methanomicrobia and a recently discovered class Methanonatronarchaeia . A wide range of polar lipids were detected across the 13 strains including dialkyl glycerol diethers (archaeols), membrane-spanning glycerol tetraethers and diether-based cardiolipins. The archaeols contained a range of core lipid structures, including combinations of C 20 and C 25 isoprenoidal alkyl chains, unsaturations, and hydroxy moieties. Several diether lipids were novel, including: (a) a phosphatidylglycerolhexose (PG-Gly) headgroup, (b) a N , N , N -trimethyl aminopentanetetrol (APT)-like lipid with a methoxy group in place of a hydroxy group on the pentanetetrol, (c) a series of polar lipids with a headgroup with elemental composition of either C 12 H 25 NO 13 S or C 12 H 25 NO 16 S 2 , and (d) novel cardiolipins containing a putative phosphatidylglycerolphosphate glycerophosphate (PGPGP) polar moiety. We found that the lipid distribution of the 13 strains could be generally separated into two groups, the methanogens (group) and the Halobacteria (class) based on the presence of specific core lipids. Within the methanogens, adaption to a high or more moderate salt concentration resulted in different ratios of glycerol dialkyl glycerol tetraethers (GDGTs) to archaeol. The methanogen Methanosalsum natronophilum AME2 T had the most complex diether lipid composition of any of the 13 strains, including hydroxy archaeol and macrocyclic archaeol which we surmise is an order-specific membrane adaption. The zwitterionic headgroups APT and APT-Me were detected only in the Methanomicrobiales member Methanocalculus alkaliphilus AMF2 T which also contained the highest level of unsaturated lipids. Only alkaliphilic members of the Natrialbales order contained PGPGP cardiolipins and the PG-Gly headgroup. The four analyzed neutrophilic members of the Halobacteria were characterized by the presence of sulfur-containing headgroups and glycolipids. The presence of cardiolipins with one or more i-C 25 alkyl chains, generally termed extended archaeol (EXT-AR), in one of the Methanonatronarchaeia strains was unexpected as only one other order of methanogenic archaea has been reported to produce EXT-AR. We examined this further by looking into the genomic potential of various archaea to produce EXT-AR.

Archaea are important players in marine biogeochemical cycles, and their membrane lipids are useful biomarkers in environmental and geobiological studies. However, many archaeal groups remain uncultured and their lipid composition unknown. Here, we aim to expand the knowledge on archaeal lipid biomarkers and determine the potential sources of those lipids in the water column of the euxinic Black Sea. The archaeal community was evaluated by 16S rRNA gene amplicon sequencing and by quantitative PCR. The archaeal intact polar lipids (IPLs) were investigated by ultra-high-pressure liquid chromatography coupled to high-resolution mass spectrometry. Our study revealed both a complex archaeal community and large changes with water depth in the IPL assemblages. In the oxic/upper suboxic waters (<105 m), the archaeal community was dominated by marine group (MG) I Thaumarchaeota, coinciding with a higher relative abundance of hexose phosphohexose crenarchaeol, a known marker for Thaumarchaeota. In the suboxic waters (80-110 m), MGI Nitrosopumilus sp. dominated and produced predominantly monohexose glycerol dibiphytanyl glycerol tetraethers (GDGTs) and hydroxy-GDGTs. Two clades of MGII Euryarchaeota were present in the oxic and upper suboxic zones in much lower abundances, preventing the detection of their specific IPLs. In the deep sulfidic waters (>110 m), archaea belonging to the DPANN Woesearchaeota, Bathyarchaeota, and ANME-1b clades dominated. Correlation analyses suggest that the IPLs GDGT-0, GDGT-1, and GDGT-2 with two phosphatidylglycerol (PG) head groups and archaeol with a PG, phosphatidylethanolamine, and phosphatidylserine head groups were produced by ANME-1b archaea. Bathyarchaeota represented 55% of the archaea in the deeper part of the euxinic zone and likely produces archaeol with phospho-dihexose and hexose-glucuronic acid head groups., (© 2018 The Authors. Geobiology Published by John Wiley & Sons Ltd.)

We have compared and assessed the suitability of several chromatographic methods for the analysis of long chain alkenones and long chain diols and the associated paleotemperature proxies (U K' 37 and LDI). We evaluated the traditional methods for the analysis of the U K' 37 and the LDI, gas chromatography (GC) - flame ionization detection (FID) and GC mass spectrometry (MS) using selected ion monitoring (SIM), respectively, and developed a new method using GC-MS/MS in multiple reaction monitoring mode (MRM) for the analysis of long chain diols as well as a method for automatic silylation of diols using a robot autosampler. Finally, we evaluated liquid chromatography (LC) methods to simultaneously measure the U K' 37 and the LDI, using ultra high performance LC (UHPLC) with low (nominal mass) resolution MS in SIM mode, and UHPLC with high resolution MS (HRMS). Detection and quantification limits and reproducibility were assessed by means of serial dilutions of culture extracts. Automated silylation by a robot autosampler showed similar reproducibility as off-line silylation while substantially decreasing sample preparation time. The novel MRM method had a slightly lower limit of quantification (LOQ; i.e. 0.3pgC 28 1,13-diol injected on-column) than the traditional method (0.5pg) and improved reproducibility while allowing more unambiguous identification of LCDs in complex matrices. For diols, UHPLC-MS using SIM had the highest LOQ (i.e. 15pg) and a comparable reproducibility as GC-MS. UHPLC-HRMS had a LOQ of ca. 1.5pg, and an improved reproducibility for diol analysis. For alkenone analysis, both UHPLC-HRMS and UHPLC-MS using SIM were 2-3 orders of magnitude more sensitive (LOQ ca. 20 and 2pgC 37:2 alkenone injected on-column, respectively) than GC-FID (LOD ca. 3ng), with a similar reproducibility of the U K' 37 index. Hence, UHPLC-HRMS allows simultaneous analysis of the U K' 37 and LDI at an increased sensitivity. In addition, it allows simultaneous measurement of TEX 86 , a temperature proxy based on the isoprenoid glycerol dialkyl glycerol tetraethers. This reduces the preparation time by excluding the need of derivatization and separation of the ketone (containing the long chain alkenones) and polar fractions (containing the long chain diols and GDGTs). However, synthetic standards are required to fully assess the accuracy of the new methods for determination of the LDI and U K' 37 ., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Denitrifying and anammox bacteria are involved in the nitrogen cycling in marine sediments but the environmental factors that regulate the relative importance of these processes are not well constrained. Here, we evaluated the abundance, diversity, and potential activity of denitrifying, anammox, and sulfide-dependent denitrifying bacteria in the sediments of the seasonally hypoxic saline Lake Grevelingen, known to harbor an active microbial community involved in sulfur oxidation pathways. Depth distributions of 16S rRNA gene, nirS gene of denitrifying and anammox bacteria, aprA gene of sulfur-oxidizing and sulfate-reducing bacteria, and ladderane lipids of anammox bacteria were studied in sediments impacted by seasonally hypoxic bottom waters. Samples were collected down to 5 cm depth (1 cm resolution) at three different locations before (March) and during summer hypoxia (August). The abundance of denitrifying bacteria did not vary despite of differences in oxygen and sulfide availability in the sediments, whereas anammox bacteria were more abundant in the summer hypoxia but in those sediments with lower sulfide concentrations. The potential activity of denitrifying and anammox bacteria as well as of sulfur-oxidizing, including sulfide-dependent denitrifiers and sulfate-reducing bacteria, was potentially inhibited by the competition for nitrate and nitrite with cable and/or Beggiatoa -like bacteria in March and by the accumulation of sulfide in the summer hypoxia. The simultaneous presence and activity of organoheterotrophic denitrifying bacteria, sulfide-dependent denitrifiers, and anammox bacteria suggests a tight network of bacteria coupling carbon-, nitrogen-, and sulfur cycling in Lake Grevelingen sediments.

Rationale: Intact polar lipids (IPLs) are the building blocks of cell membranes, and amino acid containing IPLs have been observed to be involved in response to changing environmental conditions in various species of bacteria. High-performance liquid chromatography/mass spectrometry (HPLC/MS) has become the primary method for analysis of IPLs. Many glycerol-free amino acid containing membrane lipids (AA-IPLs), which are structurally different than abundant aminophospholipids, have not been characterized using HPLC/MS. This results in many lipids remaining unrecognized in IPL analysis of microbial cultures and environmental samples, hampering the study of their occurrence and functionality., Methods: We analyzed the amino acid containing IPLs of a number of bacteria (i.e. Gluconobacter cerinus, Cyclobacterium marinus, Rhodobacter sphaeroides, and Pedobacter heparinus) in order to decipher fragmentation pathways, and explore potential novel lipid structures using HPLC/electrospray ionization ion trap MS (HPLC/ESI-IT-MS) and HPLC/high-resolution MS (HPLC/HRMS)., Results: We report differentiation between glutamine and lysine lipids with the same nominal masses, novel MS fragmentation pathways of cytolipin, the lipopeptides cerilipin and flavolipin, head group hydroxylated ornithine lipids, and the novel identification of cerilipin with a hydroxylated fatty acid., Conclusions: Non-glycerol AA lipids can be readily recognized as their fragmentation follows a clear pattern with initial dehydration or other loss from the head group, followed by fatty acid losses resulting in a diagnostic fragment ion. Higher level MSn and HRMS are valuable tools in characterizing AA lipid head group structural components.

Lipid extraction of biomass prior to stable isotope analysis is known to cause variable changes in the stable nitrogen isotopic composition (δ15N) of residual biomass. However, the underlying factors causing these changes are not yet clear. Here we address this issue by comparing the δ15N of bulk and residual biomass of several marine animal tissues (fish, crab, cockle, oyster, and polychaete), as well as the δ15N of the extracted lipids. As observed previously, lipid extraction led to a variable offset in δ15N of biomass (differences ranging from -2.3 to +1.8 ‰). Importantly, the total lipid extract (TLE) was highly depleted in 15N compared to bulk biomass, and also highly variable (differences ranging from -14 to +0.7 ‰). The TLE consisted mainly of phosphatidylcholines, a group of lipids with one nitrogen atom in the headgroup. To elucidate the cause for the 15N-depletion in the TLE, the δ15N of amino acids was determined, including serine because it is one of the main sources of nitrogen to N-containing lipids. Serine δ15N values differed by -7 to +2 ‰ from bulk biomass δ15N, and correlated well with the 15N depletion in TLEs. On average, serine was less depleted (-3‰) than the TLE (-7 ‰), possibly due to fractionation during biosynthesis of N-containing headgroups, or that other nitrogen-containing compounds, such as urea and choline, or recycled nitrogen contribute to the nitrogen isotopic composition of the TLE. The depletion in 15N of the TLE relative to biomass increased with the trophic level of the organisms.

Northern wetlands make up a substantial terrestrial carbon sink and are often dominated by decay-resistant Sphagnum mosses. Recent studies have shown that planctomycetes appear to be involved in degradation of Sphagnum-derived debris. Novel trimethylornithine (TMO) lipids have recently been characterized as abundant lipids in various Sphagnum wetland planctomycete isolates, but their occurrence in the environment has not yet been confirmed. We applied a combined intact polar lipid (IPL) and molecular analysis of peat cores collected from two northern wetlands (Saxnäs Mosse [Sweden] and Obukhovskoye [Russia]) in order to investigate the preferred niche and abundance of TMO-producing planctomycetes. TMOs were present throughout the profiles of Sphagnum bogs, but their concentration peaked at the oxic/anoxic interface, which coincided with a maximum abundance of planctomycete-specific 16S rRNA gene sequences. The sequences detected at the oxic/anoxic interface were affiliated with the Isosphaera group, while sequences present in the anoxic peat layers were related to an uncultured planctomycete group. Pyrosequencing-based analysis identified Planctomycetes as the major bacterial group at the oxic/anoxic interface at the Obukhovskoye peat (54% of total 16S rRNA gene sequence reads), followed by Acidobacteria (19% reads), while in the Saxnäs Mosse peat, Acidobacteria were dominant (46%), and Planctomycetes contributed to 6% of the total reads. The detection of abundant TMO lipids in planctomycetes isolated from peat bogs and the lack of TMO production by cultures of acidobacteria suggest that planctomycetes are the producers of TMOs in peat bogs. The higher accumulation of TMOs at the oxic/anoxic interface and the change in the planctomycete community with depth suggest that these IPLs could be synthesized as a response to changing redox conditions at the oxic/anoxic interface., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Microbial decomposition of organic matter is an essential process in the global carbon cycle. The soil bacteria Pseudopedobacter saltans and Flavobacterium johnsoniae are both able to degrade complex organic molecules, but it is not fully known how their membrane structures are adapted to their environmental niche. The membrane lipids of these species were extracted and analyzed using high performance liquid chromatography-electrospray ionization/ion trap/mass spectrometry (HPLC-ESI/IT/MS) and high resolution accurate mass/mass spectrometry (HRAM/MS). Abundant unknown intact polar lipids (IPLs) from P. saltans were isolated and further characterized using amino acid analysis and two dimensional nuclear magnetic resonance (NMR) spectroscopy. Ornithine IPLs (OLs) with variable (hydroxy) fatty acid composition were observed in both bacterial species. Lysine-containing IPLs (LLs) were also detected in both species and were characterized here for the first time using HPLC-MS. Novel LLs containing hydroxy fatty acids and novel hydroxylysine lipids with variable (hydroxy) fatty acid composition were identified in P. saltans. The confirmation of OL and LL formation in F. johnsoniae and P. saltans and the presence of OlsF putative homologs in P. saltans suggest the OlsF gene coding protein is possibly involved in OL and LL biosynthesis in both species, however, potential pathways of OL and LL hydroxylation in P. saltans are still undetermined. Triplicate cultures of P. saltans were grown at three temperature/pH combinations: 30°C/pH 7, 15°C/pH 7, and 15°C/pH 9. The fractional abundance of total amino acid containing IPLs containing hydroxylated fatty acids was significantly higher at higher temperature, and the fractional abundance of lysine-containing IPLs was significantly higher at lower temperature and higher pH. These results suggest that these amino acid-containing IPLs, including the novel hydroxylysine lipids, could be involved in temperature and pH stress response of soil bacteria.

Microbial adaptations to environmental extremes, including high temperature and low pH conditions typical of geothermal settings, are of interest in astrobiology and origin of life investigations. The lipid biomarkers preserved in silica deposits associated with six geothermal areas in the Taupo Volcanic Zone were investigated and variations in lipid composition as a function of temperature and pH were assessed. Lipid analyses reveal highly variable abundances and distributions, reflecting community composition as well as adaptations to extremes of pH and temperature. Biomarker profiles reveal three distinct microbial assemblages across the sites: the first in Champagne Pool and Loop Road, the second in Orakei Korako, Opaheke and Ngatamariki, and the third in Rotokawa. Similar lipid distributions are observed in sinters from physicochemically similar springs. Furthermore, correlation between lipid distributions and geothermal conditions is observed. The ratio of archaeol to bacterial diether abundance, bacterial diether average chain length, degree of GDGT cyclisation and C31 and C32 hopanoic acid indices typically increase with temperature. At lower pH, the ratio of archaeol to bacterial diethers, degree of GDGT cyclisation and C31 and C32 hopanoic acid indices are typically higher. No trends in fatty acid distributions with temperature or pH are evident, likely reflecting overprinting due to population influences.

Microbial processes such as nitrification and anaerobic ammonium oxidation (anammox) are important for nitrogen cycling in marine sediments. Seasonal variations of archaeal and bacterial ammonia oxidizers (AOA and AOB) and anammox bacteria, as well as the environmental factors affecting these groups, are not well studied. We have examined the seasonal and depth distribution of the abundance and potential activity of these microbial groups in coastal marine sediments of the southern North Sea. This was achieved by quantifying specific intact polar lipids as well as the abundance and gene expression of their 16S rRNA gene, the ammonia monooxygenase subunit A (amoA) gene of AOA and AOB, and the hydrazine synthase (hzsA) gene of anammox bacteria. AOA, AOB, and anammox bacteria were detected and transcriptionally active down to 12 cm sediment depth. In all seasons, the abundance of AOA was higher compared to the AOB abundance suggesting that AOA play a more dominant role in aerobic ammonia oxidation in these sediments. Anammox bacteria were abundant and active even in oxygenated and bioturbated parts of the sediment. The abundance of AOA and AOB was relatively stable with depth and over the seasonal cycle, while anammox bacteria abundance and transcriptional activity were highest in August. North Sea sediments thus seem to provide a common, stable, ecological niche for AOA, AOB, and anammox bacteria.

Recently, iso-diabolic acid (13,16-dimethyl octacosanedioic acid) has been identified as a major membrane-spanning lipid of subdivisions 1 and 3 of the Acidobacteria, a highly diverse phylum within the Bacteria. This finding pointed to the Acidobacteria as a potential source for the bacterial glycerol dialkyl glycerol tetraethers that occur ubiquitously in peat, soil, lakes, and hot springs. Here, we examined the lipid composition of seven phylogenetically divergent strains of subdivision 4 of the Acidobacteria, a bacterial group that is commonly encountered in soil. Acid hydrolysis of total cell material released iso-diabolic acid derivatives in substantial quantities (11 to 48% of all fatty acids). In contrast to subdivisions 1 and 3 of the Acidobacteria, 6 out of the 7 species of subdivision 4 (excepting "Candidatus Chloracidobacterium thermophilum") contained iso-diabolic acid ether bound to a glycerol in larger fractional abundance than iso-diabolic acid itself. This is in agreement with the analysis of intact polar lipids (IPLs) by high-performance liquid chromatography-mass spectrometry (HPLC-MS), which showed the dominance of mixed ether-ester glycerides. iso-Diabolic acid-containing IPLs were not identified, because these IPLs are not released with a Bligh-Dyer extraction, as observed before when studying lipid compositions of subdivisions 1 and 3 of the Acidobacteria. The presence of ether bonds in the membrane lipids does not seem to be an adaptation to temperature, because the five mesophilic isolates contained a larger amount of ether lipids than the thermophile "Ca. Chloracidobacterium thermophilum." Furthermore, experiments with Pyrinomonas methylaliphatogenes did not reveal a major influence of growth temperature over the 50 to 69°C range., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

We investigated the occurrence and activity of anaerobic ammonia oxidation (anammox) bacteria in sandy and muddy sand sediments of the southern North Sea. The presence of anammox bacteria was established through the detection of specific phosphocholine-monoether ladderane lipids, 16S rRNA gene, and hydrazine synthase (hzsA) genes. Anammox activity was measured in intact sediment cores (in situ rate) and in sediment slurries (potential rate) as the rate of N2 evolution from (15) N-labeled substrates and compared to the transcriptional activity of genes of anammox bacteria. The contribution of anammox to N2 production ranged between 0% and 29%. The potential rate of anammox agreed well with the abundance of anammox bacteria 16S rRNA and hzsA gene copies and the transcriptional activity of the anammox bacteria 16S rRNA gene. We found a higher abundance and activity of anammox bacteria in sediments with higher organic carbon content and also higher activity in summer than in winter. The abundance of anammox bacteria and their potential anammox rates were similar to those reported for other marine coastal sediments, suggesting that potentially they are important contributors to the nitrogen cycle in sandy sediments of shallow continental shelf areas., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Glycerol dibiphytanyl glycerol tetraether (GDGT) lipids are part of the cellular membranes of Thaumarchaeota, an archaeal phylum composed of aerobic ammonia oxidizers, and are used in the paleotemperature proxy TEX86 . GDGTs in live cells possess polar head groups and are called intact polar lipids (IPL-GDGTs). Their transformation to core lipids (CL) by cleavage of the head group was assumed to proceed rapidly after cell death, but it has been suggested that some of these IPL-GDGTs can, just like the CL-GDGTs, be preserved over geological timescales. Here, we examined IPL-GDGTs in deeply buried (0.2-186 mbsf, ~2.5 Myr) sediments from the Peru Margin. Direct measurements of the most abundant IPL-GDGT, IPL-crenarchaeol, specific for Thaumarchaeota, revealed depth profiles, which differed per head group. Shallow sediments (<1 mbsf) contained IPL-crenarchaeol with both glycosidic and phosphate head groups, as also observed in thaumarchaeal enrichment cultures, marine suspended particulate matter and marine surface sediments. However, hexose, phosphohexose-crenarchaeol is not detected anymore below 6 mbsf (~7 kyr), suggesting a high lability. In contrast, IPL-crenarchaeol with glycosidic head groups is preserved over timescales of Myr. This agrees with previous analyses of deeply buried (>1 m) marine sediments, which only reported glycosidic and no phosphate-containing IPL-GDGTs. TEX86 values of CL-GDGTs did not markedly change with depth, and the TEX86 of IPL-derived GDGTs decreased only when the proportions of monohexose- to dihexose-GDGTs changed, likely due to the enhanced preservation of the monohexose GDGTs. Our results support the hypothesis that in situ GDGT production and differential IPL degradation in sediments is not substantially affecting TEX86 paleotemperature estimations based on CL-GDGTs and indicates that likely only a small amount of IPL-GDGTs present in deeply buried sediments is part of cell membranes of active archaea. The amount of archaeal biomass in the deep biosphere based on these IPLs may have been substantially overestimated., (© 2014 John Wiley & Sons Ltd.)

Sulphoquinovosyldiacylglycerols (SQDG) are polar sulphur-containing membrane lipids, whose presence has been related to a microbial strategy to adapt to phosphate deprivation. In this study, we have targeted the sqdB gene coding the uridine 5'-diphosphate-sulphoquinovose (UDP-SQ) synthase involved in the SQDG biosynthetic pathway to assess potential microbial sources of SQDGs in the marine environment. The phylogeny of the sqdB-coding protein reveals two distinct clusters: one including green algae, higher plants and cyanobacteria, and another one comprising mainly non-photosynthetic bacteria, as well as other cyanobacteria and algal groups. Evolutionary analysis suggests that the appearance of UDP-SQ synthase occurred twice in cyanobacterial evolution, and one of those branches led to the diversification of the protein in members of the phylum Proteobacteria. A search of homologues of sqdB-proteins in marine metagenomes strongly suggested the presence of heterotrophic bacteria potential SQDG producers. Application of newly developed sqdB gene primers in the marine environment revealed a high diversity of sequences affiliated to cyanobacteria and Proteobacteria in microbial mats, while in North Sea surface water, most of the detected sqdB genes were attributed to the cyanobacterium Synechococcus sp. Lipid analysis revealed that specific SQDGs were characteristic of microbial mat depth, suggesting that SQDG lipids are associated with specific producers., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

Phospholipid-derived fatty acids (PLFAs) are commonly used to characterize microbial communities in situ and the phylogenetic positions of newly isolated microorganisms. PLFAs are obtained through separation of phospholipids from glycolipids and neutral lipids using silica column chromatography. We evaluated the performance of this separation method for the first time using direct detection of intact polar lipids (IPLs) with high-performance liquid chromatography-mass spectrometry (HPLC-MS). We show that under either standard or modified conditions, the phospholipid fraction contains not only phospholipids but also other lipid classes such as glycolipids, betaine lipids, and sulfoquinovosyldiacylglycerols. Thus, commonly reported PLFA compositions likely are not derived purely from phospholipids and perhaps may not be representative of fatty acids present in living microbes.

Northern peatlands represent a significant global carbon store and commonly originate from Sphagnum moss-dominated wetlands. These ombrotrophic ecosystems are rain fed, resulting in nutrient-poor, acidic conditions. Members of the bacterial phylum Planctomycetes are highly abundant and appear to play an important role in the decomposition of Sphagnum-derived litter in these ecosystems. High-performance liquid chromatography coupled to high-resolution accurate-mass mass spectrometry (HPLC-HRAM/MS) analysis of lipid extracts of four isolated planctomycetes from wetlands of European north Russia revealed novel ornithine membrane lipids (OLs) that are mono-, di-, and trimethylated at the ε-nitrogen position of the ornithine head group. Nuclear magnetic resonance (NMR) analysis of the isolated trimethylornithine lipid confirmed the structural identification. Similar fatty acid distributions between mono-, di-, and trimethylornithine lipids suggest that the three lipid classes are biosynthetically linked, as in the sequential methylation of the terminal nitrogen in phosphatidylethanolamine to produce phosphatidylcholine. The mono-, di-, and trimethylornithine lipids described here represent the first report of methylation of the ornithine head groups in biological membranes. Various bacteria are known to produce OLs under phosphorus limitation or fatty-acid-hydroxylated OLs under thermal or acid stress. The sequential methylation of OLs, leading to a charged choline-like moiety in the trimethylornithine lipid head group, may be an adaptation to provide membrane stability under acidic conditions without the use of scarce phosphate in nutrient-poor ombrotrophic wetlands.

An efficient asymmetric synthesis of cyclo-archaeol and β-glucosyl cyclo-archaeol is presented employing catalytic asymmetric conjugate addition and catalytic epoxide ring opening as the key steps. Their occurrence in deep sea hydrothermal vents has been confirmed by chromatographic comparison with natural samples.

A novel sulfate-reducing bacterium designated OPF15(T) was isolated from Obsidian Pool, Yellowstone National Park, Wyoming. The phylogeny of 16S rRNA and functional genes (dsrAB) placed the organism within the family Thermodesulfobacteriaceae. The organism displayed hyperthermophilic temperature requirements for growth with a range of 70-90 °C and an optimum of 83 °C. Optimal pH was around 6.5-7.0 and the organism required the presence of H2 or formate as an electron donor and CO2 as a carbon source. Electron acceptors supporting growth included sulfate, thiosulfate, and elemental sulfur. Lactate, acetate, pyruvate, benzoate, oleic acid, and ethanol did not serve as electron donors. Membrane lipid analysis revealed diacyl glycerols and acyl/ether glycerols which ranged from C14:0 to C20:0. Alkyl chains present in acyl/ether and diether glycerol lipids ranged from C16:0 to C18:0. Straight, iso- and anteiso-configurations were found for all lipid types. The presence of OPF15(T) was also shown to increase cellulose consumption during co-cultivation with Caldicellulosiruptor obsidiansis, a fermentative, cellulolytic extreme thermophile isolated from the same environment. On the basis of phylogenetic, phenotypic, and structural analyses, Thermodesulfobacterium geofontis sp. nov. is proposed as a new species with OPF15(T) representing the type strain.

The intact polar lipid (IPL) composition of twelve hydrothermal vent deposits from the Rainbow (RHF) and Lucky Strike hydrothermal fields (LSHF) has been investigated in order to assess its utility as a proxy for microbial community composition associated with deep-sea hydrothermal locations. Gene-based culture-independent surveys of the microbial populations of the same vent deposits have shown that microbial populations are different in the two locations and appear to be controlled by the geochemical and geological processes that drive hydrothermal circulation. Large differences in the IPL composition between these two sites are evident. In the ultramafic-hosted RHF, mainly archaeal-IPLs were identified, including those known to be produced by hyperthermophilic Euryarchaeota. More specifically, polyglycosyl derivatives of archaeol and macrocyclic archaeol indicate the presence of hyperthermophilic methanogenic archaea in the vent deposits, which are related to members of the Methanocaldococcaceae or Methanococcaceae. In contrast, bacterial IPLs dominate IPL distributions from LSHF, suggesting that bacteria are more predominant at LSHF than at RHF. Bacterial Diacyl glycerol (DAG) IPLs containing phosphocholine, phosphoethanolamine or phosphoglycerol head groups were identified at both vent fields. In some vent deposits from LSHF ornithine lipids and IPLs containing phosphoaminopentanetetrol head groups were also observed. By comparison with previously characterized bacterial communities at the sites, it is likely the DAG-IPLs observed derive from Epsilon- and Gammaproteobacteria. Variation in the relative amounts of archaeal versus bacterial IPLs appears to indicate differences in the microbial community between vent sites. Overall, IPL distributions appear to be consistent with gene-based surveys., (© 2012 Blackwell Publishing Ltd.)

The heterocysts of limnetic nitrogen-fixing filamentous cyanobacteria contain unique glycolipids in their cell wall that create the distinctive gas impermeability of the heterocyst cell wall as well as serve as biomarker lipids for these microbes. It has been assumed that marine free-living and endosymbiotic cyanobacteria synthesize the same glycolipids although they have not been investigated in any detail. Here we report the glycolipid composition of several marine free-living heterocystous cyanobacteria as well as the heterocystous endosymbiont Richelia intracellularis found in the biogeochemically important diatoms Hemiaulus hauckii and Hemiaulus membranaceus. In the marine cyanobacteria Nostoc muscorum and Calothrix sp., we detected the same glycolipids as found in freshwater representatives of these genera. However, we did not detect these glycolipids in the Hemiaulus-Richelia association. Instead, we identified glycolipids which comprised a C₅ sugar, ribose, rather than the C₆ sugars normally encountered in glycolipids of free-living cyanobacteria. In addition, the glycolipids had slightly longer chain lengths (C₃₀ and C₃₂ versus C₂₆ and C₂₈) in the aglycone moiety. The different glycolipid composition of the marine endosymbotic heterocystous cyanobacteria compared to their free-living counterparts may be an adaptation to the high intracellular O₂ concentrations within their host. These glycolipids may provide unique tracers for the presence of these microbes in marine environments and permit exploration of the evolutionary origins of these symbioses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Ecological studies of thaumarchaeota often apply glycerol dibiphytanyl glycerol tetraether (GDGT)-based intact membrane lipids. However, these components have only been characterized for thaumarchaeota from aquatic environments. Thaumarchaeota have been shown to play an important role in the nitrogen cycle in soil as ammonium oxidizers, and GDGTs are common lipids encountered in soil. We report the core and intact polar lipid (IPL) GDGTs produced by three newly available thaumarchaeota isolated from grassland soil in Austria ("Nitrososphaera viennensis," group I.1b) and enriched from agricultural soils in South Korea ("Candidatus Nitrosoarchaeum koreensis" MY1, group I.1a; and "Candidatus Nitrososphaera" strain JG1, group I.1b). The soil thaumarchaeota all synthesize crenarchaeol as their major core GDGT, in agreement with the fact that crenarchaeol has also been detected in thaumarchaeota from aquatic environments. The crenarchaeol regioisomer apparently is produced in significant quantities only by soil thaumarchaeota of the I.1b subgroup. In addition, GDGTs with 0 to 4 cyclopentane moieties and GDGTs containing an additional hydroxyl group were detected. The IPL head groups of their membrane lipids comprised mainly monohexose, dihexose, trihexose, phosphohexose, and hexose-phosphohexose moieties. The hexose-phosphohexose head group bound to crenarchaeol occurred in all soil thaumarchaeota, and this IPL is at present the only lipid that is detected in all thaumarchaeota analyzed so far. This specificity and its lability indicate that it is the most suitable biomarker lipid to trace living thaumarchaeota. This study, in combination with previous studies, also suggests that hydroxylated GDGTs occur in the I.1a, but not in the I.1b, subgroup of the thaumarchaeota.

Ammonia-oxidizing archaea (AOA) and anaerobic ammonia-oxidizing (anammox) bacteria have emerged as significant factors in the marine nitrogen cycle and are responsible for the oxidation of ammonium to nitrite and dinitrogen gas, respectively. Potential for an interaction between these groups exists; however, their distributions are rarely determined in tandem. Here we have examined the vertical distribution of AOA and anammox bacteria through the Arabian Sea oxygen minimum zone (OMZ), one of the most intense and vertically exaggerated OMZs in the global ocean, using a unique combination of intact polar lipid (IPL) and gene-based analyses, at both DNA and RNA levels. To screen for AOA-specific IPLs, we developed a high-performance liquid chromatography/mass spectrometry/mass spectrometry method targeting hexose-phosphohexose (HPH) crenarchaeol, a common IPL of cultivated AOA. HPH-crenarchaeol showed highest abundances in the upper OMZ transition zone at oxygen concentrations of ca. 5  μM, coincident with peaks in both thaumarchaeotal 16S rDNA and amoA gene abundances and gene expression. In contrast, concentrations of anammox-specific IPLs peaked within the core of the OMZ at 600  m, where oxygen reached the lowest concentrations, and coincided with peak anammox 16S rDNA and the hydrazine oxidoreductase (hzo) gene abundances and their expression. Taken together, the data reveal a unique depth distribution of abundant AOA and anammox bacteria and the segregation of their respective niches by >400  m, suggesting no direct coupling of their metabolisms at the time and site of sampling in the Arabian Sea OMZ.

The diazotrophic community in microbial mats growing along the shore of the North Sea barrier island Schiermonnikoog (The Netherlands) was studied using microscopy, lipid biomarkers, stable carbon (δ(13) C(TOC) ) and nitrogen (δ(15) N) isotopes as well as by constructing and analyzing 16S rRNA gene libraries. Depending on their position on the littoral gradient, two types of mats were identified, which showed distinct differences regarding the structure, development and composition of the microbial community. Intertidal microbial mats showed a low species diversity with filamentous non-heterocystous Cyanobacteria providing the main mat structure. In contrast, supratidal microbial mats showed a distinct vertical zonation and a high degree of species diversity. Morphotypes of non-heterocystous Cyanobacteria were recognized as the main structural component in these mats. In addition, unicellular Cyanobacteria were frequently observed, whereas filamentous heterocystous Cyanobacteria occurred only in low numbers. Besides the apparent visual dominance of cyanobacterial morphotpyes, 16S rRNA gene libraries indicated that both microbial mat types also included members of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides group as well as diatoms. Bulk δ(15) N isotopes of the microbial mats ranged from +6.1‰ in the lower intertidal to -1.2‰ in the supratidal zone, indicating a shift from predominantly nitrate utilization to nitrogen fixation along the littoral gradient. This conclusion was supported by the presence of heterocyst glycolipids, representing lipid biomarkers for nitrogen-fixing heterocystous Cyanobacteria, in supratidal but not in intertidal microbial mats. The availability of combined nitrogen species might thus be a key factor in controlling and regulating the distribution of the diazotrophic microbial community of Schiermonnikoog., (© 2011 Blackwell Publishing Ltd.)

The distribution of membrane lipids of 17 different strains representing 13 species of subdivisions 1 and 3 of the phylum Acidobacteria, a highly diverse phylum of the Bacteria, were examined by hydrolysis and gas chromatography-mass spectrometry (MS) and by high-performance liquid chromatography-MS of intact polar lipids. Upon both acid and base hydrolyses of total cell material, the uncommon membrane-spanning lipid 13,16-dimethyl octacosanedioic acid (iso-diabolic acid) was released in substantial amounts (22 to 43% of the total fatty acids) from all of the acidobacteria studied. This lipid has previously been encountered only in thermophilic Thermoanaerobacter species but bears a structural resemblance to the alkyl chains of bacterial glycerol dialkyl glycerol tetraethers (GDGTs) that occur ubiquitously in peat and soil and are suspected to be produced by acidobacteria. As reported previously, most species also contained iso-C(15) and C(16:1ω7C) as major fatty acids but the presence of iso-diabolic acid was unnoticed in previous studies, most probably because the complex lipid that contained this moiety was not extractable from the cells; it could only be released by hydrolysis. Direct analysis of intact polar lipids in the Bligh-Dyer extract of three acidobacterial strains, indeed, did not reveal any membrane-spanning lipids containing iso-diabolic acid. In 3 of the 17 strains, ether-bound iso-diabolic acid was detected after hydrolysis of the cells, including one branched GDGT containing iso-diabolic acid-derived alkyl chains. Since the GDGT distribution in soils is much more complex, branched GDGTs in soil likely also originate from other (acido)bacteria capable of biosynthesizing these components.

Anaerobic ammonium oxidation (anammox) is an important process for nitrogen removal in marine pelagic and benthic environments and represents a major sink in the global nitrogen cycle. We applied a suite of complementary methods for the detection and enumeration of anammox activity and anammox bacteria in marine sediments of the Gullmar Fjord, and compared the results obtained with each technique. (15) N labelling experiments showed that nitrogen removal through N2 production was essentially limited to the upper 2 cm of the sediment, where anammox contributed 23-47% of the total production. The presence of marine anammox bacteria belonging to the genus 'Candidatus Scalindua' was shown by 16S rRNA gene sequence comparison. FISH counts of anammox bacteria correlated well with anammox activity, while quantitative PCR may have underestimated the number of anammox bacterial 16S rRNA gene copies at this site. Potential nitrogen conversion by anammox ranged from 0.6 to 4.8 fmol N cell(-1) day(-1) , in agreement with previous measurements in the marine environment and in bioreactors. Finally, intact ladderane glycerophospholipid concentrations better reflected anammox activity and abundance than ladderane core lipid concentrations, most likely because the core lipid fraction contained a substantial fossil component, especially deeper in the sediment., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Glycerol dibiphytanyl glycerol tetraether (GDGT)-based intact membrane lipids are increasingly being used as complements to conventional molecular methods in ecological studies of ammonia-oxidizing archaea (AOA) in the marine environment. However, the few studies that have been done on the detailed lipid structures synthesized by AOA in (enrichment) culture are based on species enriched from nonmarine environments, i.e., a hot spring, an aquarium filter, and a sponge. Here we have analyzed core and intact polar lipid (IPL)-GDGTs synthesized by three newly available AOA enriched directly from marine sediments taken from the San Francisco Bay estuary ("Candidatus Nitrosoarchaeum limnia"), and coastal marine sediments from Svalbard, Norway, and South Korea. Like previously screened AOA, the sedimentary AOA all synthesize crenarchaeol (a GDGT containing a cyclohexane moiety and four cyclopentane moieties) as a major core GDGT, thereby supporting the hypothesis that crenarchaeol is a biomarker lipid for AOA. The IPL headgroups synthesized by sedimentary AOA comprised mainly monohexose, dihexose, phosphohexose, and hexose-phosphohexose moieties. The hexose-phosphohexose headgroup bound to crenarchaeol was common to all enrichments and, in fact, the only IPL common to every AOA enrichment analyzed to date. This apparent specificity, in combination with its inferred lability, suggests that it may be the most suitable biomarker lipid to trace living AOA. GDGTs bound to headgroups with a mass of 180 Da of unknown structure appear to be specific to the marine group I.1a AOA: they were synthesized by all three sedimentary AOA and "Candidatus Nitrosopumilus maritimus"; however, they were absent in the group I.1b AOA "Candidatus Nitrososphaera gargensis."

Lipid biomarkers are widely used to study the earliest life on Earth and have been invoked as potential astrobiological markers, but few studies have assessed their survival and persistence in geothermal settings. Here, we investigate lipid preservation in active and inactive geothermal silica sinters, with ages of up to 900 years, from Champagne Pool, Waiotapu, New Zealand. Analyses revealed a wide range of bacterial biomarkers, including free and bound fatty acids, 1,2-di-O-alkylglycerols (diethers), and various hopanoids. Dominant archaeal lipids include archaeol and glycerol dialkyl glycerol tetraethers (GDGTs). The predominance of generally similar biomarker groups in all sinters suggests a stable microbial community throughout Champagne Pool's history and indicates that incorporated lipids can be well preserved. Moreover, subtle differences in lipid distributions suggest that past changes in environmental conditions can be elucidated. In this case, higher archaeol abundances relative to the bacterial diethers, a greater proportion of cyclic GDGTs, the high average chain length of the bacterial diethers, and greater concentrations of hopanoic acids in the older sinters all suggest hotter conditions at Champagne Pool in the past.

N(2)-fixing cyanobacteria play an essential role in sustaining primary productivity in contemporary oceans and freshwater systems. However, the significance of N(2)-fixing cyanobacteria in past nitrogen cycling is difficult to establish as their preservation potential is relatively poor and specific biological markers are presently lacking. Heterocystous N(2)-fixing cyanobacteria synthesize unique long-chain glycolipids in the cell envelope covering the heterocyst cell to protect the oxygen-sensitive nitrogenase enzyme. We found that these heterocyst glycolipids are remarkably well preserved in (ancient) lacustrine and marine sediments, unambiguously indicating the (past) presence of N(2)-fixing heterocystous cyanobacteria. Analysis of Pleistocene sediments of the eastern Mediterranean Sea showed that heterocystous cyanobacteria, likely as epiphytes in symbiosis with planktonic diatoms, were particularly abundant during deposition of sapropels. Eocene Arctic Ocean sediments deposited at a time of large Azolla blooms contained glycolipids typical for heterocystous cyanobacteria presently living in symbiosis with the freshwater fern Azolla, indicating that this symbiosis already existed in that time. Our study thus suggests that heterocystous cyanobacteria played a major role in adding "new" fixed nitrogen to surface waters in past stratified oceans.