Phylogenetic diversity of nitrogenase (nifH) genes in deep-sea and hydrothermal vent environments of the Juan de Fuca Ridge

The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid. Dissolved N2 gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid. In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene nifH from hydrothermal vent fluid. A total of 120 nifH sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal. Subseafloor nifH genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers. Other nifH genes unique to the vent samples include proteobacteria and divergent ARCHAEA: All of the nifH genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon Methanococcus thermolithotrophicus (77 to 83% amino acid similarity). These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of nifH genes detected in hydrothermal vent fluid: nifH genes from an anaerobic, hot subseafloor and nifH genes from cold, oxygenated deep seawater.     

Phylogenetic Diversity of Nitrogenase (nifH) Genes in Deep-Sea and Hydrothermal Vent Environments of the Juan de Fuca Ridge

The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid. Dissolved N₂ gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid. In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene nifH from hydrothermal vent fluid. A total of 120 nifH sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal. Subseafloor nifH genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers. Other nifH genes unique to the vent samples include proteobacteria and divergent Archaea. All of the nifH genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon Methanococcus thermolithotrophicus (77 to 83% amino acid similarity). These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of nifH genes detected in hydrothermal vent fluid: nifH genes from an anaerobic, hot subseafloor and nifH genes from cold, oxygenated deep seawater.     

Mercury adaptation among bacteria from a deep-sea hydrothermal vent

Since deep-sea hydrothermal vent fluids are enriched with toxic metals, it was hypothesized that (i) the biota in the vicinity of a vent is adapted to life in the presence of toxic metals and (ii) metal toxicity is modulated by the steep physical-chemical gradients that occur when anoxic, hot fluids are mixed with cold oxygenated seawater. We collected bacterial biomass at different distances from a diffuse flow vent at 9 degrees N on the East Pacific Rise and tested these hypotheses by examining the effect of mercuric mercury [Hg(II)] on vent bacteria. Four of six moderate thermophiles, most of which were vent isolates belonging to the genus Alcanivorax, and six of eight mesophiles from the vent plume were resistant to >10 microM Hg(II) and reduced it to elemental mercury [Hg(0)]. However, four psychrophiles that were isolated from a nearby inactive sulfide structure were Hg(II) sensitive. A neighbor-joining tree constructed from the deduced amino acids of a PCR-amplified fragment of merA, the gene encoding the mercuric reductase (MR), showed that sequences obtained from the vent moderate thermophiles formed a unique cluster (bootstrap value, 100) in the MR phylogenetic tree, which expanded the known diversity of this locus. The temperature optimum for Hg(II) reduction by resting cells and MR activity in crude cell extracts of a vent moderate thermophile corresponded to its optimal growth temperature, 45 degrees C. However, the optimal temperature for activity of the MR encoded by transposon Tn501 was found to be 55 to 65 degrees C, suggesting that, in spite of its original isolation from a mesophile, this MR is a thermophilic enzyme that may represent a relic of early evolution in high-temperature environments. Results showing that there is enrichment of Hg(II) resistance among vent bacteria suggest that these bacteria have an ecological role in mercury detoxification in the vent environment and, together with the thermophilicity of MR, point to geothermal environments as a likely niche for the evolution of bacterial mercury resistance.     

Enhanced Photocatalytic Performance of ZnS for Reversible Amination of α-oxo Acids by Hydrothermal Treatment

To understand how life could have originated on early Earth, it is essential to know what biomolecules and metabolic pathways are shared by extant organisms and what organic compounds and their chemical reaction channels were likely to have been primordially available during the initial phase of the formation of prebiotic metabolism. In a previous study, we demonstrated for the first time the reversible amination of α-oxo acids on the surface of photo-illuminated ZnS. The sulfide mineral is a typical component at the periphery of submarine hydrothermal vents which has been frequently argued as a very attractive venue for the origin of life. In this work, in order to simulate more closely the precipitation environments of ZnS in the vent systems, we treated newly-precipitated ZnS with hydrothermal conditions and found that its photocatalytic power was significantly enhanced because the relative crystallinity of the treated sample was markedly increased with increasing temperature. Since the reported experimental conditions are believed to have been prevalent in shallow-water hydrothermal vents of early Earth and the reversible amination of α-oxo acids is a key metabolic pathway in all extant life forms, the results of this work provide a prototypical model of the prebiotic amino acid redox metabolism. The amino acid dehydrogenase-like chemistry on photo-irradiated ZnS surfaces may advance our understanding of the establishment of archaic non-enzymatic metabolic systems.     

Mercury Adaptation among Bacteria from a Deep-Sea Hydrothermal Vent

Since deep-sea hydrothermal vent fluids are enriched with toxic metals, it was hypothesized that (i) the biota in the vicinity of a vent is adapted to life in the presence of toxic metals and (ii) metal toxicity is modulated by the steep physical-chemical gradients that occur when anoxic, hot fluids are mixed with cold oxygenated seawater. We collected bacterial biomass at different distances from a diffuse flow vent at 9°N on the East Pacific Rise and tested these hypotheses by examining the effect of mercuric mercury [Hg(II)] on vent bacteria. Four of six moderate thermophiles, most of which were vent isolates belonging to the genus Alcanivorax, and six of eight mesophiles from the vent plume were resistant to >10 μM Hg(II) and reduced it to elemental mercury [Hg(0)]. However, four psychrophiles that were isolated from a nearby inactive sulfide structure were Hg(II) sensitive. A neighbor-joining tree constructed from the deduced amino acids of a PCR-amplified fragment of merA, the gene encoding the mercuric reductase (MR), showed that sequences obtained from the vent moderate thermophiles formed a unique cluster (bootstrap value, 100) in the MR phylogenetic tree, which expanded the known diversity of this locus. The temperature optimum for Hg(II) reduction by resting cells and MR activity in crude cell extracts of a vent moderate thermophile corresponded to its optimal growth temperature, 45°C. However, the optimal temperature for activity of the MR encoded by transposon Tn501 was found to be 55 to 65°C, suggesting that, in spite of its original isolation from a mesophile, this MR is a thermophilic enzyme that may represent a relic of early evolution in high-temperature environments. Results showing that there is enrichment of Hg(II) resistance among vent bacteria suggest that these bacteria have an ecological role in mercury detoxification in the vent environment and, together with the thermophilicity of MR, point to geothermal environments as a likely niche for the evolution of bacterial mercury resistance.     

The Role of Submarine Hydrothermal Systems in the Synthesis of Amino Acids

There is little consensus regarding the plausibility of organic synthesis in submarine hydrothermal systems (SHSs) and its possible relevance to the origin of life. The primary reason for the persistence of this debate is that most experimental high temperature and high-pressure organic synthesis studies have neglected important geochemical constraints with respect to source material composition. We report here the results of experiments exploring the potential for amino acid synthesis at high temperature from synthetic seawater solutions of varying composition. The synthesis of amino acids was examined as a function of temperature, heating time, starting material composition and concentration. Using very favorable reactant conditions (high concentrations of reactive, reduced species), small amounts of a limited set of amino acids are generated at moderate temperature conditions (∼125–175°C) over short heating times of a few days, but even these products are significantly decomposed after exposure times of approximately 1 week. The high concentration dependence observed for these synthetic reactions are demonstrated by the fact that a 10-fold drop in concentration results in orders of magnitude lower yields of amino acids. There may be other synthetic mechanisms not studied herein that merit investigation, but the results are likely to be similar. We conclude that although amino acids can be generated from simple likely environmentally available precursors under SHS conditions, the equilibrium at high temperatures characteristic of SHSs favors net amino acid degradation rather than synthesis, and that synthesis at lower temperatures may be more favorable.     

Fatty acid composition in deep hydrothermal vent symbiotic bivalves.

Fatty acids in deep hydrothermal vent bivalves have been analyzed. Their composition is completely different from that of a littoral mussel collected in the Mediterranean sea. The distribution of fatty acids in the littoral mussel is characterized by a predominance of polyunsaturated fatty acids (20:5n-3, 22:6n-3) reflecting the planktonic origin of the food. Vent bivalve fatty acid distribution is dominated by an abundance of the monounsaturated acids (double bond in the n-7 position) 16:1n-7, 18:1n-7, and 20:1n-7 which are clearly of bacterial origin and give an indication of the symbiotic bacterial activity in the bivalves. Differences between the fatty acid composition of the bivalves from two hydrothermal sites (13 degrees N and Galapagos) and differences between the mantle and the gill were observed and are discussed with respect to vent activities at the two sites and species metabolic capacities as a function of ecological conditions.     

Lipid Biomarkers Reveal Microbial Communities in Hydrothermal Chimney Structures from the 49.6°E Hydrothermal Vent Field at the Southwest Indian Ocean Ridge

Lipid biomarkers were investigated to reveal the microbial life preserved in sulfide and Si-rich chimney from the 49.6°E hydrothermal vent field. In sulfide chimney, iso-/anteiso-fatty acids and H-shaped glycerol dialkyl glycerol tetraethers are the main microbial biomarkers. In Si-rich chimney, monounsaturated fatty acids (C16:1n7, C18:1n7) are the main bacterial biomarkers detected, and crenarchaeol and its isomer are relatively abundant (up to 25% of glycerol dialkyl glycerol tetraethers) archaeol biomarkers. Composition of lipid biomarkers reveals the diversity of microbial communities in different types of chimney structures. Sulfate-reducing bacteria and hyperthermophilic archaea were considered to be the majority microbial life in sulfide chimney, and sulfur-oxidizing bacteria were abundant in Si-rich chimney while archaea in Si-rich chimney and mainly attributed to Thaumarchaeota, which were predominately ammonia oxidizers. Our result suggested that fluid temperature and gaseous components could be the main constraints for the diversity of microbial communities in hydrothermal chimney structures in 49.6°E hydrothermal vent field.     

Stable-Carbon-Isotope Composition of Fatty Acids in Hydrothermal Vent Mussels Containing Methanotrophic and Thiotrophic Bacterial Endosymbionts

Fatty acid biomarker analysis coupled with gas chromatography-isotope ratio mass spectrometry was used to confirm the presence of methanotrophic and thiotrophic bacterial endosymbionts in the tissues of a hydrothermal vent mussel (Bathymodiolus sp.), collected from the Menez Gwen vent field on the mid-Atlantic ridge. Monounsaturated (n-8) fatty acids, which are diagnostic of methanotrophic bacteria, were detected in all three types of tissues examined (gill, posterior adductor, and mantle), although levels were highest in gill tissues where the bacteria were found. Stable-carbon-isotope compositions (δ-¹³C per mille relative to that of Peedee belemnite) of fatty acids for all three tissues ranged from −24.9 to −34.9‰, which encompasses the range predicted for both thiotroph- and methanotroph-based nutrition. The data suggest that these thio- and methanotrophic bacterial endosymbionts are equally important in the nutrition of the vent mussel at this particular vent site.     

Experimentally induced endosymbiont loss and re-acquirement in the hydrothermal vent bivalve Bathymodiolus azoricus

Invertebrates harbouring endosymbiotic chemoautotroph bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. In these species mechanisms of symbiont transmission are likely to be key elements of dispersal strategies that remained partially unresolved because the early life stages are not available for developmental studies. To study cessation and re-establishment of symbiosis in the host gill a laboratory experiment was conducted over 45 days in a controlled set-up (LabHorta) that endeavour re-creation of the hydrothermal vent chemical environment. Our animal model was the vent bivalve Bathymodiolus azoricus from the Menez Gwen vent site of the Mid Atlantic Ridge (MAR). Animals were exposed to conditions lacking inorganic S supply for 30 days, which is vital for their symbionts, and then re-acclimatized in sulphide-supplied seawater for an additional 15 days.Gradual disappearance of bacteria from the symbiont-bearing gill cells was observed in animals kept in seawater free of dissolved sulphide for up to 30 days, and was evidenced by histological, ultrastructural observations and Polymerase Chain Reaction tests. Following re-acclimatisation in S-supplied seawater, proliferation of sulphur-bacteria in the gill bacteriocytes confirms the functionality of our sulfide-feeding system in supporting chemoautotrophic symbionts. It may also indicate a horizontal endosymbiont acquisition, i.e. from the environment to the host by means of phagocytosis-like mechanism involving special “pit-like” structures on the apical cell membrane.The present work reports the first laboratory set-up successfully used to maintain the hydrothermal vent bivalve B. azoricus for prolonged periods of time by supplying inorganic sulphur as an energy source for its bacterial endosymbionts. Survival of symbiont bacteria is a critical factor influencing the host physiology and thus the methods reported here represent great potential for future studies of host-symbiont dynamics and for post-capture experimental investigations.     

Analysis by high-performance liquid chromatography of free amino acids extracted from needles of drought-stressed and shaded Pinus ponderosa seedlings

Free amino acid concentrations in needles of drought-stressed and shaded seedlings of ponderosa pine (Pinus ponderosa Dougl. ex Laws.) were analyzed to determine the influence of low irradiance on a biochemical response to drought stress. A convenient method was developed for separating and quantitating amino acids in needle extracts using derivatization with dansyl chloride and reversed-phase high-performance liquid chromatography (HPLC). Twenty-one amino acids were separated on an Ultrasphere ODS CIS column and detected with a fluorescence spectrophotometer. As little as 10 pmol of each were detected with good peak separation and reproducible retention times. The results of HPLC analysis showed that drought and shading induced an increase in total amino acid concentrations in needles; shading had the greater effect. Arginine and proline concentrations increased most in needles of drought-stressed seedlings and remained high in unshaded seedlings recovering from drought 48 h after rewatering. Arginine and glutamine increased most in the shaded seedlings, which did not survive severe drought. The large increase in arginine in both drought-stressed and severely shaded seedlings suggests that sequestering and storage of ammonia are important when stress reduces carbon fixation.     

Characterization of Bacterial Communities in Deep-Sea Hydrothermal Vents from Three Oceanic Regions

Deep-sea hydrothermal vents are considered to be one of the most spectacular ecosystems on Earth. Microorganisms form the basis of the food chain in vents controlling the vent communities. However, the diversity of bacterial communities in deep-sea hydrothermal vents from different oceans remains largely unknown. In this study, the pyrosequencing of 16S rRNA gene was used to characterize the bacterial communities of the venting sulfide, seawater, and tubeworm trophosome from East Pacific Rise, South Atlantic Ridge, and Southwest Indian Ridge, respectively. A total of 23,767 operational taxonomic units (OTUs) were assigned into 42 different phyla. Although Proteobacteria, Actinobacteria, and Bacteroidetes were the predominant phyla in all vents, differences of bacterial diversity were observed among different vents from three oceanic regions. The sulfides of East Pacific Rise possessed the most diverse bacterial communities. The bacterial diversities of venting seawater were much lower than those of vent sulfides. The symbiotic bacteria of tubeworm Ridgeia piscesae were included in the bacterial community of vent sulfides, suggesting their significant ecological functions as the primary producers in the deep-sea hydrothermal vent ecosystems. Therefore, our study presented a comprehensive view of bacterial communities in deep-sea hydrothermal vents from different oceans.     

Stratification analysis of the Óhalász Ox-bow of the River Tisza (Kisköre Reservoir,Hungary)

The present study was carried out in Palaeochori Bay, south eastern coast of Milos, where outflow of hot gas bubbles and hydrothermal water seepage was observed. The study tests hypotheses about relationships between seawater temperature and species with warm-water affinities in areas close to shallow-water vents. We predicted that if temperature plays a major role in influencing abundance of species with warm-water affinities, then there should be more thermophilous species in areas with higher seawater temperature. Time series of seawater temperature were recorded in Palaeochori Bay and Pollonia Bay where hot emission was not observed. Temperature, water pressure and conductivity were measured every 30 min from 19 June 1996 to 16 June 1997. Fourteen qualitative samples of benthic flora were collected by SCUBA diving in six rocky sites. Photosamples of benthic assemblages were collected using an UW camera equipped with a wide-angle lens (15 mm) and an electronic flash unit. A constant area of 0.7 m² was sampled. Quantitative estimates of abundance of algal species were performed as percent cover. This paper shows that patterns of fluctuation in seawater temperature in Palaeochori were different from the control where no venting was found. In Palaeochori, algal assemblages were dominated by species with warm-water affinities that were not found elsewhere. These results are consistent with the hypothesis that temperature plays a major role in influencing abundance of species with warm-water affinities. According to our results, hydrothermal vents are potential oases that experience biological-physical coupling not yet clearly understood. They are important in affecting migration of alloctonous thermophilous species suggesting a possible role of vent areas as stepping stones for species migration.     

Co-ordination of leaf minor amino acid contents in crop species: significance and interpretation

The question of whether general control of amino acid synthesis exists in plants remains to be resolved. It is not known whether there is overall co-ordination of the biosynthesis of amino acids that are formed through distinct pathways. In this work, amino acid contents were measured in a large number of samples taken from wheat, potato and barley leaves under different photosynthetic conditions. The variability in total soluble amino acid contents between samples was approximately 6-fold in wheat and potato. Subtracting the major amino acids from the total soluble amino acids showed that the variability in summed minor amino acid contents was approximately 20-fold. This variability was not correlated with short-term changes in primary carbon and nitrogen metabolism, and only poorly correlated with total leaf amino acids. By contrast, striking linear relationships between the contents of most minor amino acids were observed, demonstrating that the contents of many minor amino acids vary in concert. These observations show that amino acid contents are co-ordinated across biosynthetic families. While these data might be interpreted as an indication of cross-pathway regulation of the expression of key biosynthetic enzymes, the impact of factors such as protein degradation and storage cannot be ignored.     

Prebiotic Synthesis of Glycine from Ethanolamine in Simulated Archean Alkaline Hydrothermal Vents

Submarine hydrothermal vents are generally considered as the likely habitats for the origin and evolution of early life on Earth. In recent years, a novel hydrothermal system in Archean subseafloor has been proposed. In this model, highly alkaline and high temperature hydrothermal fluids were generated in basalt-hosted hydrothermal vents, where H₂ and CO₂ could be abundantly provided. These extreme conditions could have played an irreplaceable role in the early evolution of life. Nevertheless, sufficient information has not yet been obtained for the abiotic synthesis of amino acids, which are indispensable components of life, at high temperature and alkaline condition. This study aims to propose a new method for the synthesis of glycine in simulated Archean submarine alkaline vent systems. We investigated the formation of glycine from ethanolamine under conditions of high temperature (80–160 °C) and highly alkaline solutions (pH = 9.70). Experiments were performed in an anaerobic environment under mild pressure (0.1–8.0 MPa) at the same time. The results suggested that the formation of glycine from ethanolamine occurred rapidly and efficiently in the presence of metal powders, and was favored by high temperatures and high pressures. The experiment provides a new pathway for prebiotic glycine formation and points out the phenomenal influence of high-temperature alkaline hydrothermal vents in origin of life in the early ocean.     

Temporal changes in archaeal diversity and chemistry in a mid-ocean ridge subseafloor habitat

The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles. To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000. The archaeal population was divided into particle-attached (>3-microm-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms. To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I Crenarchaeota and Group II Euryarchaeota, both of which were also present in vent fluids. The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic Methanococcales, as well as many uncultured Euryarchaeota, some of which have been identified in other vent environments. The particle-attached fraction consistently showed greater diversity than the free-living fraction. The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community. The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.     

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.     

Temporal Changes in Archaeal Diversity and Chemistry in a Mid-Ocean Ridge Subseafloor Habitat

The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles. To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000. The archaeal population was divided into particle-attached (>3-μm-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms. To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I Crenarchaeota and Group II Euryarchaeota, both of which were also present in vent fluids. The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic Methanococcales, as well as many uncultured Euryarchaeota, some of which have been identified in other vent environments. The particle-attached fraction consistently showed greater diversity than the free-living fraction. The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community. The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.     

A metabolic study to decipher amino acid catabolism-directed biofuel synthesis in Acetoanaerobium sticklandii DSM 519

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia-producing anaerobe. It has the ability to produce organic solvents and acids from protein catabolism through Stickland reactions and specialized pathways. Nevertheless, its protein catabolism-directed biofuel production has not yet been understood. The present study aimed to decipher such growth-associated metabolic potential of this organism at different growth phases using metabolic profiling. A seed culture of this organism was grown separately in metabolic assay media supplemented with gelatin and or a mixture of amino acids. The extracellular metabolites produced by this organism were qualitatively analyzed by gas chromatography–mass spectrometry platform. The residual amino acids after protein degradation and amino acids assimilation were identified and quantitatively measured by high-performance liquid chromatography (HPLC). Organic solvents and acids produced by this organism were detected and the quantity of them determined with HPLC. Metabolic profiling data confirmed the presence of amino acid catabolic products including tyramine, cadaverine, methylamine, and putrescine in fermented broth. It also found products including short-chain fatty acids and organic solvents of the Stickland reactions. It reported that amino acids were more appropriate for its growth yield compared to gelatin. Results of quantitative analysis of amino acids indicated that many amino acids either from gelatin or amino acid mixture were catabolised at a log-growth phase. Glycine and proline were poorly consumed in all growth phases. This study revealed that apart from Stickland reactions, a specialized system was established in A. sticklandii for protein catabolism-directed biofuel production. Acetone–butanol–ethanol (ABE), acetic acid, and butyric acid were the most important biofuel components produced by this organism. The production of these components was achieved much more on gelatin than amino acids. Thus, A. sticklandii is suggested herein as a potential organism to produce butyric acid along with ABE from protein-based wastes (gelatin) in bio-energy sectors.