Membrane Lipid Composition and Amino Acid Excretion Patterns of Methanothermococcus okinawensis Grown in the Presence of Inhibitors Detected in the Enceladian Plume.

Lipids and amino acids are regarded as important biomarkers for the search for extraterrestrial life in the Solar System. Such biomarkers may be used to trace methanogenic life on other planets or moons in the Solar System, such as Saturn's icy moon Enceladus. However, little is known about the environmental conditions shaping the synthesis of lipids and amino acids. Here, we present the lipid production and amino acid excretion patterns of the methanogenic archaeon Methanothermococcus okinawensis after exposing it to different multivariate concentrations of the inhibitors ammonium, formaldehyde, and methanol present in the Enceladian plume. M. okinawensis shows different patterns of lipid and amino acids excretion, depending on the amount of these inhibitors in the growth medium. While methanol did not show a significant impact on growth, lipid or amino acid production rates, ammonium and formaldehyde strongly affected these parameters. These findings are important for understanding the eco-physiology of methanogens on Earth and have implications for the use of biomarkers as possible signs of extraterrestrial life for future space missions in the Solar System.

Transseptal course of anomalous left main coronary artery originating from single right coronary orifice presenting as unstable angina.

Transseptal course of coronary artery has often been described as a benign entity; however, this report and literature analysis provides growing evidence of high risk of serious cardiovascular events in this anomaly. We present a case of unstable angina in a patient with anomalous common origin of left and right coronary arteries from a single coronary ostium at the right sinus of Valsalva, with subsequent transseptal course of the left main artery, review of relevant literature, and discussion of possible management options.

Endemic hydrothermal vent species identified in the open ocean seed bank.

Hydrothermal vent systems host microbial communities among which several microorganisms have been considered endemic to this type of habitat. It is still unclear how these organisms colonize geographically distant hydrothermal environments. Based on 16S rRNA gene sequences, we compare the bacterial communities of sixteen Atlantic hydrothermal vent samples with our own and publicly available global open ocean samples. Analysing sequences obtained from 63 million 16S rRNA genes, the genera we could identify in the open ocean waters contained 99.9% of the vent reads. This suggests that previously observed vent exclusiveness is, in most cases, probably an artefact of lower sequencing depth. These findings are a further step towards elucidating the role of the open ocean as a seed bank. They can explain the predicament of how species expected to be endemic to vent systems are able to colonize geographically distant hydrothermal habitats and contribute to our understanding of whether 'everything is really everywhere'.

Methanogenic capabilities of ANME-archaea deduced from (13) C-labelling approaches.

Anaerobic methanotrophic archaea (ANME) are ubiquitous in marine sediments where sulfate dependent anaerobic oxidation of methane (AOM) occurs. Despite considerable progress in the understanding of AOM, physiological details are still widely unresolved. We investigated two distinct microbial mat samples from the Black Sea that were dominated by either ANME-1 or ANME-2. The (13) C lipid stable isotope probing (SIP) method using labelled substances, namely methane, bicarbonate, acetate, and methanol, was applied, and the substrate-dependent methanogenic capabilities were tested. Our data provide strong evidence for a versatile physiology of both, ANME-1 and ANME-2. Considerable methane production rates (MPRs) from CO2 -reduction were observed, particularly from ANME-2 dominated samples and in the presence of methane, which supports the hypothesis of a co-occurrence of methanotrophy and methanogenesis in the AOM systems (AOM/MPR up to 2:1). The experiments also revealed strong methylotrophic capabilities through (13) C-assimilation from labelled methanol, which was independent of the presence of methane. Additionally, high MPRs from methanol were detected in both of the mat samples. As demonstrated by the (13) C-uptake into lipids, ANME-1 was found to thrive also under methane free conditions. Finally, C35 -isoprenoid hydrocarbons were identified as new lipid biomarkers for ANME-1, most likely functioning as a hydrogen sink during methanogenesis.

Hydrogen is an energy source for hydrothermal vent symbioses.

The discovery of deep-sea hydrothermal vents in 1977 revolutionized our understanding of the energy sources that fuel primary productivity on Earth. Hydrothermal vent ecosystems are dominated by animals that live in symbiosis with chemosynthetic bacteria. So far, only two energy sources have been shown to power chemosynthetic symbioses: reduced sulphur compounds and methane. Using metagenome sequencing, single-gene fluorescence in situ hybridization, immunohistochemistry, shipboard incubations and in situ mass spectrometry, we show here that the symbionts of the hydrothermal vent mussel Bathymodiolus from the Mid-Atlantic Ridge use hydrogen to power primary production. In addition, we show that the symbionts of Bathymodiolus mussels from Pacific vents have hupL, the key gene for hydrogen oxidation. Furthermore, the symbionts of other vent animals such as the tubeworm Riftia pachyptila and the shrimp Rimicaris exoculata also have hupL. We propose that the ability to use hydrogen as an energy source is widespread in hydrothermal vent symbioses, particularly at sites where hydrogen is abundant.

Driving forces behind the biotope structures in two low-temperature hydrothermal venting sites on the southern Mid-Atlantic Ridge.

Although it has been more than 30 years since the discovery of deep-sea hydrothermal vents, comprehending the interconnections between hydrothermal venting and microbial life remains a challenge. Here we investigate abiotic-biotic linkages in low-temperature hydrothermal biotopes at Desperate and Lilliput on the southern Mid-Atlantic Ridge. Both sites are basalt-hosted and fluids exhibit the expected chemical signatures. However, contrasting crustal permeabilities have been proposed, supporting pervasive mixing at Desperate but restricting circulation at Lilliput. In Desperate fluids, sulfide and O2 were readily available but H2 hardly detectable. Under incubation conditions (oxic unamended, sulfide-spiked, oxic and anoxic H2 -spiked at 18°C), only sulfide oxidation by Thiomicrospira fuelled biomass synthesis. Microbial phylogenies from Desperate incubation experiments resembled those of the natural samples suggesting that the incubation conditions mimicked the environment. In Lilliput fluids, O2 was limited, whereas sulfide and H2 were enriched. Autotrophy appeared to be stimulated by residual sulfide and by amended H2 . Yet, based on bacterial phylogenies only conditions in anoxic H2 -spiked Lilliput incubations appeared similar to parts of the Lilliput habitat. In anoxic H2 -spiked Lilliput enrichments Campylobacteraceae likely supported biomass production through H2 oxidation. We argue that the diverging circulation patterns arising from different subseafloor permeabilities act as major driving forces shaping these biotope structures.

Geochemical constraints on the diversity and activity of H2 -oxidizing microorganisms in diffuse hydrothermal fluids from a basalt- and an ultramafic-hosted vent.

Mixing processes of reduced hydrothermal fluids with oxygenated seawater and fluid-rock reactions contribute to the chemical signatures of diffuse venting and likely determine the geochemical constraints on microbial life. We examined the influence of fluid chemistry on microbial diversity and activity by sampling diffuse fluids emanating through mussel beds at two contrasting hydrothermal vents. The H(2) concentration was very low at the basalt-hosted Clueless site, and mixing models suggest O(2) availability throughout much of the habitat. In contrast, effluents from the ultramafic-hosted Quest site were considerably enriched in H(2) , while O(2) is likely limited to the mussel layer. Only two different hydrogenase genes were identified in clone libraries from the H(2) -poor Clueless fluids, but these fluids exhibited the highest H(2) uptake rates in H(2) -spiked incubations (oxic conditions, at 18 °C). In contrast, a phylogenetically diverse H(2) -oxidizing potential was associated with distinct thermal conditions in the H(2) -rich Quest fluids, but under oxic conditions, H(2) uptake rates were extremely low. Significant stimulation of CO(2) fixation rates by H(2) addition was solely illustrated in Quest incubations (P-value <0.02), but only in conjunction with anoxic conditions (at 18 °C). We conclude that the factors contributing toward differences in the diversity and activity of H(2) oxidizers at these sites include H(2) and O(2) availability.

A novel, multi-layered methanotrophic microbial mat system growing on the sediment of the Black Sea.

A novel microbially diverse type of 1- to 5-cm-thick mat performing anaerobic oxidation of methane (AOM) and covering several square metres of the seafloor was discovered in the Black Sea at 180 m water depth. Contrary to other AOM-mat systems of the Black Sea these floating mats are not associated to free gas and are not stabilized by authigenic carbonates. However, supply of methane is ensured by the horizontal orientation of the mats acting as a cover of methane enriched fluids ascending from the underlying sediments. Thorough investigation of their community composition by molecular microbiology and lipid biomarkers, metabolic activities and elemental composition showed that the mats provide a clearly structured system with extracellular polymeric substances (EPS) building the framework of the mats. The top black zone, showing high rates of AOM (15 mumol g(dw) (-1) day(-1)), was dominated by ANME-2, while the following equally active pink layer was dominated by ANME-1 Archaea. The lowest AOM activity (2 mumol g(dw) (-1) day(-1)) and cell numbers were found in the greyish middle part delimited towards the sediment by a second pink, ANME-1-dominated and sometimes a black outer layer (ANME-2). Our work clearly shows that the different microbial populations are established along defined chemical gradients such as methane, sulfate or sulfide.

The influence of ultramafic rocks on microbial communities at the Logatchev hydrothermal field, located 15 degrees N on the Mid-Atlantic Ridge.

The ultramafic-hosted Logatchev hydrothermal field (LHF) on the Mid-Atlantic Ridge is characterized by high hydrogen and methane contents in the subseafloor, which support a specialized microbial community of phylogenetically diverse, hydrogen-oxidizing chemolithoautotrophs. We compared the prokaryotic communities of three sites located in the LHF and encountered a predominance of archaeal sequences affiliated with methanogenic Methanococcales at all three. However, the bacterial composition varied in accordance with differences in fluid chemistry between the three sites investigated. An increase in hydrogen seemed to coincide with the diversification of hydrogen-oxidizing bacteria. This might indicate that the host rock indirectly selects this specific group of bacteria. However, next to hydrogen availability further factors are evident (e.g. mixing of hot reduced hydrothermal fluids with cold oxygenated seawater), which have a significant impact on the distribution of microorganisms.

Microbial CO(2) fixation and sulfur cycling associated with low-temperature emissions at the Lilliput hydrothermal field, southern Mid-Atlantic Ridge (9 degrees S).

Lilliput was discovered in 2005 as the southernmost known hydrothermal field along the Mid-Atlantic Ridge. It is exceptional in that it lacks high-temperature venting probably because of a thickened crust. The absence of thermophilic and hyperthermophilic prokaryotes in emissions supports the argument against the presence of a hot subsurface at Lilliput, as is typically suggested for diffuse emissions from areas of high-temperature venting. The high phylogenetic diversity and novelty of bacteria observed could be because of the low-temperature influence, the distinct location of the hydrothermal field or the Bathymodiolus assemblages covering the sites of discharge. The low-temperature fluids at the Lilliput are characterized by lowered pH and slightly elevated hydrogen (16 nM) and methane ( approximately 2.6 microM) contents compared with ambient seawater. No typical hydrogen and methane oxidizing prokaryotes were detected. The higher diversity of reverse tricarboxylic acid genes and the form II RubisCO genes of the Calvin Benson-Bassham (CBB) cycle compared with the form I RubisCO genes of the CBB cycle suggests that the chemoautotrophic community is better adapted to low oxygen concentrations. Thiomicrospira spp. and Epsilonproteobacteria dominated the autotrophic community. Sulfide is the most abundant inorganic energy source (0.5 mM). Diverse bacteria were associated with sulfur cycling, including Gamma-, Delta- and Epsilonproteobacteria, with the latter being the most abundant bacteria according to fluorescence in situ hybridization. With members of various Candidate Divisions constituting for 25% of clone library sequences we suggest that their role in vent ecosystems might be more important than previously assumed and propose potential mechanisms they might be involved in at the Lilliput hydrothermal field.

Biosynthesis of hopanoids by sulfate-reducing bacteria (genus Desulfovibrio).

Sulfate reduction accounts for about a half of the remineralization of organic carbon in anoxic marine shelf regions. Moreover, it was already a major microbial process in the very early ocean at least 2.4 billion years before the present. Here we demonstrate for the first time the capability of sulfate-reducing bacteria (SRB) to biosynthesize hopanoids, compounds that are quantitatively important and widely distributed biomarkers in recent and fossil sediments dating back to the late Archean. We found high concentrations (9.8-12.3 mg per gram of dry cells) of non-extended and extended bacteriohopanoids (bacteriohopanetetrol, aminobacteriohopanetriol, aminobacteriohopanetetrol) in pure cultures of SRB belonging to the widely distributed genus Desulfovibrio. Biohopanoids were found--considered as membrane rigidifiers--in more than 50% of bacterial species analysed so far. However, their biosynthesis appeared to be restricted to aerobes or facultative anaerobes with a very few recently described exceptions. Consequently, findings of sedimentary hopanoids are often used as indication for oxygenated settings. Nevertheless, our findings shed new light on the presence of hopanoids in specific anoxic settings and suggests that SRB are substantial sources of this quantitatively important lipid class in recent but also past anoxic environments.

Subsurface microbial methanotrophic mats in the Black Sea.

A nodule-shaped microbial mat was found subsurface in sediments of a gas seep in the anoxic Black Sea. This mat was dominated by ANME-1 archaea and consumed methane and sulfate simultaneously. We propose that such subsurface mats represent the initial stage of previously investigated microbial reefs.

In vitro study of lipid biosynthesis in an anaerobically methane-oxidizing microbial mat.

The anaerobic oxidation of methane (AOM) is a key process in the global methane cycle, and the majority of methane formed in marine sediments is oxidized in this way. Here we present results of an in vitro 13CH4 labeling study (delta13CH4, approximately 5,400 per thousand) in which microorganisms that perform AOM in a microbial mat from the Black Sea were used. During 316 days of incubation, the 13C uptake into the mat biomass increased steadily, and there were remarkable differences for individual bacterial and archaeal lipid compounds. The greatest shifts were observed for bacterial fatty acids (e.g., hexadec-11-enoic acid [16:1Delta11]; difference between the delta13C at the start and the end of the experiment [Deltadelta13C(start-end)], approximately 160 per thousand). In contrast, bacterial glycerol diethers exhibited only slight changes in delta13C (Deltadelta13C(start-end), approximately 10 per thousand). Differences were also found for individual archaeal lipids. Relatively high uptake of methane-derived carbon was observed for archaeol (Deltadelta13C(start-end), approximately 25 per thousand), a monounsaturated archaeol, and biphytanes, whereas for sn-2-hydroxyarchaeol there was considerably less change in the delta13C (Deltadelta13C(start-end), approximately 2 per thousand). Moreover, an increase in the uptake of 13C for compounds with a higher number of double bonds within a suite of polyunsaturated 2,6,10,15,19-pentamethyleicosenes indicated that in methanotrophic archaea there is a biosynthetic pathway similar to that proposed for methanogenic archaea. The presence of group-specific biomarkers (for ANME-1 and ANME-2 associations) and the observation that there were differences in 13C uptake into specific lipid compounds confirmed that multiple phylogenetically distinct microorganisms participate to various extents in biomass formation linked to AOM. However, the greater 13C uptake into the lipids of the sulfate-reducing bacteria (SRB) than into the lipids of archaea supports the hypothesis that there is autotrophic growth of SRB on small methane-derived carbon compounds supplied by the methane oxidizers.

Membrane lipid patterns typify distinct anaerobic methanotrophic consortia.

The anaerobic oxidation of methane (AOM) is one of the major sinks of this substantial greenhouse gas in marine environments. Recent investigations have shown that diverse communities of anaerobic archaea and sulfate-reducing bacteria are involved in AOM. Most of the relevant archaea are assigned to two distinct phylogenetic clusters, ANME-1 and ANME-2. A suite of specific (13)C-depleted lipids demonstrating the presence of consortia mediating AOM in fossil and recent environments has been established. Here we report on substantial differences in the lipid composition of microbial consortia sampled from distinct compartments of AOM-driven carbonate reefs growing in the northwestern Black Sea. Communities in which the dominant archaea are from the ANME-1 cluster yield internally cyclized tetraether lipids typical of thermophiles. Those in which ANME-2 archaea are dominant yield sn-2-hydroxyarchaeol accompanied by crocetane and crocetenes. The bacterial lipids from these communities are also distinct even though the sulfate-reducing bacteria all belong to the Desulfosarcina/Desulfococcus group. Nonisoprenoidal glycerol diethers are predominantly associated with ANME-1-dominated communities. Communities with ANME-2 yield mainly conventional, ester-linked diglycerides. ANME-1 archaea and associated sulfate-reducing bacteria seem to be enabled to use low concentrations of methane and to grow within a broad range of temperatures. Our results offer a tool for the study of recent and especially of fossil methane environments.

Hydrogen and carbon isotope fractionation during anaerobic biodegradation of aromatic hydrocarbons--a field study.

The aquifer of a former manufactured gas plant site, highly contaminated by dissolved monocyclic, heterocyclic, and polycyclic aromatic hydrocarbons, was studied to evaluate the applicability of carbon and hydrogen isotope fractionation to prove ongoing biodegradation of these compounds even in complex aquifer settings. The loss of toluene, o-xylene, p,m-xylene, and 2-methylnaphthalene was accompanied by a considerable carbon isotope fractionation. Additionally, a strong 2H enrichment in residual o-xylene was detected. All isotope fractionations observed could be related to established biochemical degradation mechanisms, each involving a C-H bond cleavage in the rate-determining step. In contrast, other compounds such as 1-methylnaphthalene, methylbenzofuran, and acenaphthene exhibited a uniform stable carbon isotope composition. However, a decrease in concentration for these compounds was observed in the flowpath of the aquifer. High threshold concentrations of acenaphthene downgradient indicate that this contaminant is, if at all, only marginally biodegraded. Detailed analyses of xylenes provided support that compound specific isotope analyses and subsequent application of the Rayleigh model may provide a valuable basis to distinguish between different biodegradation mechanisms as well as dissolution processes in heterogeneous aquifers.

Microbial reefs in the Black Sea fueled by anaerobic oxidation of methane.

Massive microbial mats covering up to 4-meter-high carbonate buildups prosper at methane seeps in anoxic waters of the northwestern Black Sea shelf. Strong 13C depletions indicate an incorporation of methane carbon into carbonates, bulk biomass, and specific lipids. The mats mainly consist of densely aggregated archaea (phylogenetic ANME-1 cluster) and sulfate-reducing bacteria (Desulfosarcina/Desulfococcus group). If incubated in vitro, these mats perform anaerobic oxidation of methane coupled to sulfate reduction. Obviously, anaerobic microbial consortia can generate both carbonate precipitation and substantial biomass accumulation, which has implications for our understanding of carbon cycling during earlier periods of Earth's history.