Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

Growth and methane oxidation rates of anaerobic methanotrophic archaea in a continuous-flow bioreactor

Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.     

Growth and Methane Oxidation Rates of Anaerobic Methanotrophic Archaea in a Continuous-Flow Bioreactor

Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.     

Diversity and distribution of methanotrophic archaea at cold seeps

In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to Methanococcoides spp.) and to the Crenarchaeota of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.     

Nitrate-based niche differentiation by distinct sulfate-reducing bacteria involved in the anaerobic oxidation of methane

Diverse associations between methanotrophic archaea (ANME) and sulfate-reducing bacterial groups (SRB) often co-occur in marine methane seeps; however, the ecophysiology of these different symbiotic associations has not been examined. Here, we applied a combination of molecular, geochemical and Fluorescence in situ hybridization (FISH) coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS) analyses of in situ seep sediments and methane-amended sediment incubations from diverse locations (Eel River Basin, Hydrate Ridge and Costa Rican Margin seeps) to investigate the distribution and physiology of a newly identified subgroup of the Desulfobulbaceae (seepDBB) found in consortia with ANME-2c archaea, and compared these with the more commonly observed associations between the same ANME partner and the Desulfobacteraceae (DSS). FISH analyses revealed aggregates of seepDBB cells in association with ANME-2 from both environmental samples and laboratory incubations that are distinct in their structure relative to co-occurring ANME/DSS consortia. ANME/seepDBB aggregates were most abundant in shallow sediment depths below sulfide-oxidizing microbial mats. Depth profiles of ANME/seepDBB aggregate abundance revealed a positive correlation with elevated porewater nitrate relative to ANME/DSS aggregates in all seep sites examined. This relationship with nitrate was supported by sediment microcosm experiments, in which the abundance of ANME/seepDBB was greater in nitrate-amended incubations relative to the unamended control. FISH-NanoSIMS additionally revealed significantly higher ¹⁵N-nitrate incorporation levels in individual aggregates of ANME/seepDBB relative to ANME/DSS aggregates from the same incubation. These combined results suggest that nitrate is a geochemical effector of ANME/seepDBB aggregate distribution, and provides a unique niche for these consortia through their utilization of a greater range of nitrogen substrates than the ANME/DSS.     

Diversity and Distribution of Methanotrophic Archaea at Cold Seeps

In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to Methanococcoides spp.) and to the Crenarchaeota of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.     

Microbial interactions in the anaerobic oxidation of methane: model simulations constrained by process rates and activity patterns

Proposed syntrophic interactions between the archaeal and bacterial cells mediating anaerobic oxidation of methane coupled with sulfate reduction include electron transfer through (1) the exchange of H₂ or small organic molecules between methane-oxidizing archaea and sulfate-reducing bacteria, (2) the delivery of disulfide from methane-oxidizing archaea to bacteria for disproportionation and (3) direct interspecies electron transfer. Each of these mechanisms was implemented in a reactive transport model. The simulated activities across different arrangements of archaeal and bacterial cells and aggregate sizes were compared to empirical data for AOM rates and intra-aggregate spatial patterns of cell-specific anabolic activity determined by FISH-nanoSIMS. Simulation results showed that rates for chemical diffusion by mechanism (1) were limited by the build-up of metabolites, while mechanisms (2) and (3) yielded cell specific rates and archaeal activity distributions that were consistent with observations from single cell resolved FISH-nanoSIMS analyses. The novel integration of both intra-aggregate and environmental data provided powerful constraints on the model results, but the similarities in model outcomes for mechanisms (2) and (3) highlight the need for additional observational data (e.g. genomic or physiological) on electron transfer and metabolic functioning of these globally important methanotrophic consortia.     

Translocation of amino acids from stem nodules of Sesbania rostrata demonstrated by GC-MS in planta 15N isotope dilution

We report a novel use of the ¹⁵N dilution technique to detail the translocation of amino compounds in the legume Sesbania rostrata . The conventional ¹⁵N dilution technique follows the dilution of ¹⁵N within a labelled plant, as ¹⁴N₂ is fixed by symbiotic bacteria. In our experiments, stem-nodulated Sesbania rostrata were enriched by feeding with ¹⁵N ammonium nitrate for 2 weeks, followed by a 1 week period where the only N available to the plants was via nitrogen fixation of atmospheric N₂. We measured the composition, concentration and ¹⁵N enrichment of amino compounds in various plant tissues, both above and below the stem nodules, using GC-MS and isotopic abundance mass spectrometry techniques. Approximately 28% of the total N in the stem nodules was derived from internal plant sources. The ureides allantoic acid and allantoin were not abundant in xylem, leaf or nodule tissues. The amides asparagine and glutamine were the major export products from stem nodules although a wide range of other amino compounds are also synthesized. Amino acids within the nodules had a low level of enrichment, demonstrating that a small fraction (≈ 11%) was derived from outside the nodules, and significant cycling of N (28% of xylem N) through the root system was revealed by measurements of ¹⁵N distribution and amino acid concentrations.     

Effects of Ketone Bodies on Astrocyte Amino Acid Metabolism

Abstract: The effects of acetoacetate and 3-hydroxybutyrate on glial amino acid metabolism were studied in primary cultures of astrocytes. The exchange of nitrogen among amino acids was measured with ¹⁵N as a metabolic probe and gas chromatography-mass spectrometry as a tool with which to quantify isotope abundance. Addition of either acetoacetate or 3-hydroxybutyrate (5 m M ) to the incubation medium did not alter the initial rate of appearance of [¹⁵N]glutamate in the glia, but it did inhibit transamination of glutamate to [¹⁵N]aspartate. Addition of acetoacetate also inhibited formation of [2-¹⁵N]glutamine, but 3-hydroxybutyrate had a stimulatory effect. The presence in the medium of sodium acetate (5 m M ) was also associated with diminished production of [¹⁵N]aspartate and [2-¹⁵N]glutamine with [¹⁵N]glutamate as precursor. Studies with [2-¹⁵N]glutamine as precursor indicated that treatment of the astrocytes with ketone bodies did not alter flux through the glutaminase pathway. Nor did the presence of the ketone bodies reduce significantly the flux of nitrogen from [¹⁵N]GABA to [2-¹⁵N]glutamine when the former species served as a metabolic tracer. The concentration of internal citrate increased in the presence of acetoacetate, 3-hydroxybutyrate, and acetate. Studies with purified sheep brain glutamine synthetase showed that citrate inhibited this enzyme. These findings are considered in terms of the known anticonvulsant effect of a ketogenic diet.     

Photosynthetic 14CO2 fixation and [15N]-ammonia assimilation during UV-B radiation of Lithodesmium variabile

Uptake of [¹⁵N]-ammonia was more sensitive to UV-B exposure than the total ¹⁴CO₂ fixation rate of Lithodesmium variabile Takano. Short-term UV-B radiation (15 min) had practically no effect on the kinetics of [¹⁵N]-ammonia, whereas there was an effect on [¹⁴C]-bicarbonate uptake rate. A significant reduction was found after 30 and 60 min UV-B stress. The time course of photosynthetic uptake of ¹⁵NH₄Cl at several wavelengths was markedly depressed at shorter wavelengths (irradiation with WG 280). A short-term (11 min) exposure to ultraviolet radiation had no influence on the [¹⁴C]-labeled photosynthetic products. However, the [¹⁵N]-label of several amino acids and the ratio of [¹⁵N]-glutamine to [¹⁵N]-glutamic acid varied after irradiation with different ultraviolet wavebands. The results are discussed with reference to UV damage to the key enzymes of nitrogen metabolism.     

Absence or masking of metabolic fractionations of 13C in a freshwater benthic food web

1. Although marine research has indicated that metabolic fractionations of ¹³C due to differences in organismal trophic position and proximal composition can complicate the isotopic interpretation of energy flow pathways, such potentially confounding problems have never been examined in freshwater benthic food webs.
2. The δ¹³C values of animals comprising a littoral benthic food web composited from four Canadian Shield lakes showed no relationship with either individual trophic position (δ¹⁵N) or lipid content (C/N ratios).
3. Differences in the relative incorporation of autochthonous and allochthonous energy sources by freshwater benthic organisms will alter their δ¹³C and δ¹⁵N values, thereby masking any possibility of observing ¹³C trophic enrichment.
4. Removal of the possibly confounding influences of lipids through either empirical correction or by analytical extraction may be unnecessary in studies of freshwater benthic food webs. Likewise, a priori adjustments in δ¹³C for freshwater benthic organisms in order to accommodate trophic fractionations which are presumed to occur, based on data from marine offshore food webs, may also be inappropriate.     

Absence or masking of metabolic fractionations of 13C in a freshwater benthic food web

1. Although marine research has indicated that metabolic fractionations of ¹³C due to differences in organismal trophic position and proximal composition can complicate the isotopic interpretation of energy flow pathways, such potentially confounding problems have never been examined in freshwater benthic food webs.
2. The δ¹³C values of animals comprising a littoral benthic food web composited from four Canadian Shield lakes showed no relationship with either individual trophic position (δ¹⁵N) or lipid content (C/N ratios).
3. Differences in the relative incorporation of autochthonous and allochthonous energy sources by freshwater benthic organisms will alter their δ¹³C and δ¹⁵N values, thereby masking any possibility of observing ¹³C trophic enrichment.
4. Removal of the possibly confounding influences of lipids through either empirical correction or by analytical extraction may be unnecessary in studies of freshwater benthic food webs. Likewise, a priori adjustments in δ¹³C for freshwater benthic organisms in order to accommodate trophic fractionations which are presumed to occur, based on data from marine offshore food webs, may also be inappropriate.     

Natural 15N enrichment of amide-exporting legume nodules

The natural ¹⁵N abundance of amide-exporting nodules was compared to that of shoots in 12 plant species. Nodules were statistically less abundant in ¹⁵N than shoots in one of three cultivars of Pisum sativum L., in Vicia faba L. and in Medicago sativa L., but the ¹⁵N depletion of nodules was very samall. Nodules were statistically more abundant in ¹⁵N than shoots in Trifolium pratense L., depending on time during the growing season, Cyamopsis tetragonaloba L. Taub. and 7 Lupinus species, but the enrichment was small except for C. tetragonalova and 6 Lupinus species. Nodules of 3 Lupinus species infected with Rhizobium lupini isolated from Lupinus subcarnosa Hook, were only slightly enriched in ¹⁵N, but nodules of two of these species were substantially enriched in ¹⁵N when infected with a mix of other Rhizobium lupini strains. The third species, L. texensis Hook., was not infected by this mix of strains. Differences in ¹⁵N abundance between nodules and other tissues of amide-exporting and ureide-exporting nodules from several studies are tabulated. All ureide-exporting nodules in this tabulation are enriched in ¹⁵N. Amide-exporting nodules are considerably more variable in this regard. These results confirm that events associated with ureide synthesis or transport cannot be the sole cause of the substantial ¹⁵N enrichment seen in nodules.     

Extensive carbon isotopic heterogeneity among methane seep microbiota

To assess and study the heterogeneity of δ¹³C values for seep microorganisms of the Eel River Basin, we studied two principally different sample sets: sediments from push cores and artificial surfaces colonized over a 14 month in situ incubation. In a single sediment core, the δ¹³C compositions of methane seep-associated microorganisms were measured and the relative activity of several metabolisms was determined using radiotracers. We observed a large range of archaeal δ¹³C values (> 50‰) in this microbial community. The δ¹³C of ANME-1 rods ranged from −24‰ to −87‰. The δ¹³C of ANME-2 sarcina ranged from −18‰ to −75‰. Initial measurements of shell aggregates were as heavy as −19.5‰ with none observed to be lighter than −57‰. Subsequent measurements on shell aggregates trended lighter reaching values as ¹³C-depleted as −73‰. The observed isotopic trends found for mixed aggregates were similar to those found for shell aggregates in that the initial measurements were often enriched and the subsequent analyses were more ¹³C-depleted (with values as light as −56‰). The isotopic heterogeneity and trends observed within taxonomic groups suggest that ANME-1 and ANME-2 sarcina are capable of both methanogenesis and methanotrophy. In situ microbial growth was investigated by incubating a series of slides and silicon (Si) wafers for 14 months in seep sediment. The experiment showed ubiquitous growth of bacterial filaments (mean δ¹³C = −38 ± 3‰), suggesting that this bacterial morphotype was capable of rapid colonization and growth.     

Improvement of the 15N dilution method for estimation of absorption of NOx by plants supplied with 15N-labelled fertilizer

To develop further the methods for estimation of NOx absorption by plants supplied with ¹⁵N-labelled fertilizer, we proposed a new calculation method, total N fixed method (TNF), and compared with the ¹⁵N dilution method and the classical mass balance method (MB).
Hydroponically grown soybean plants were supplied with ¹⁵N-labelled nitrate and exposed to 200–250 nl l⁻¹ NO₂ for 7 d. The proportions of the N derived from NO₂ to total N in exposed plants were estimated by the three methods.
The reported rates of NO₂ absorption by several plant species, estimated by the ¹⁵N dilution method, were recalculated using the TNF method. The results of the two methods were compared and showed that: (1) The ¹⁵N dilution method overestimated the content of NO₂-N in exposed plants compared with the MB method whilst the TNF method produced estimations of NO₂-N closer to those by the MB method when the plants were supplied with 5 m M nitrate. (2) The differences in estimations between the MB method and either the ¹⁵N dilution method or the TNF method increased with decreasing supply of ¹⁵N-labelled nitrate to roots.     

Identification of the dominant sulfate-reducing bacterial partner of anaerobic methanotrophs of the ANME-2 clade

The anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor is mediated by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Whereas three clades of ANME have been repeatedly studied with respect to phylogeny, key genes and genomic capabilities, little is known about their sulfate-reducing partner. In order to identify the partner of anaerobic methanotrophs of the ANME-2 clade, bacterial 16S rRNA gene libraries were constructed from cultures highly enriched for ANME-2a and ANME-2c in consortia with Deltaproteobacteria of the Desulfosarcina/Desulfococcus group (DSS). Phylogenetic analysis of those and publicly available sequences from AOM sites supported the hypothesis by Knittel and colleagues that the DSS partner belongs to the diverse SEEP-SRB1 cluster. Six subclusters of SEEP-SRB1, SEEP-SRB1a to SEEP-SRB1f, were proposed and specific oligonucleotide probes were designed. Using fluorescence in situ hybridization on samples from six different AOM sites, SEEP-SRB1a was identified as sulfate-reducing partner in up to 95% of total ANME-2 consortia. SEEP-SRB1a cells exhibited a rod-shaped, vibrioid, or coccoid morphology and were found to be associated with subgroups ANME-2a and ANME-2c. Moreover, SEEP-SRB1a was also detected in 8% to 23% of ANME-3 consortia in Haakon Mosby Mud Volcano sediments, previously described to be predominantly associated with SRB of the Desulfobulbus group. SEEP-SRB1a contributed to only 0.3% to 0.7% of all single cells in almost all samples indicating that these bacteria are highly adapted to a symbiotic relationship with ANME-2.     

15N natural abundance during early and late succession in a middle-European dry acidic grassland

δ¹⁵N and total nitrogen content of above- and belowground tissues of 13 plant species from two successional stages (open pioneer community and ruderal grass stage) of a dry acidic grassland in Southern Germany were analysed, in order to evaluate whether resource use partitioning by niche separation and N input by N₂-fixing legumes are potential determinants for species coexistence and successional changes. Within each stage, plants from plots with different legume cover were compared. Soil inorganic N content, total plant biomass and δ¹⁵N values of bulk plant material were significantly lower in the pioneer stage than in the ruderal grass community. The observed δ¹⁵N differences were rather species- than site-specific. Within both stages, there were also species-specific differences in isotopic composition between above- and belowground plant dry matter. Species-specific δ¹⁵N signatures may theoretically be explained by (i) isotopic fractionation during microbial-mediated soil N transformations; (ii) isotopic fractionation during plant N uptake or fractionation during plant–mycorrhiza transfer processes; (iii) differences in metabolic pathways and isotopic fractionation within the plant; or (iv) partitioning of available N resources (or pools) among plant groups or differential use of the same resources by different species, which seems to be the most probable route in the present case. A significant influence of N₂-fixing legumes on the N balance of the surrounding plant community was not detectable. This was confirmed by the results of an independent in situ removal experiment, showing that after 3 years there were no measurable differences in the frequency distribution between plots with and without N₂-fixing legumes.     

In vitro cell growth of marine archaeal-bacterial consortia during anaerobic oxidation of methane with sulfate

Anoxic sediment from a methane hydrate area (Hydrate Ridge, north-east Pacific; water depth 780 m) was incubated in a long-term laboratory experiment with semi-continuous supply of pressurized [1.4 MPa (14 atm)] methane and sulfate to attempt in vitro propagation of the indigenous consortia of archaea (ANME-2) and bacteria (DSS, Desulfosarcina/Desulfococcus cluster) to which anaerobic oxidation of methane (AOM) with sulfate has been attributed. During 24 months of incubation, the rate of AOM (measured as methane-dependent sulfide formation) increased from 20 to 230 micromol day(-1) (g sediment dry weight)(-1) and the number of aggregates (determined by microscopic counts) from 0.5 x 10(8) to 5.7 x 10(8) (g sediment dry weight)(-1). Fluorescence in situ hybridization targeting 16S rRNA of both partners showed that the newly grown consortia contained central archaeal clusters and peripheral bacterial layers, both with the same morphology and phylogenetic affiliation as in the original sediment. The development of the AOM rate and the total consortia biovolume over time indicated that the consortia grew with a doubling time of approximately 7 months (growth rate 0.003 day(-1)) under the given conditions. The molar growth yield of AOM was approximately 0.6 g cell dry weight (mol CH(4) oxidized)(-1); according to this, only 1% of the consumed methane is channelled into synthesis of consortia biomass. Concentrations of biomarker lipids previously attributed to ANME-2 archaea (e.g. sn-2-hydroxyarchaeol, archaeol, crocetane, pentamethylicosatriene) and Desulfosarcina-like bacteria [e.g. hexadecenoic-11 acid (16:1omega5c), 11,12-methylene-hexadecanoic acid (cy17:0omega5,6)] strongly increased over time (some of them over-proportionally to consortia biovolume), suggesting that they are useful biomarkers to detect active anaerobic methanotrophic consortia in sediments.     

Fractionation of δ15N and δ13C for Atlantic salmon and its intestinal cestode Eubothrium crassum

Stable isotopes of nitrogen (δ¹⁵N) and carbon (δ¹³C) were measured for Atlantic salmon Salmo salar and their intestinal cestode, Eubothrium crassum , sharing the same diet. Atlantic salmon muscle tissues were enriched in ¹⁵N and depleted in ¹³C compared to their prey (sprat Sprattus sprattus sprattus ) and their intestinal cestode. There was no significant difference in δ¹⁵N or δ¹³C between E. crassum and the sprat. Differences in nutrient uptake and intestine physiology between Atlantic salmon and E. crassum are discussed, as well as how these may give rise to different fractionations of stable isotopes between a host and its parasites. Furthermore, Atlantic salmon contained a significantly higher lipid content than their prey, which may partly explain differences in δ¹³C values between the host and its cestode. In addition, cestodes inhabiting lipid-rich hosts were also lipid rich. Larger Atlantic salmon were enriched in ¹⁵N compared to smaller fish. Cestodes inhabiting large hosts were also enriched in ¹⁵N compared to parasites living in smaller hosts. The last two results were explained by larger fish possibly feeding from a higher trophic level, or from larger and older prey, that resulted in both a higher lipid content and an enrichment in ¹⁵N.     

Natural 13C and 15N abundance of field-collected fungi and their ecological implications

The natural abundance of ¹³C and ¹⁵N was measured in basidiocarps of at least 115 species in 88 genera of ectomycorrhizal, wood-decomposing and litter-decomposing fungi from Japan and Malaysia. The natural abundance of ¹³C and ¹⁵N was also measured in leaves, litter, soil and wood from three different sites. ¹⁵N and ¹³C were enriched in ectomycorrhizal and wood-decomposing fungi, respectively, relative to their substrates. Ectomycorrhizal and wood-decomposing fungi could be distinguished on the basis of their δ¹³C and δ¹⁵N signatures. Although there was high variability in the isotopic composition of fungi, the following isotope- enrichment factors (ε, mean±SD) of the fungi relative to substrates were observed:
ε_{ectomycorrhizal fungi/litter} = 6.1±0.4‰¹⁵N
ε_{ectomycorrhizal fungi/wood} = 1.4±0.8‰¹³C
ε_{wood-decomposing fungi/wood} = −0.6±0.7‰¹⁵N
ε_{wood-decomposing fungi/wood} = 3.5±0.9‰¹³C
The basis of isotope fractionation in C metabolism from wood to wood-decomposing fungus is discussed.