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The sole unifying feature of the incredibly diverse Archaea is their isoprenoid‐based ether‐linked lipid membranes. Unique lipid membrane composition, including an abundance of membrane‐spanning tetraether lipids, impart resistance to extreme conditions. Many questions remain, however, regarding the synthesis and modification of tetraether lipids and how dynamic changes to archaeal lipid membrane composition support hyperthermophily. Tetraether membranes, termed glycerol dibiphytanyl glycerol tetraethers (GDGTs), are generated by tetraether synthase (Tes) by joining the tails of two bilayer lipids known as archaeol. GDGTs are often further specialized through the addition of cyclopentane rings by GDGT ring synthase (Grs). A positive correlation between relative GDGT abundance and entry into stationary phase growth has been observed, but the physiological impact of inhibiting GDGT synthesis has not previously been reported. Here, we demonstrate that the model hyperthermophile Thermococcus kodakarensis remains viable when Tes (TK2145) or Grs (TK0167) are deleted, permitting phenotypic and lipid analyses at different temperatures. The absence of cyclopentane rings in GDGTs does not impact growth in T. kodakarensis, but an overabundance of rings due to ectopic Grs expression is highly fitness negative at supra‐optimal temperatures. In contrast, deletion of Tes resulted in the loss of all GDGTs, cyclization of archaeol, and loss of viability upon transition to the stationary phase in this model archaea. These results demonstrate the critical roles of highly specialized, dynamic, isoprenoid‐based lipid membranes for archaeal survival at high temperatures. Plain Language Summary: The preponderance of tetraether membrane‐spanning lipids in many archaeal clades indicates the fitness advantage of unique membranes for archaeal survival. The inability to rationally control the synthesis of tetraether and cyclized tetraether‐lipids has limited investigations into how dynamic shifts in lipid composition support archaeal growth and phase transitions. Employing the facile genetic system of the model hyperthermophilic archaeon Thermococcus kodakarensis that naturally synthesizes abundant tetraether lipids, we identified and targeted the genes necessary for tetraether lipid synthesis and derivatization. While impairing tetraether lipid biosynthesis is possible, the lack of specialized lipids dramatically impairs long‐term survival, supporting a critical role for dynamic lipidome changes in supporting archaeal life in the extremes. [ABSTRACT FROM AUTHOR]

Organic biomarker distribution and stable isotope composition was used to investigate biogeochemical carbon cycling in the anoxic basin of Lake Untersee, Antarctica. Phospholipid fatty acid (PLFA) concentrations indicative of microbial abundances were low in the oxic water column overlying the basin but rose in the suboxic transition zone and further increased within the underlying anoxic water, with the highest abundances near the sediment water interface. Archaeol (up to 24.8 mg/kg) and glycerol dialkyl glycerol tetraethers were only detected within the deepest water sample and sediment. High methane (CH4) concentrations (ca. 172 mg/L or 11 mmol/L) were observed in the deepest water samples and produced via hydrogenotrophy (CO2-reduction) based on methane isotopes and highly 13C-enriched dissolved inorganic carbon. Methane concentration slowly decreased away from the sediment, across the anoxic water column and then decreased rapidly at the oxic/anoxic interface (78–68 m). Here a ca. 10‰ increase in δ13CCH4 values combined with δ13CPLFA values that decreased as CH4 concentrations rapidly declined indicated an aerobic methanotrophy fueled microbial community. Findings suggest that upward methane diffusion drives microbial productivity within the suboxic/anoxic zones resulting in the observed high PLFA biomass. Subsequent sinking of detrital material from these communities supports heterotrophic microbes throughout the anoxic water column and potentially supplied nutrients to support phototrophy in the upper suboxic transition zone, itself contributing to sinking detrital material and accumulation of sedimentary organic material. Notably, while a clear biosignature of methane oxidation is present in suboxic zone PLFA, this signature is not recognizable within the sediments. [ABSTRACT FROM AUTHOR]

Abstract As a hallmark of Archaea, their cell membranes are comprised of ether lipids. However, Archaea‐type ether lipids have recently been identified in Bacteria as well, with a somewhat different composition: In Bacillales, sn‐glycerol 1‐phosphate is etherified with one C35 isoprenoid chain, which is longer than the typical C20 chain in Archaea, and instead of a second isoprenoid chain, the product heptaprenylglyceryl phosphate becomes dephosphorylated and afterward diacetylated by the O‐acetyltransferase YvoF. Interestingly, database searches have revealed YvoF homologs in Halobacteria (Archaea), too. Here, we demonstrate that YvoF from Haloferax volcanii can acetylate geranylgeranylglycerol in vitro. Additionally, we present the first‐time identification of acetylated diether lipids in H. volcanii and Halobacterium salinarum by mass spectrometry. A variety of different acetylated lipids, namely acetylated archaeol, and acetylated archaetidylglycerol, were found, suggesting that halobacterial YvoF has a broad substrate range. We suppose that the acetyl group might serve to modify the polarity of the lipid headgroup, with still unknown biological effects.

Archaeosomes are liposomes formulated using total polar lipids (TPLs) or semi-synthetic glycolipids derived from archaea. Conventional archaeosomes with entrapped antigen exhibit robust adjuvant activity as demonstrated by increased antigen-specific humoral and cell-mediated responses and enhanced protective immunity in various murine infection and cancer models. However, antigen entrapment efficiency can vary greatly resulting in antigen loss during formulation and variable antigen:lipid ratios. In order to circumvent this, we recently developed an admixed archaeosome formulation composed of a single semi-synthetic archaeal lipid (SLA, sulfated lactosylarchaeol) which can induce similarly robust adjuvant activity as an encapsulated formulation. Herein, we evaluate and compare the mechanisms involved in the induction of early innate and antigen-specific responses by both admixed (Adm) and encapsulated (Enc) SLA archaeosomes. We demonstrate that both archaeosome formulations result in increased immune cell infiltration, enhanced antigen retention at injection site and increased antigen uptake by antigen-presenting cells and other immune cell types, including neutrophils and monocytes following intramuscular injection to mice using ovalbumin as a model antigen. In vitro studies demonstrate SLA in either formulation is preferentially taken up by macrophages. Although the encapsulated formulation was better able to induce antigen-specific CD8+ T cell activation by dendritic cells in vitro, both encapsulated and admixed formulations gave equivalently enhanced protection from tumor challenge when tested in vivo using a B16-OVA melanoma model. Despite some differences in the immunostimulatory profile relative to the SLA (Enc) formulation, SLA (Adm) induces strong in vivo immunogenicity and efficacy, while offering an ease of formulation.

During Messinian time, the Mediterranean underwent hydrological modifications culminating 5.97 Ma ago with the Messinian salinity crisis (MSC). Evaporite deposition and alleged annihilation of most marine eukaryotes were taken as evidence of the establishment of basin-wide hypersalinity followed by desiccation. However, the palaeoenvironmental conditions during the MSC are still a matter of debate, chiefly because most of its sedimentary record is buried below the abyssal plains of the present-day Mediterranean Sea. To shed light on environmental change at the advent and during the early phase of the MSC, we investigated the Govone section from the Piedmont Basin (NW Italy) using a multidisciplinary approach (organic geochemical, petrographic, and carbon and oxygen stable isotope analyses). The Govone section archives the onset of the crisis in a succession of organic-rich shales and dolomite-rich marls. The MSC part of the succession represents the deep-water equivalent of sulphate evaporites deposited at the basin margins during the first phase of the crisis. Our study reveals that the onset of the MSC was marked by the intensification of water-column stratification, rather than the establishment of widespread hypersaline conditions. A chemocline divided the water column into an oxygen-depleted, denser and more saline bottom layer and an oxygenated, upper seawater layer influenced by freshwater inflow. Vertical oscillations of the chemocline controlled the stratigraphic architecture of the sediments pertaining to the first stage of the MSC. Accordingly, temporal and spatial changes of water masses with different redox chemistries must be considered when interpreting the MSC event. [ABSTRACT FROM AUTHOR]

• Near-surface sediments at a giant Calyptogena colony have abundant archaeal lipids. • Low diether δ13C values are characteristic of an origin from anaerobic methanotrophic archaea. • The δ13 C values of crocetane were also characteristic of an ANME origin. • Most archaeal lipids were from ANME associated with a past methane release event. • Higher δ13 C values of PMI suggest derivation from both ANME and methanogens. To investigate the possibility that dissociation of subsurface methane hydrate (MH) in the eastern Nankai Trough, offshore of Japan, led to the formation of a giant colony of the bivalve Calyptogena (currently mostly dead), the carbon isotope ratios (δ13C) of archaeal lipids and methane were measured in near-surface core sediments at Daini-Tenryu Knoll. The irregular variation of porewater methane δ13C with depth (from −75 ‰ to −26 ‰) suggested that originally low δ13C microbial methane was degraded in different proportions by anaerobic methane oxidation. Consistent with this inference, biomarkers of anaerobic methanotrophic archaea (ANME), namely, crocetane (2,6,11,15-tetramethylhexadecane), PMI (2,6,10,15,19-pentamethylicosane), and diethers (archaeol and hydroxyarchaeols), were detected in lipid extracts. The low diether δ13C values (−121 ‰ to −104 ‰) were characteristic of ANME, but less variable than the methane δ13C values, and the relationships between diethers and methane δ13C values deviated from regression lines derived using worldwide data from modern methane seep sites. In contrast, δ13C values of the ANME source methane predicted from those regression lines and the diether δ13C values agreed well with methane δ13C values in MH samples obtained by nearby deep drilling. This result strongly suggests that most of the diethers were produced by ANME that proliferated during a past massive methane release event associated with MH dissociation. The crocetane δ13C value, measured in a mixture with phytane and estimated from the correlation of the δ13C of the mixture with the mole fraction of crocetane, was about −127 ‰. More than half of the PMI δ13C values were greater than −100 ‰, suggesting the background presence of fossil PMI from methanogens. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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 C20 and C25 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 C12H25NO13S or C12H25NO16S2, 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 AME2T 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 AMF2T 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-C25 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Archaeosomes composed of archaeal total polar lipids (TPL) or semi-synthetic analog vesicles have been used as vaccine adjuvants and delivery systems in animal models for many years. Typically administered by intramuscular or subcutaneous injections, archaeosomes can induce robust, long-lasting humoral and cell-mediated immune responses against entrapped antigens and provide protection in murine models of infectious disease and cancer. Herein, we evaluated various archaeosomes for transdermal delivery, since this route may help eliminate needle-stick injuries and needle re-use, and therefore increase patient compliance. Archaeosomes composed of TPL from different archaea (Halobacterium salinarum, Methanobrevibacter smithii, Haloferax volcanii) and various semi-synthetic glycolipid combinations were evaluated for their ability to diffuse across the skin barrier using an ex vivo pig skin model and the results were compared to conventional synthetic ester liposomes. Physicochemical characteristics were determined for selected formulations including vesicle size, size distribution, zeta potential, fluidity, antigen (ovalbumin) incorporation efficiency and release. Archaeosomes, in particular those composed of M. smithii TPL or the synthetic glycolipid sulfated S-lactosylarchaeol (SLA) mixed with uncharged glycolipid lactosyl archaeol (LA), appeared to be effective carriers for ovalbumin, achieving much better antigen distribution and vesicle accumulation in the skin epidermis than conventional liposomes. The enhanced skin permeation of archaeosomes may be attributed to their chemical structure and physicochemical properties such as particle size, surface charge, stability, and fluidity of their lipid bilayer. [ABSTRACT FROM AUTHOR]

The targeting of mcrA or 16S rRNA genes by quantitative PCR (qPCR) has become the dominant method for quantifying methanogens in rumen. There are considerable discrepancies between estimates based on different primer sets, and the literature is equivocal about the relationship with methane production. There are a number of problems with qPCR, including low primer specificity, multiple copies of genes and multiple genomes per cell. Accordingly, we have investigated alternative markers for methanogens, on the basis of the distinctive ether lipids of archaeal cell membranes. The membranes of Archaea contain dialkyl glycerol ethers such as 2,3-diphytanayl-O-sn-glycerol (archaeol), and glycerol dialkyl glycerol tetraethers (GDGTs) such as caldarchaeol (GDGT-0) in different proportions. The relationships between estimates of methanogen abundance using qPCR and archaeol measurements varied across primers. Studies in other ecosystems have identified environmental effects on the profile of ether lipids in Archaea. There is a long history of analysing easily accessible samples, such as faeces, urine and milk, to provide information about digestion and metabolism in livestock without the need for intrusive procedures. Purine derivatives in urine and odd-chain fatty acids in milk have been used to study rumen function. The association between volatile fatty acid proportions and methane production is probably the basis for empirical relationships between milk fatty acid profiles and methane production. However, these studies have not yet identified consistent predictors. We have evaluated the relationship between faecal archaeol concentration and methane production across a range of diets in studies on beef and dairy cattle. Faecal archaeol is diagnostic for ruminant faeces being below the limit of detection in faeces from non-ruminant herbivores. The relationship between faecal archaeol and methane production was significant when comparing treatment means across diets, but appears to be subject to considerable between-animal variation. This variation was also evident in the weak relationship between archaeol concentrations in rumen digesta and faeces. We speculate that variation in the distribution and kinetics of methanogens in the rumen may affect the survival and functioning of Archaea in the rumen and therefore contribute to genetic variation in methane production. Indeed, variation in the relationship between the numbers of micro-organisms present in the rumen and those leaving the rumen may explain variation in relationships between methane production and both milk fatty acid profiles and faecal archaeol. As a result, microbial markers in the faeces and milk are unlikely to relate well back to methanogenesis in the rumen. This work has also highlighted the need to describe methanogen abundance in all rumen fractions and this may explain the difficulty interpreting results on the basis of samples taken using stomach tubes or rumenocentesis.

Archaeol (1,2-di-O-phytanyl-sn-glycerol) is a cell membrane lipid component of methanogens that has the potential to be used as a biomarker for methane production in ruminants. However, its analysis via gas chromatography-mass spectrometry (GC-MS) is challenging because of its molecular size and structure. In this study, 2 different sample preparation methods were tested, Soxhlet and sonication-aided extraction, and the methods were compared for extraction efficiency using the internal standard (IS; 1,2-di-o-hexadecyl-rac-glycerol). The extraction efficiency of the Soxhlet method for fecal archaeol was twice that of sonication. With the use of a high-temperature GC column, the retention times of IS and archaeol were 17.6 and 19.4 min, respectively, with a total run time of only 25 min. The molecule ions m/z 611.4 (IS) and m/z 725.8 (archaeol), or alternatively the fragment ion of the glycerol moiety m/z 130.0, were used for identification and quantification via GC-MS in positive chemical ionization mode. The intra-assay coefficients of variation for fecal archaeol measurements were 1.3% (m/z 725.8) and 2.1% (m/z 130.0) (n = 3), respectively. Fecal archaeol quantifications did not differ between the use of the molecule or glycerol moiety ions (paired t-test, n = 156). Archaeol concentrations tended to be 3.3% greater in samples stored at -20°C before drying compared with samples that were immediately dried after collection (paired t-test, n = 5). The detection limit of archaeol was 0.5 μg/g of fecal dry matter (DM); no archaeol could be detected in feed samples. In different fractions of rumen fluid, archaeol levels ranged from 1.9 to 24.0 μg/g of DM. In 10 cows fed the same grass and corn silage/hay-based ration, diurnal variations of fecal archaeol levels (5 time points over 2 d) were cow dependent and ranged from 26.2 to 77.2 μg/g of DM (mean 48.4 μg/g of DM). Thus, within-animal variation in cows on the same diet was between 4 and 27%. We suggest that this finding is related to the amount and time of the latest feed intake event before the fecal sampling. Feeding pattern can determine the passage rate of digesta through the alimentary tract and thus the duration of contact time of archaea with their substrate. [ABSTRACT FROM AUTHOR]

Archaea play an important role in global carbon and nitrogen cycles. Archaeal lipids, such as isoprenoid glycerol diakyl glycerol tetraethers (iGDGTs), are important biomarkers tracing change in archaeal community structure and biogeochemical processes in the natural environments. In this research, the spatial distributions of archaeal lipids in the surface sediments of the Jiulong River (JR) and the Jiulong River estuary (JRE) were examined. GDGT-0 (containing zero cyclopentyl ring) and crenarchaeol were the most abundant iGDGTs in the JR and JRE. From the rivers to the estuary, the total iGDGTs, GDGT-0, crenarchaeol and archaeol concentrations showed significant spatial variation; in particular, GDGT-0 and archaeol in the river may be predominantly derived in situ from methanogens, whereas crenarchaeol in the estuary mainly derived in situ from Thaumarchaeota. We inferred that archaeal community was dominated by methanogens in the Jiulong River and by Thaumarchaeota in the Jiulong River estuary, which are consistent with change in archaeal community structure observed in other estuarine environments. [ABSTRACT FROM AUTHOR]

Preservation of organic matter (OM) in the geosphere has a direct impact on carbon bioavailability, the carbon cycle and the formation of fossil fuels. We have examined some of the processes that lead to the preservation of OM by characterising insoluble OM in a Sphagnum peat bog. We focussed on the partitioning of prokaryotic biomarkers between solvent-extractable and insoluble OM fractions and how that partitioning changed with depth. The insoluble organic matter (IOM) was examined using stepwise chemical degradation involving base and acid hydrolysis. Iso- and anteiso- C 15 and C 17 fatty acids (FAs), hopanoic acid and bishomohopanol, and branched glycerol dialkyl glycerol tetraethers (GDGTs) – diagnostic for Bacteria – were targeted as well as archaeol and isoprenoidal GDGTs – diagnostic for Archaea. High percentages of these compounds – up to 65% – occur in IOM pools, indicating that archaeal- and bacterially derived OM is prone to insolubilization even in shallow sediments (< 5 cm depth). Differences in functionalities – likely related to intact polar (IP) head groups – seem to determine the insolubilization of prokaryotic lipids; specifically, we propose that compounds bearing rapidly degradable phosphate-based polar head groups are less likely to be incorporated into the insoluble OM pools. Collectively, these data indicate that microbial membrane lipids are rapidly incorporated into peat IOM during early diagenesis. We suggest that this is due to an inherently recalcitrant character of some prokaryotic cells which affords protection to their membrane lipids. However, this large proportion of insoluble prokaryotic lipids does not appear to be entirely stable at shallow burial depths, indicating that the peat IOM is a dynamic reservoir, at least during early diagenesis. [ABSTRACT FROM AUTHOR]

The archaeon Thermococcus barophilus, one of the most extreme members of hyperthermophilic piezophiles known thus far, is able to grow at temperatures up to 103C and pressures up to 80 MPa. We analyzed the membrane lipids of T. barophilus by high performance liquid chromatography-mass spectrometry as a function of pressure and temperature. In contrast to previous reports, we show that under optimal growth conditions (40 MPa, 85C) the membrane spanning tetraether lipid GDGT-0 (sometimes called caldarchaeol) is a major membrane lipid of T. barophilus together with archaeol. Increasing pressure and decreasing temperature lead to an increase of the proportion of archaeol. Reversely, a higher proportion of GDGT-0 is observed under low pressure and high temperature conditions. Noticeably, pressure and temperature fluctuations also impact the level of unsaturation of apolar lipids having an irregular polyisoprenoid carbon skeleton (unsaturated lycopane derivatives), suggesting a structural role for these neutral lipids in the membrane of T. barophilus. Whether these apolar lipids insert in the membrane or not remains to be addressed. However, our results raise questions about the structure of the membrane in this archaeon and other Archaea harboring a mixture of di- and tetraether lipids. [ABSTRACT FROM AUTHOR]

Four representatives of methanogenic Euryarchaeota ( Methanosarcina mazei strain Gö1, Methanosphaera stadtmanae , Methanobrevibacter smithii and Methanosaeta thermophila ), the hyperthermophilic euryarchaeon Thermococcus kodakarensis and the halophilic euryarchaeon Haloferax volcanii were studied for their glycerol ether lipid composition. The predominant core membrane lipid in all of them was archaeol, which was accompanied by variable quantities of sn -2-hydroxyarchaeol in the methanogens M. mazei (Methanosarcinales) and M. stadtmanae (Methanobacteriales). All methanogenic and hyperthermophilic Euryarchaeota also contained comparatively high abundances of the glycerol dialkyl glycerol tetraether without a cyclopentane moiety (GDGT-0). The methanoarchaeon M. stadtmanae , in addition to GDGT-0, contained GDGT core lipid structures with 1–4 cyclopentane moieties (GDGTs 1–4). We also found minor amounts of a glycerol trialkyl glycerol tetraether (GTGT) and a glycerol dialkanol diether (GDD), both of which did not contain cyclopentane moieties, as well as methylated and dimethylated GDGT-0 in all the archaea with the exception of H. volcanii . Like its GDGT distribution, M. stadtmanae showed an extended range of GDD structures with up to two cyclopentane ring systems. Our results thus indicate that both methanogenic and hyperthermophilic Euryarchaeota may represent source organisms of GTGT-0, GDDs and methylated-GDGTs in natural environments. All the latter components have recently been reported to be ubiquitously distributed in marine sediments but their biological origin is largely unknown. Moreover, a suite of unsaturated GDGTs without a cyclopentane moiety and up to four double bonds in the hyperthermophile T. kodakarensis was tentatively assigned. [ABSTRACT FROM AUTHOR]

The occurrence and distribution of archaeal and bacterial glycerol dialkyl glycerol tetraether lipids (GDGTs) in continental saline environments have been rarely investigated. Here, the abundance and distribution of archaeal isoprenoid GDGTs (iGDGTs) and archaeol, and of bacterial branched GDGTs (brGDGTs) in four tropical water ponds of contrasting salinity in two islands from the French West Indies, Grande-Terre and La Désirade, have been determined. The sedimentary distribution of the GDGTs strongly differed between the two islands. Caldarchaeol was largely predominant among iGDGTs in the (hyper)saline ponds from Grande-Terre, suggesting a substantial contribution of iGDGTs derived from methanogenic Archaea. In contrast, both caldarchaeol and crenarchaeol were present in high relative abundance in the low salinity ponds from La Désirade, suggesting that iGDGTs were derived from mixed archaeal communities. In addition, the relative proportion of the most methylated brGDGTs was much higher in Grande-Terre ponds than in La Désirade ponds. The applicability of different proxies based on GDGTs and archaeol was tested for these specific environments. The relative abundance of archaeol vs. caldarchaeol (ACE index) was comparable for the four ponds, independent of salinity, showing that the ACE index might not necessarily track salinity change. Moreover, the relative proportion of caldarchaeol vs. total iGDGTs was unexpectedly observed to increase with salinity, suggesting production of this compound by halophilic Archaea. The supposed high abundance of methanogenic Archaea in Grande-Terre ponds prevented the application of TEX 86 as a temperature proxy, whereas TEX 86 could be successfully used for local temperature reconstruction in La Désirade ponds. BrGDGTs seem to be produced predominantly in situ (water column and/or sediment) in hypersaline ponds from Grande-Terre, but in La Désirade ponds likely result from a mixture of soil and aquatic sources. In Grande-Terre ponds, brGDGT-derived temperature estimates generated using either soil or lacustrine calibrations were much lower than expected. The mismatch between expected and estimated temperature might be explained by the presence of halophilic microbial communities producing specific brGDGT distributions in the saline ponds from Grande-Terre. The study shows that the sources of brGDGTs, iGDGTs and archaeol (i) may strongly differ in aquatic environments of varying salinity, even at a regional scale, and (ii) have to be constrained before tetraether-derived proxies in such settings can be confidently applied. [ABSTRACT FROM AUTHOR]

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 C and C hopanoic acid indices typically increase with temperature. At lower pH, the ratio of archaeol to bacterial diethers, degree of GDGT cyclisation and C and C 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. [ABSTRACT FROM AUTHOR]

We examined methane (CH4) oxidation pathways and associated methanotrophic communities in the water column of Lake Kivu using abundance and isotopic compositions of CH4 and phospholipid fatty acids (PLFA), distributions of glycerol dialkyl glycerol tetraethers, and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) analysis. The carbon isotopic data of CH4 indicate that aerobic CH4 oxidation is the predominant pathway of microbial CH4 consumption with an isotopic fractionation factor ( α) of 1.022-1.038. A small amount of CH4 is oxidized anaerobically, with an α of 1.002-1.006. Aerobic CH4 oxidation is mediated by type II methane-oxidizing bacteria (type II MOB) based on the 13C depletion ( δ13C of −40.5‰ to −43.7‰) of diagnostic C18:1 ω7 fatty acids in the surface waters. CARD-FISH images and PLFA components C16:1 ω7 and C16:1 ω5 indicate the presence of type I MOB in the methane and nutrient-rich deep-water region. 13C depletion of C16:1 ω7 and C16:1 ω5 ( δ13C, ∼ −40‰ to −50.6‰) in the lake water below 52 m suggests the involvement of type I MOB in methane oxidation in the anoxic deep-water regions of the lake. A novel cluster of anaerobic methane-oxidizing archaea (ANME) rather than the known ANME-1 and ANME-2 appear to be involved in anaerobic oxidation of methane (AOM). Sulfate reducing bacteria are associated with AOM in the lake based on the 13C depletion ( δ13C, −38.2‰ to −45.0‰) of anteiso-methyl-C15:0 fatty acid. Methane constitutes an important carbon and energy source (up to 38%) for the heterotrophic and autotrophic communities in the lake. [ABSTRACT FROM AUTHOR]

Although the methane in marine methane hydrates is mainly of microbial origin, information about the distribution of methanogens in subseafloor sediments is limited. To address this issue, we analyzed sediment core samples from two sites in the Nankai Trough, off the Pacific coast of central Japan, including those bearing methane hydrates from depths > 100 m below the seafloor (mbsf), for isopranyl ether-linked polar lipids (i.e. with polar head groups of phosphate, sugar, or both) as biomarkers of archaea, including methanogens. In most samples, including the deepest (381 mbsf), archaeol, and sn -2- and sn -3-hydroxyarchaeols were detected as their hydrolyzed derivatives. Concentrations of these three archaeal lipids correlated strongly with each other, suggesting a common biological source. The δ 13 C values of phytane derived from the phytanyl groups in the archaeal lipids were distinctly higher than those of methane, indicating that methanogens rather than anaerobic methanotrophic archaea were the major biological source. Depth profiles of polar sn -2-hydroxyarchaeol concentration were consistent with those of the potential methane production activity previously estimated from incubation of core sediments from the same sites. This observation, together with results of previous studies showing the presence of sn -2-hydroxyarchaeol mainly in shallow young sediments, strongly suggests that this polar lipid is a valid biomarker for in situ methanogens in sediments. There was a strong correlation between the concentration of polar sn -2-hydroxyarchaeol and that of total organic carbon, suggesting that bulk organic matter concentration is a primary control on the distribution of methanogens in sediments. [ABSTRACT FROM AUTHOR]

ABSTRACT Permafrost peatlands are both an important source of atmospheric CH4 and a substantial sink for atmospheric CO2. Climate change can affect this balance, with higher temperatures resulting in the conversion of permafrost soils to wetlands and associated accelerated mineralisation and increased CH4 emission. To better understand the impact of such processes on methanogen populations, we investigated the anaerobic decay of soil carbon in a low Arctic, discontinuous permafrost peatland. Cores were collected monthly from sedge and Sphagnum mires in north Sweden during the summer of 2006. We determined CH4 concentrations and production potentials, together with variations in the size of the methanogenic community as indicated by concentrations of archaeal lipid biomarkers (phosphorylated archaeol, archaeol and hydroxyarchaeol). Concentrations of methanogen biomarkers generally were higher at the sedge site, increased with depth for all sites and months, and were usually below the detection limits in shallow (<10 cm) Sphagnum peat. The distribution of biomarkers reflects the strong influence of water table depth on anaerobic conditions and methanogen populations, while differences in biomarker concentrations can be explained by differences in vegetation cover and pH. However, methanogen populations inferred from biomarker data show a decoupling from in-situ CH4 production over the season and from CH4 production potential, suggesting that other factors such as the availability of labile organic substrates can influence methanogen abundance. Archaeal lipid biomarkers appear to offer a potential new means to investigate permafrost biogeochemical processes but the interpretation of signals remains complex. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The distinctive membrane lipids of the archaea can contain a wide range of chemical structures. The membrane lipid composition of mminal methanogenic archaea has not yet been characterized. In this study, we analyzed proportions of the core archaeal membrane lipids dialkyl glycerol diethers (DGDG) and glycerol dialkyl glycerol tetraether (GDGT). We analyzed the feces of beef steers consuming diets that promoted differences in mminal conditions that were either favorable (i.e., grass silage) or challenging (i.e., concentrates) for the methanogenic archaea. There was significantly less total ether lipid in the feces of cattle consuming the concentrate diet in comparison to the grass silage diet (97 vs. 218 mg/kg DM, respectively), reflecting the inhibitory effect of dietary concentrate on methanogens. Additionally, the proportion of fecal ether lipids as GDGT was much greater in feces from cattle consuming the concentrate diet than in feces from cattle fed grass silage (90% vs. 67% GDGT). A possible explanation for this adaptation is that membrane lipids composited of GDGT lipids are less permeable to protons, thereby protecting the methanogens against low mminal pH and helping to maintain the chemiosmotic potential (which is important for ATP production, methanogenesis, and growth). The greater proportion of fecal ether lipids as GDGT may reflect adaptation of membrane lipids within the same species, a shift toward methanogens that have a greater proportion of GDGT (e.g., Thermoplasmata), or both. The effect of ruminal environment on membrane composition means that it will be important to consider the production of both DGDG and GDGT lipids when developing a proxy for methanogenesis. [ABSTRACT FROM AUTHOR]

Abstract: The quantification of archaeol, a methanogen membrane lipid, may provide an alternative method to estimate methanogen abundance. The focus of this study was to determine the location of methanogens in the ruminant digestive tract using this biomarker. Archaeol was quantified in samples obtained from four lactating cows with rumen cannulae that grazed on either white clover (WC) or perennial ryegrass (PRG) in a changeover design study with three 3-week periods. Faeces were collected over the final 5d of each period and total rumen contents (TRC) were obtained on the final 2 days (day 1: 9am; day 2: 3pm). Solid-associated microbes (SAM) and liquid-associated microbes (LAM) were also isolated from the TRC. Concentrations of archaeol in the TRC showed a significant diet by time interaction, which may be related to diurnal grazing patterns and different rumen conditions associated with PRG or WC diets. There was significantly more archaeol associated with SAM than LAM, which may reflect difficulties of methanogen proliferation in the liquid phase. Faeces had higher concentrations of archaeol than SAM and LAM which was unexpected, although, losses of methanogens may have occurred during isolation (i.e., attachment to protozoa and very small particles), or the methanogens associated with SAM may have been underestimated. There was no significant relationship between faecal and TRC archaeol concentrations. Finally, there was a significant positive relationship between rumen pH and concentrations of archaeol in SAM and LAM, which may be caused by pH and/or WC diet effects. In conclusion, archaeol is potentially a useful alternative marker for determining the abundance of methanogens in the ruminant digestive tract. This work has also highlighted the difficulties associated with methanogen quantification from microbial isolates, and the need for more representative rumen sampling in future studies. [Copyright &y& Elsevier]

Quantitative real-time PCR (qPCR) has become a popular method for estimation of meth-anogen abundance in the ruminant digestive tract. However, there is no established method in terms of primer choice and quantification, which means that results are variable and not directly comparable between studies. Archaeol has been proposed as an alternative marker for methanogen abundance, as it is ubiquitous in methanogenic Archaea, and can be quantified by gas chromatography-mass spectrometry (GC-MS). The aim of this experiment was to compare total metha-nogen populations estimated using the new archaeol approach with estimates based on qPCR. Specific prim-er sets and probes were used to detect dominant luminal methanogen species Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanosphaera stadt-manae, and total methanogen populations. There was variation in the relationships among total methanogen abundance estimates based on archaeol and qPCR. In addition, the universal methanogen primers appeared to preferentially amplify genes from M. smithii. Archaeol had the strongest relationship with the dominant rumen methanogen M. ruminantium, whereas the total metha-nogen primers had a comparatively weak relationship with archaeol. Archaeol analysis was a useful adjunct to molecular biology methods, but it seems that a valid specific primer for M. ruminantium would be more use-ful than a biased primer for total methanogens. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess archaeol, a membrane lipid present in methanogenic Archaea, in cattle feces as a molecular proxy for methanogenesis in the rumen. Feces from 16 heifers either in early lactation [71 d in milk (DIM)] or mid lactation (120 DIM), consuming a diet consisting of 30/70 grass silage/ concentrates [dry matter (DM) basis], were analyzed for archaeol. To prepare the feces for analysis, total lipids were obtained by Bligh-Dyer extraction, polar head groups were removed by acid methanolysis, an alcohol fraction was obtained by column chromatography, and finally, the alcohol fraction was trimethylsilylated before analysis by gas chromatography-mass spectrometry. Archaeol was quantified by comparison to an internal standard. A highly significant positive relationship was found between fecal archaeol concentration (mg/kg of DM) and methane output (g/kg of DM intake). A highly significant effect of stage of lactation on this relationship was observed. The significant relationship was surprising, given the lack of agreement between methane and total methanogens in previous studies using molecular biology techniques. Variation in the relationship between fecal archaeol concentrations and methane output could be attributed to differences in the methane-producing capability per cell and the selective retention of methanogens in the rumen. The effect of stage of lactation may have been due to differences in DM intake, affecting rumen passage rates and, thus, methanogen populations and activities. [ABSTRACT FROM AUTHOR]

Abstract: This study evaluated concentrations of archaeol, a lipid biomarker for Archaea and, presumably, methanogens in cattle faeces. Twelve continental cross-bred steers were allocated at random to receive either a concentrate based diet or a grass silage based diet. Lipids were extracted from dried faeces, separated into specific compound classes and analysed by gas chromatography/mass spectrometry, and CH4 production was estimated using the sulphur hexafluoride (SF6) technique. Steers fed the grass silage based diet consumed less feed (9.15 versus 11.43kg dry matter (DM)/d; SED=0.824; P<0.05), emitted more CH4 (341 versus 174g/d; SED=60.5; P<0.05) and produced faeces with higher concentrations of archaeol (30.6 versus 5.1mg/kg DM; SED=5.42; P<0.001) than those fed the concentrate based diet. Other dialkyl glycerol lipids, such as hydroxyarchaeol, macrocyclic archaeol and unsaturated archaeol analogues, were not detected. Since the feeds contained no archaeol, it must have been produced in the ruminant digestive tract, most likely by Archaea in the rumen. The relationship between CH4 emission and faecal archaeol concentration for individual animals was weak. Possible explanations include the inherent limitations of the SF6 technique, the sampling regime, selective retention of Archaea in the rumen and possible degradation of the archaeol during gut transit and post excretion. Despite these limitations, faecal lipid biomarker analysis shows potential as a new method to study ruminant methanogens. This paper is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors: K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson. [Copyright &y& Elsevier]