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Pulses of labile carbon cause transient decoupling of fermentation and respiration in permeable sediments
Limnology and Oceanography
Abstract
Dihydrogen (H 2 ) is an important intermediate in anaerobic microbial processes, and concentrations are tightly controlled by thermodynamic limits of consumption and production. However, recent studies reported unusual H 2 accumulation in permeable marine sediments under anoxic conditions, suggesting decoupling of fermentation and sulfate reduction, the dominant respiratory process in anoxic permeable marine sediments. Yet, the extent, prevalence and potential triggers for such H 2 accumulation and decoupling remain unknown. We surveyed H 2 concentrations in situ at different settings of permeable sand and found that H 2 accumulation was only observed during a coral spawning event on the Great Barrier Reef. A flume experiment with organic matter addition to the water column showed a rapid accumulation of hydrogen within the sediment. Laboratory experiments were used to explore the effect of oxygen exposure, physical disturbance and organic matter inputs on H 2 accumulation. Oxygen exposure had little effect on H 2 accumulation in permeable sediments suggesting both fermenters and sulfate reducers survive and rapidly resume activity after exposure to oxygen. Mild physical disturbance mimicking sediment resuspension had little effect on H 2 accumulation; however, vigorous shaking led to a transient accumulation of H 2 and release of dissolved organic carbon suggesting mechanical disturbance and cell destruction led to organic matter release and transient decoupling of fermenters and sulfate reducers. In summary, the highly dynamic nature of permeable sediments and its microbial community allows for rapid but transient decoupling of fermentation and respiration after a C pulse, leading to high H 2 levels in the sediment.
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The microbial community composition and biogeochemical dynamics of coastal permeable (sand) sediments differs from cohesive (mud) sediments. Tide-and wave-driven hydrodynamic disturbance causes spatiotemporal variations in oxygen levels, which select for microbial generalists and disrupt redox cascades. In this work, we profiled microbial communities and biogeochemical dynamics in sediment profiles from three sites varying in their exposure to hydrodynamic disturbance. Strong variations in sediment geochemistry, biogeochemical activities, and microbial abundance, composition, and capabilities were observed between the sites. Most of these variations, except for microbial abundance and diversity, significantly correlated with the relative disturbance level of each sample. In line with previous findings, metabolically flexible habitat generalists (e.g., Flavobacteriaceae, Woeseaiceae, Rhodobacteraceae) dominated in all samples. However, we present evidence that aerobic specialists such as ammonia-oxidizing archaea (Nitrosopumilaceae) were more abundant and active in more disturbed samples, whereas bacteria capable of sulfate reduction (e.g., uncultured Desulfobacterales), dissimilatory nitrate reduction to ammonium (DNRA; e.g., Ignavibacteriaceae), and sulfide-dependent chemolithoautotrophy (e.g., Sulfurovaceae) were enriched and active in less disturbed samples. These findings are supported by insights from nine deeply sequenced metagenomes and 169 derived metagenome-assembled genomes. Altogether, these findings suggest that hydrodynamic disturbance is a critical factor controlling microbial community assembly and biogeochemical processes in coastal sediments. Moreover, they strengthen our understanding of the relationships between microbial composition and biogeochemical processes in these unique environments.
Atmospheric trace gases such as dihydrogen (H2), carbon monoxide (CO) and methane (CH4) play important roles in microbial metabolism and biogeochemical cycles. Analysis of these gases at trace levels requires reliable storage of discrete samples of low volume. While commercial sampling vials such as Exetainers® have been tested for CH4 and other greenhouse gases, no information on reliable storage is available for H2 and CO. We show that vials sealed with butyl rubber stoppers are not suitable for storing H2 and CO due to release of these gases from rubber material. Treating butyl septa with NaOH reduced trace-gas release, but contamination was still substantial, with H2 and CO mixing ratios in air samples increasing by a factor of 3 and 10 after 30 d of storage in conventional 12 mL Exetainers. All tested materials showed a near-linear increase in H2 and CO mixing ratios, indicating a zero-order reaction and material degradation as the underlying cause. Among the rubber materials tested, silicone showed the lowest potential for H2 and CO release. We thus propose modifying Exetainers by closing them with a silicone plug to minimise contamination and sealing them with a stainless-steel bolt and O-ring as a secondary diffusion barrier for long-term storage. Such modified Exetainers exhibited stable mixing ratios of H2 and CH4 exceeding 60 d of storage at atmospheric and elevated (10 ppm) mixing ratios. The increase of CO was still measurable but was 9 times lower than in conventional Exetainers with treated septa; this can be corrected for due to its linearity by storing a standard gas alongside the samples. The proposed modification is inexpensive, scalable and robust, and thus it enables reliable storage of large numbers of low-volume gas samples from remote field locations.
Atmospheric trace gases such as dihydrogen (H2), carbon monoxide (CO) and methane (CH4) play important roles in microbial metabolism and biogeochemical cycles. Analysis of these gases at trace levels requires reliable storage of discrete samples of low volume. While commercial sampling vials such as Exetainers® have been tested for CH4 and other greenhouse gases, no information on reliable storage is available for H2 and CO. We show that vials sealed with butyl rubber stoppers are not suitable for storing H2 and CO due to release of these gases from rubber material. Treating butyl septa with NaOH reduced trace gas release, but contamination was still substantial, with H2 and CO concentrations in air samples increasing by a factor of 3 and 10 after 30 days of storage in conventional 12 mL Exetainers. Among the rubber materials tested, silicone showed the lowest potential for H2 and CO release. We thus propose to modify Exetainers by closing them with a silicone plug, and sealing them with a stainless steel bolt and O-ring for long-term storage. Such modified Exetainers exhibited stable concentrations of H2 and CH4 exceeding 60 days of storage at atmospheric and elevated (10 ppm) concentrations. The increase of CO was still measurable, but nine times lower than in conventional Exetainers with treated septa, and can be corrected for due to its linearity by storing a standard gas alongside the samples. The proposed modification is inexpensive, scalable and robust, and thus enables reliable storage of large numbers of low-volume gas samples from remote field locations.
We investigated the impact of kelp deposition on the geochemistry and microbial community composition of beach sands on the island of Helgoland (North Sea). The composition of the microbial community at a beach with regular kelp deposition appeared shaped by this regular input of organic material, as indicated by significantly higher proportions of aerobic degraders, fermenters, and sulfur cycling microorganisms. Rapid degradation of deposited kelp by this community leads to high levels of dissolved organic and inorganic carbon and nutrients, a lower pH and anoxia. Aerobic respiration, fermentation, Fe‐ and SO42− reduction, and methanogenesis were strongly enhanced, with SO42− reduction being the main process in kelp degradation. SO42− reduction rates increased 20‐ to 25‐fold upon addition of kelp. The main route of electrons from kelp to SO42− was not via CO and H2, as expected, but via organic fermentation products. O2 supply by the tides was not sufficient and reduced intermediates escaped from the sediment with tidal water retraction. The resulting extremely high levels of free sulfide (>10 mmol L−1) lead to abundant filamentous growth of sulfur‐oxidizing bacteria largely composed of a rare O2‐adapted Sulfurovum lacking the expected denitrification genes. Our results show that regular kelp deposition strongly enhances the thermodynamic disequilibrium in the beach sand habitat, leading to a dramatic enhancement of the sulfur cycle.
Most aerobic bacteria exist in dormant states within natural environments. In these states, they endure adverse environmental conditions such as nutrient starvation by decreasing metabolic expenditure and using alternative energy sources. In this study, we investigated the energy sources that support persistence of two aerobic thermophilic strains of the environmentally widespread but understudied phylum Chloroflexi. A transcriptome study revealed that Thermomicrobium roseum (class Chloroflexia) extensively remodels its respiratory chain upon entry into stationary phase due to nutrient limitation. Whereas primary dehydrogenases associated with heterotrophic respiration were downregulated, putative operons encoding enzymes involved in molecular hydrogen (H 2), carbon monoxide (CO), and sulfur compound oxidation were significantly upregulated. Gas chromatography and microsensor experiments showed that T. roseum aerobically respires H 2 and CO at a range of environmentally relevant concentrations to sub-atmospheric levels. Phylogenetic analysis suggests that the hydrogenases and carbon monoxide dehydrogenases mediating these processes are widely distributed in Chloroflexi genomes and have probably been horizontally acquired on more than one occasion. Consistently, we confirmed that the sporulating isolate Thermogemmatispora sp. T81 (class Ktedonobacteria) also oxidises atmospheric H 2 and CO during persistence, though further studies are required to determine if these findings extend to mesophilic strains. This study provides axenic culture evidence that atmospheric CO supports bacterial persistence and reports the third phylum, following Actinobacteria and Acidobacteria, to be experimentally shown to mediate the biogeochemically and ecologically important process of atmospheric H 2 oxidation. This adds to the growing body of evidence that atmospheric trace gases are dependable energy sources for bacterial persistence.
Permeable (sandy) sediments cover half of the continental margin and are major regulators of oceanic carbon cycling. The microbial communities within these highly dynamic sediments frequently shift between oxic and anoxic states, and hence are less stratified than those in cohesive (muddy) sediments. A major question is, therefore, how these communities maintain metabolism during oxic–anoxic transitions. Here, we show that molecular hydrogen (H2) accumulates in silicate sand sediments due to decoupling of bacterial fermentation and respiration processes following anoxia. In situ measurements show that H2 is 250-fold supersaturated in the water column overlying these sediments and has an isotopic composition consistent with fermentative production. Genome-resolved shotgun metagenomic profiling suggests that the sands harbour diverse and specialized microbial communities with a high abundance of [NiFe]-hydrogenase genes. Hydrogenase profiles predict that H2 is primarily produced by facultatively fermentative bacteria, including the dominant gammaproteobacterial family Woeseiaceae, and can be consumed by aerobic respiratory bacteria. Flow-through reactor and slurry experiments consistently demonstrate that H2 is rapidly produced by fermentation following anoxia, immediately consumed by aerobic respiration following reaeration and consumed by sulfate reduction only during prolonged anoxia. Hydrogenotrophic sulfur, nitrate and nitrite reducers were also detected, although contrary to previous hypotheses there was limited capacity for microalgal fermentation. In combination, these experiments confirm that fermentation dominates anoxic carbon mineralization in these permeable sediments and, in contrast to the case in cohesive sediments, is largely uncoupled from anaerobic respiration. Frequent changes in oxygen availability in these sediments may have selected for metabolically flexible bacteria while excluding strict anaerobes.
Molecular hydrogen (H2) is the second most abundant reduced trace gas (after methane) in the atmosphere, but its biogeochemical cycle is not well understood. Our study focuses on the soil production and uptake of H2 and the associated isotope effects. Air samples from a grass field and a forest site in the Netherlands were collected using soil chambers. The results show that uptake and emission of H2 occurred simultaneously at all sampling sites, with strongest emission at the grassland sites where clover (N2 fixing legume) was present. The H2 mole fraction and deuterium content were measured in the laboratory to determine the isotopic fractionation factor during H2 soil uptake (αsoil) and the isotopic signature of H2 that is simultaneously emitted from the soil (δDsoil). By considering all net-uptake experiments, an overall fractionation factor for deposition of αsoil = kHD/kHH = 0.945 ± 0.004 (95 % CI) was obtained. The difference in mean αsoil between the forest soil 0.937 ± 0.008 and the grassland 0.951 ± 0.025 is not statistically significant. For two experiments, the removal of soil cover increased the deposition velocity (vd) and αsoil simultaneously, but a general positive correlation between vd and αsoil was not found in this study. When the data are evaluated with a model of simultaneous production and uptake, the isotopic composition of H2 that is emitted at the grassland site is calculated as δDsoil = (-530 ± 40) ‰. This is less deuterium-depleted than what is expected from isotope equilibrium between H2O and H2.
For the anaerobic remineralization of organic matter in marine sediments, sulfate reduction coupled to fermentation plays a key role. Here, we enriched sulfate-reducing/fermentative communities from intertidal sediments under defined conditions in continuous culture. We transiently exposed the cultures to oxygen or nitrate twice daily and investigated the community response. Chemical measurements, provisional genomes and transcriptomic profiles revealed trophic networks of microbial populations. Sulfate reducers coexisted with facultative nitrate reducers or aerobes enabling the community to adjust to nitrate or oxygen pulses. Exposure to oxygen and nitrate impacted the community structure, but did not suppress fermentation or sulfate reduction as community functions, highlighting their stability under dynamic conditions. The most abundant sulfate reducer in all cultures, related to Desulfotignum balticum, appeared to have coupled both acetate- and hydrogen oxidation to sulfate reduction. We describe a novel representative of the widespread uncultured candidate phylum Fermentibacteria (formerly candidate division Hyd24-12). For this strictly anaerobic, obligate fermentative bacterium, we propose the name “USabulitectum silens” and identify it as a partner of sulfate reducers in marine sediments. Overall, we provide insights into the function of fermentative, as well as sulfate-reducing microbial communities and their adaptation to a dynamic environment. This article is protected by copyright. All rights reserved.
Biologically produced molecular hydrogen (H2) is characterized by a very strong depletion in deuterium. Although the biological source to the atmosphere is small compared to photochemical or combustion sources, it makes an important contribution to the global isotope budget of molecular hydrogen (H2). Large uncertainties exist in the quantification of the individual production and degradation processes that contribute to the atmospheric budget, and isotope measurements are a tool to distinguish the contributions from the different sources. Measurements of δD from the various H2 sources are scarce and for biologically produced H2 only very few measurements exist. Here the first systematic study of the isotopic composition of biologically produced H2 is presented. We investigated δD of H2 produced in a biogas plant, covering different treatments of biogas production, and from several H2 producing microorganisms such as bacteria or green algae. A Keeling plot analysis provides a robust overall source signature of δD = –712‰ (±13‰) for the samples from the biogas reactor (at 38 °C, δDH2O = 73.4‰), with a fractionation constant ϵH2−H2O of –689‰ (±20‰). The pure culture samples from different microorganisms give a mean source signature of δD = –728‰ (±39‰), and a fractionation constant ϵH2−H2O of –711‰ (±45‰) between H2 and the water, respectively. The results confirm the massive deuterium depletion of biologically produced H2 as was predicted by calculation of the thermodynamic fractionation factors for hydrogen exchange between H2 and water vapor. As expected for a thermodynamic equilibrium, the fractionation factor is largely independent of the substrates used and the H2 production conditions. The predicted equilibrium fractionation coefficient is positively correlated with temperature and we measured a change of 2.2‰/°C between 45 °C and 60 °C. This is in general agreement with the theoretical predictions. Our best estimate for ϵH2−H2O at a temperature of 20 °C is –728‰ for biologically produced H2, and we suggest using this value in future global H2 isotope budget calculations and models.
Oceans are a net source of molecular hydrogen (N2) to the atmosphere, where nitrogen (N2) fixation is assumed to be the main biological production pathway besides photochemical production from organic material. The sources can be distinguished using isotope measurements because of clearly differing isotopic signatures of the produced hydrogen.
Here we present the first ship-borne measurements of atmospheric molecular H2 mixing ratio and isotopic composition at the West African coast of Mauritania (16–25° W, 17–24° N). This area is one of the biologically most active regions of the world's oceans with seasonal upwelling events and characterized by strongly differing hydrographical/biological properties and phytoplankton community structures. The aim of this study was to identify areas of H2 production and distinguish H2 sources by isotopic signatures of atmospheric H2. Besides this a diurnal cycle of atmospheric H2 was investigated. For this more than 100 air samples were taken during two cruises in February 2007 and 2008, respectively. During both cruises a transect from the Cape Verde Island towards the Mauritanian Coast was sampled. In 2007 additionally four days were sampled with a high resolution of one sample per hour.
Our results clearly indicate the influence of local sources and suggest the Banc d'Arguin as a pool for precursors for photochemical H2 production, whereas N2 fixation could not be identified as a H2 source during these two cruises. With our experimental setup we could demonstrate that variability in diurnal cycles is probably influenced and biased by released precursors for photochemical H2 production and the origin of air masses. This means for further investigations that just measuring the mixing ratio of H2 is insufficient to explain the variability of a diurnal cycle and support is needed, e.g. by isotopic measurements. However, measurements of H2 mixing ratios, which are easy to conduct online during ship cruises could be a helpful tool to easily identify production areas of biological precursors such as VOC's for further investigations.
Interest in atmospheric hydrogen (H2) has been growing in recent years with the prospect of H2 being a potential alternative to fossil fuels as an energy carrier. This has intensified research for a quantitative understanding of the atmospheric hydrogen cycle and its total budget, including the expansion of the global atmospheric measurement network. However, inconsistencies in published observational data constitute a major limitation in exploring such data sets. The discrepancies can be mainly attributed to difficulties in the calibration of the measurements. In this study various factors that may interfere with accurate quantification of atmospheric H2 were investigated including drifts of standard gases in high pressure cylinders. As an experimental basis a procedure to generate precise mixtures of H2 within the atmospheric concentration range was established. Application of this method has enabled a thorough linearity characterization of the commonly used GC-HgO reduction detector. We discovered that the detector response was sensitive to the composition of the matrix gas. Addressing these systematic errors, an accurate calibration scale has been generated defined by thirteen standards with dry air mole fractions ranging from 139–1226 nmol mol−1. The new scale has been accepted as the official World Meteorological Organisation's (WMO) Global Atmospheric Watch (GAW) H2 mole fraction scale.
Link to full text:
http://www.atmos-chem-phys.net/15/13003/2015/acp-15-13003-2015.html
Molecular hydrogen (H2) is the second most abundant reduced trace gas (after methane) in the atmosphere, but its biogeochemical cycle is not well understood. Our study focuses on the soil production and uptake of H2 and the associated isotope effects. Air samples from a grass field and a forest site in the Netherlands were collected using soil chambers. The results show that uptake and emission of H2 occurred simultaneously at all sampling sites, with strongest emission at the grassland sites where clover (N2 fixing legume) was present. The H2 mole fraction and deuterium content were measured in the laboratory to determine the isotopic fractionation factor during H2 soil uptake (αsoil) and the isotopic signature of H2 that is simultaneously emitted from the soil (δDsoil). By considering all net-uptake experiments, an overall fractionation factor for deposition of αsoil = kHD/kHH = 0.945 ± 0.004 (95 % CI) was obtained. The difference in mean αsoil between the forest soil 0.937 ± 0.008 and the grassland 0.951 ± 0.025 is not statistically significant. For two experiments, the removal of soil cover increased the deposition velocity (vd) and αsoil simultaneously, but a general positive correlation between vd and αsoil was not found in this study. When the data are evaluated with a model of simultaneous production and uptake, the isotopic composition of H2 that is emitted at the grassland site is calculated as δDsoil = (−530 ± 40) ‰. This is less deuterium-depleted than what is expected from isotope equilibrium between H2O and H2.
Many atmospheric chemicals occur in the gas
phase as well as in liquid cloud droplets and aerosol particles.
Therefore, it is necessary to understand the distribution
between the phases. According to Henry's law, the equilibrium
ratio between the abundances in the gas phase and in
the aqueous phase is constant for a dilute solution. Henry's
law constants of trace gases of potential importance in environmental
chemistry have been collected and converted into
a uniform format. The compilation contains 17 350 values of
Henry's law constants for 4632 species, collected from 689
references. It is also available at http://www.henrys-law.org.
The gutless marine worm Olavius algarvensis lives in symbiosis with chemosynthetic bacteria that provide nutrition by fixing CO2 into biomass using reduced sulfur compounds as energy sources. A recent metaproteomic analysis of the O. algarvensis symbiosis indicated that carbon monoxide (CO) and hydrogen (H2 ) might also be used as energy sources. We provide direct evidence that the O. algarvensis symbiosis consumes CO and H2 . Single cell imaging using nanoSIMS revealed that one of the symbionts, the γ3-symbiont, uses the energy from CO oxidation to fix CO2 . Pore water analysis revealed considerable in-situ-concentrations of CO and H2 in the O. algarvensis environment, Mediterranean seagrass sediments. Pore water H2 concentrations (89 - 2147 nM) were up to two orders of magnitude higher than in seawater, and up to 36-fold higher than previously known from shallow-water marine sediments. Pore water CO concentrations (17 - 51 nM) were twice as high as in the overlying seawater (no literature data from other shallow-water sediments are available for comparison). Ex-situ incubation experiments showed that dead seagrass rhizomes produced large amounts of CO. CO production from decaying plant material could thus be a significant energy source for microbial primary production in seagrass sediments.
This article is protected by copyright. All rights reserved.
Oceans are a net source of molecular hydrogen (N2) to the
atmosphere, where nitrogen (N2) fixation is assumed to be the
main biological production pathway besides photochemical production from
organic material. The sources can be distinguished using isotope
measurements because of clearly differing isotopic signatures of the
produced hydrogen. Here we present the first ship-borne
measurements of atmospheric molecular H2 mixing ratio and
isotopic composition at the West African coast of Mauritania (16-25°
W, 17-24° N). This area is one of the biologically most active
regions of the world's oceans with seasonal upwelling events and
characterized by strongly differing hydrographical/biological properties
and phytoplankton community structures. The aim of this study was to
identify areas of H2 production and distinguish H2
sources by isotopic signatures of atmospheric H2. Besides
this a diurnal cycle of atmospheric H2 was investigated. For
this more than 100 air samples were taken during two cruises in February
2007 and 2008, respectively. During both cruises a transect from the
Cape Verde Island towards the Mauritanian Coast was sampled. In 2007
additionally four days were sampled with a high resolution of one sample
per hour. Our results clearly indicate the influence of
local sources and suggest the Banc d'Arguin as a pool for precursors for
photochemical H2 production, whereas N2 fixation
could not be identified as a H2 source during these two
cruises. With our experimental setup we could demonstrate that
variability in diurnal cycles is probably influenced and biased by
released precursors for photochemical H2 production and the
origin of air masses. This means for further investigations that just
measuring the mixing ratio of H2 is insufficient to explain
the variability of a diurnal cycle and support is needed, e.g. by
isotopic measurements. However, measurements of H2 mixing
ratios, which are easy to conduct online during ship cruises could be a
helpful tool to easily identify production areas of biological
precursors such as VOC's for further investigations.
In this study we show for the first time the microscale (mm) 2- and 3-dimensional spatial distribution and abundance of prokaryotes, viruses, and oxygen in a tidal sediment. Prokaryotes and viruses were highly heterogeneously distributed with patches of elevated abundances surrounded by areas of ca. 3-fold lower abundance within distances of <2 mm. Abundances of prokaryotes and viruses ranged from 1.3 x 10(9) to 4.2 x 10(9) cells cm(-3) and 4.1 x 10(9) to 13.1 x 10(9) viruses cm-3, respectively. The results showed oxygen concentration and uptake rates to be heterogeneously distributed at the same spatial scale, with the oxygen penetration depth varying from 1.5 to 5.8 mm and with an average (+/- SD) diffusive oxygen uptake of 18.9 +/- 6.4 mmol m(-2) d(-1). Locally, prokaryotes, viruses, and oxygen were found to be positively, negatively, or not correlated, but overall no significant relationship was detected. The lack of consistent spatial correlation between viruses and prokaryotes was explained by a temporal experiment using organic carbon-enriched homogenized sediment samples. Enhancement in metabolic activity and the proliferation of prokaryotes and viruses were not completely phased. These results suggest that local nourishment is likely to be an important driver of a high small-scale heterogeneity in abundance and dynamics of benthic viruses and prokaryotes. This is expected to influence the rates and regulation of benthic virus-host inter actions and thus microbial biogeochemical cycling.
Permeable sediments comprise the majority of shelf sediments, yet the rates of denitrification remain highly uncertain in these environments. Computational models are increasingly being used to understand the dynamics of denitrification in permeable sediments, which are complex environments to study experimentally. The realistic implementation of such models requires reliable experimentally derived data on the kinetics of denitrification. Here we undertook measurements of denitrification kinetics as a function of nitrate concentration in carefully controlled flow through reactor experiments on sediments taken from six shallow coastal sites in Port Phillip Bay, Victoria, Australia. The results showed that denitrification commenced rapidly (within 30 min) after the onset of anoxia and the kinetics could be well described by Michaelis–Menten kinetics with half saturation constants (apparent Km) ranging between 1.5 and 19.8 μM, and maximum denitrification rate (Vmax) were in the range of 0.9–7.5 nmol mL−1 h−1. The production of N2 through anaerobic ammonium oxidation (anammox) was generally found to be less than 10 % of denitrification. Vmax were in the same range as previously reported in cohesive sediments despite organic carbon contents one order of magnitude lower for the sediments studied here. The ratio of sediment O2 consumption to Vmax was in the range of 0.02–0.09, and was on average much lower than the theoretical ratio of 0.8. As a consequence, models implemented with the theoretical ratio of 0.8 are likely to overestimate denitrification by a factor of ~3. The most likely explanation for this is that the microbial community is not able to instantaneously shift or optimally use a particular electron acceptor in the highly dynamic redox environment experienced in permeable sediments. In contrast to previous studies, we did not observe any significant rates of oxic denitrification.
The sandy sediments that blanket the inner shelf are situated in a zone where nutrient input from land and strong mixing produce maximum primary production and tight coupling between water column and sedimentary processes. The high permeability of the shelf sands renders them susceptible to pressure gradients generated by hydrodynamic and biological forces that modulate spatial and temporal patterns of water circulation through these sediments. The resulting dynamic three-dimensional patterns of particle and solute distribution generate a broad spectrum of biogeochemical reaction zones that facilitate effective decomposition of the pelagic and benthic primary production products. The intricate coupling between the water column and sediment makes it challenging to quantify the production and decomposition processes and the resultant fluxes in permeable shelf sands. Recent technical developments have led to insights into the high biogeochemical and biological activity of these permeable sediments and their role in the global cycles of matter. Expected final online publication date for the Annual Review of Marine Science Volume 6 is January 03, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Biologically produced molecular hydrogen (H2) is characterized by a very strong depletion
in deuterium. Although the biological source to the atmosphere is small compared
to photochemical or combustion sources, it makes an important contribution to
the global isotope budget of molecular hydrogen (H2). Large uncertainties exist in the
quantification of the individual production and degradation processes that contribute to
the atmospheric budget, and isotope measurements are a tool to distinguish the contributions
from the different sources. Measurements of δD from the various H2 sources
are scarce and for biologically produced H2 only very few measurements exist. Here
the first systematic study of the isotopic composition of biologically produced H2 is
presented.
We investigated δD of H2 produced in a biogas plant, covering different treatments
of biogas production, and from several H2 producing microorganisms such as bacteria
or green algae. A Keeling plot analysis provides a robust overall source sig
nature of δD = –712‰ (±13‰) for the samples from the biogas reactor (at 38 °C,
δDH2O = 73.4 ‰), with a fractionation constant εH2−H2O of –689‰ (±20 ‰). The pure
culture samples from different microorganisms give a mean source signature of δD =
–728‰ (± 39 ‰), and a fractionation constant εH2−H2O of –711‰ (± 45 ‰) between
H2 and the water, respectively. The results confirm the massive deuterium depletion
of biologically produced H2 as was predicted by calculation of the thermodynamic fractionation
factors for hydrogen exchange between H2 and water vapor. As expected
for a thermodynamic equilibrium, the fractionation factor is largely independent of the
substrates used and the H2 production conditions. The predicted equilibrium fractionation
coefficient is positively correlated with temperature and we measured a change
25 of 2.2‰/°C between 45 °C and 60 °C. This is in general agreement with the theoretical predictions.
The addition of 20 mM-molybdate to sediment slurry in order to inhibit sulphate-reducing bacteria increased the amount of methane formed. Only a small proportion (7.8%) of the total methane came from the H2 + CO2 pathway, while methanogenesis from acetate was negligible. Conversion of-14C-labelled formaldehyde, methanol and methionine to 14CH4 by sediment slurry in the presence of molybdate showed that these were potential precursors of the additional methane, although lack of adequate analytical techniques precluded establishment of the quantitative significance of this turnover; [14C]formate was not converted to 14CH4. It is suggested that inhibition of sulphate-reducing bacteria by molybdate immediately increased methanogenesis from formaldehyde, methanol and methionine, and to some extent from H2 + CO2. There was also evidence for longer term development of an increased methanogenic bacterial population when there was no competition from the sulphate-reducing bacteria for available nutrients.
During the annual synchronous release of gametes by corals, a large amount of energy- rich organic material is released to the reef environment. In November 2001, we studied a minor spawning event at Heron Island in the Great Barrier Reef (GBR), Australia. Laboratory experiments showed that egg release by the staghorn coral Acropora millepora amounted to 19 ± 15 g dry mass (mean ± SE, n = 8) per m 2 coral surface. Carbon content reached 60.1 ± 4.0% and nitrogen content 3.6 ± 0.4% of the egg dry mass. During this minor spawning period, Acropora corals from the reef crest released 7 g C and 0.4 g N as eggs m -2 reef. In situ experiments (n = 11) using stirred benthic chamber measurements revealed that the sedimentary O2 consumption (SOC) of the lagoon sedi- ments increased sharply immediately after the coral spawning. Extreme SOC rates of 230 mmol O2 m -2 d -1 were reached 2 d after the event, exceeding the pre-spawning rate by a factor of 2.5. This maximum was followed by a steep decrease in SOC rates that gradually levelled off and reached pre- spawning values 11 d after the event. The immediate and strong response of SOC shows that the coral spawning event provides a strong food impulse to the benthic food chain. Our results demon- strate high decomposition efficiency of permeable carbonate reef sands and underline the role of these sediments as a biocatalytical recycling system in the oligotrophic reef environment.
We conducted four field campaigns to evaluate benthic O2 consumption and the effect of advective pore-water flow in nearshore permeable sediments in the North Sea and Baltic Sea. Advective pore-water transport had a marked effect on the benthic exchange of O2 and TCO2 in benthic chamber incubations, with the rates of exchange increasing by a factor of up to 2.5 when imposing flushing rates of 100-300 L m22 d21, compared to settings with diffusive exchange only. Estimates of in situ exchange rates using oxygen penetration and volumetric O2 consumption and TCO2 production rates were within the range measured in the chambers. The contribution of advection to solute exchange was highly variable and dependent on sediment topography. Advective processes also had a pronounced influence on the in situ distribution of O2 within the sediment, with characteristic two- dimensional patterns of O2 distribution across ripples, and also deep subsurface O2 pools, being observed. Mineralization pathways were predominantly aerobic when benthic mineralization rates were low and advective pore-water flow high as a result of well-developed sediment topography. By contrast, mineralization proceeded predominantly through sulfate reduction when benthic mineralization rates were high and advective pore-water flow low as a result of poorly developed topography. Previous studies of benthic mineralization in shallow sandy sediments have generally ignored these dynamics and, hence, have overlooked crucial aspects of permeable sediment function in coastal ecosystems.
Benthic O 2 availability regulates many important biogeochemical processes and has crucial implications for the biology and ecology of benthic communities. Further, the benthic O 2 exchange rate represents the most widely used proxy for quantifying mineralization and primary production of marine sediments. Consequently, numerous researchers have investigated the benthic O 2 dynamics in a wide range of environments. On the basis of case studies Á from abyssal sediments to microbial phototrophic communities Á I hereby try to review the current status on what we know about controls that interrelate with the O 2 dynamics of marine sediments. This includes factors like: sedimentation rates, bottom water O 2 concentrations, diffusive boundary layers, fauna activity, light, temperature, and sediment permeability. The investigation of benthic O 2 dynamics represents a challenge in resolving variations on temporal and spatial scales covering several orders of magnitude. Such an effort requires the use of several complementary measuring techniques and modeling approaches. Recent technical developments (improved chamber approaches, O 2 optodes, eddy-correlation, benthic observatories) and advances in diagenetic modeling have facilitated our abilities to resolve and interpret benthic O 2 dynamics. However, all approaches have limitations and caveats that must be carefully evaluated during data interpretation. Much has been learned during the last decades but there are still many unanswered questions that need to be addressed in order to fully understand benthic O 2 dynamics and the role of sediments for marine carbon cycling.
Interest in atmospheric hydrogen (H2) has been growing in recent years with the prospect of H2 being a potential alternative to fossil fuels as an energy carrier. This has intensified research for a quantitative understanding of the atmospheric hydrogen cycle and its total budget, including the expansion of the global atmospheric measurement network. However, inconsistencies in published observational data constitute a major limitation in exploring such data sets. The discrepancies can be mainly attributed to difficulties in the calibration of the measurements. In this study various factors that may interfere with accurate quantification of atmospheric H2 were investigated including drifts of standard gases in high pressure cylinders. As an experimental basis a procedure to generate precise mixtures of H2 within the atmospheric concentration range was established. Application of this method has enabled a thorough linearity characterization of the commonly used GC-HgO reduction detector. We discovered that the detector response was sensitive to the composition of the matrix gas. Addressing these systematic errors, a new calibration scale has been generated defined by thirteen standards with dry air mole fractions ranging from 139-1226 nmol mol-1. This new scale has been accepted as the official World Meteorological Organisation's (WMO) Global Atmospheric Watch (GAW) H2 mole fraction scale.
With in situ and laboratory chamber incubations we demonstrate that coral mucus, an important component of particulate organic matter in reef ecosystems, is a valuable substrate for microbial communities in the water column and sandy sediments of coral reefs. The addition of coral mucus to the water of benthic chambers placed on lagoon sands in the coral cay Heron Island, Australia, resulted in a rapid and significant increase in both O-2 consumption and DIC production in the chambers. The permeable coral sands permitted the transport of mucus into the sediment with interfacial water flows, resulting in the mucus being mainly (>90%) degraded in the sediment and not in the water column of the chambers. A low ratio of 0.48 (in situ) to 0.64 (laboratory) for O-2 consumption/DIC production after the addition of coral mucus, and high sulfate reduction rates (SRR) in natural sediments which were exposed to coral mucus, suggest a large contribution of anaerobic processes to the degradation of coral mucus. Oxygen penetrated less than 5 mm deep into these sediments. The microbial reaction to mucus addition was rapid, with a calculated in situ C turnover rate ranging from 7 to 18% h(-1). The degradation of coral mucus showed a dependency on the permeability of the carbonate sediments, with faster degradation and remineralization in coarse sands. This indicates the importance of permeable reef sediments for the trapping and degradation of organic matter. We suggest that coral mucus may have a function as a carrier of energy to the benthic microbial consumers.
Coral mass-spawning represents a spectacular annual, short-term, fertilization event of many oligotrophic reef communities. The spawning event in 2005 at Heron Island, Great Barrier Reef, was followed by an intense bloom of benthic dinoflagellates. Within a day from the first observed spawning, the primary production of the water column and the benthic compartment increased by factors of 4 and 2.5, respectively. However, the phototrophic communities were intensively grazed by macrozoans, and after 4–5 d the net photosynthesis (P) returned to the pre-spawning background level. The heterotrophic activity (R) mirrored the phototrophic response: a short term of elevated activity was followed by a rapid decline. However, the net autotrophic microbial communities exhibited a marked increase in the P :R ratio just after coral mass-spawning, indicating a preferential phototrophic recycling of nutrients rather than a microbial exploitation of the release of labile organic carbon. The heterotrophic and phototrophic activity of the benthic community exceeded the pelagic activity by ~2- and ~5-fold, respectively, underlining the importance of benthic activity for coral reef ecosystem function. Mass balance calculations indicated an efficient recycling of spawn-derived nitrogen (N) and carbon (C) within the benthic reef community. This was presumably facilitated by advective solute transport within the coarse, permeable, carbonate sand.
Zooxanthellae, endosymbiotic algae of reef-building corals, substantially contribute to the high gross primary production of coral reefs, but corals exude up to half of the carbon assimilated by their zooxanthellae as mucus. Here we show that released coral mucus efficiently traps organic matter from the water column and rapidly carries energy and nutrients to the reef lagoon sediment, which acts as a biocatalytic mineralizing filter. In the Great Barrier Reef, the dominant genus of hard corals, Acropora, exudes up to 4.8 litres of mucus per square metre of reef area per day. Between 56% and 80% of this mucus dissolves in the reef water, which is filtered through the lagoon sands. Here, coral mucus is degraded at a turnover rate of at least 7% per hour. Detached undissolved mucus traps suspended particles, increasing its initial organic carbon and nitrogen content by three orders of magnitude within 2 h. Tidal currents concentrate these mucus aggregates into the lagoon, where they rapidly settle. Coral mucus provides light energy harvested by the zooxanthellae and trapped particles to the heterotrophic reef community, thereby establishing a recycling loop that supports benthic life, while reducing loss of energy and nutrients from the reef ecosystem.
The identification of nitrogen sources and cycling processes is critical to the management of nitrogen pollution. Here, we used both stable (δ15N-NO3-, δ18O-NO3-, δ15N-NH4+) and radiogenic (222Rn) isotopes together with nitrogen concentrations to evaluate the relative importance of point (i.e. sewage) and diffuse sources (i.e. agricultural-derived NO3- from groundwater, drains and creeks) in driving nitrogen dynamic in a shallow coastal embayment, Port Phillip Bay (PPB) in Victoria, Australia. This study is an exemplar of nitrogen-limited coastal systems around the world where nitrogen contamination is prevalent and where constraining it may be challenging. In addition to surrounding land use, we found that the distributions of NO3- and NH4+ in the bay were closely linked to the presence of drift algae. Highest NO3- and NH4+ concentrations were 315 μmol L-1 and 2140 μmol L-1, respectively. Based on the isotopic signatures of NO3- (δ15N: 0.17 to 21‰; δ18O: 3 to 26‰) and NH4+ (δ15N: 30 to 39‰) in PPB, the high nitrogen concentrations were attributed to three major sources which varied between winter and summer; (1) nitrified sewage effluent and drift algae derived NH4+ mainly during winter, (2) NO3- mixture from atmospheric deposition, drains and creeks predominantly observed during summer and (3) groundwater and sewage derived NO3- during both surveys. The isotopic composition of NO3- also suggested the removal of agriculture-derived NO3- through denitrification was prevalent during transport. This study highlights the role of terrestrial-coastal interactions on nitrogen dynamics and illustrates the importance of submarine groundwater discharge as a prominent pathway of diffuse NO3- inputs. Quantifying the relative contributions of multiple NO3- input pathways, however, require more extensive efforts and is an important avenue for future research.
Due to decreases in seawater pH resulting from ocean acidification, permeable calcium carbonate reef sands are predicted to be net dissolving by 2050. However, the rate of dissolution and factors that control this rate remain poorly understood. Experiments performed in benthic chambers predict that reefs will become net dissolving when the aragonite saturation state (Ωa) in sea water falls below ∼ 3, as underlying reef sediments start net dissolution due to lower saturation states in the pore water. We used flow-through reactors to investigate the rate of dissolution at various Ωa at the pore scale. The sediment became net dissolving at Ωa = 1.68 – 2.25, which is significantly greater than 1. This indicates that the bulk pore water does not represent conditions at the site of dissolution, and dissolution probably occurs in microniches inside porous sand grains. Measured dissolution rates were much higher under oxic conditions than anoxic conditions, but were not affected by the addition of carbonic anhydrase. Analysis of δ13C-CO2 produced in the flow-through reactors revealed a bias in the conventional alkalinity anomaly method under anoxic conditions, showing that some of the CO2 attributed to metabolism by may actually be derived from carbonate dissolution. This deviation likely originates from alkalinity consumption by fermentation, which masks the alkalinity generated by dissolution. Therefore, dissolution rates determined by alkalinity changes in reef sands with anaerobic metabolisms may underestimate actual values.
Large areas of the oceanic shelf are composed of sandy sediments through which reactive solutes are transported via porewater advection fueling active microbial communities. The advective oxygen transport in permeable sands of the North Sea was investigated under in situ conditions using a new benthic observatory to assess the dynamic interaction of hydrodynamics, sediment morphodynamics, and oxygen penetration depth. During 16 deployments, concurrent measurement of current velocity, sediment topography, and porewater oxygen concentration were carried out. In all cases the oxyclines were found at depths of 1–6 cm, correlating with the topography of stationary and migrating bedforms (ripples). Different conditions in terms of bottom water currents and bedform migration led to fluctuating oxygen penetration depths and, hence, highly variable redox conditions in up to 2.5 cm thick layers beneath the surface. Volumetric oxygen consumption rates of surface sediments were measured on board in flow-through reactors. Bedform migration was found to reduce consumption rates by up to , presumably caused by the washout of organic carbon that is otherwise trapped in the pore space of the sediment. Based on the observations we found oxygen penetration depths to be largely controlled by oxygen consumption rates, grain size, and current velocity. These controlling variables are summarized by an adapted Damköhler number which allows for prediction of oxygen penetretion depths based on a simple scaling law. By integrating the oxygen consumption rates over the oxygen penetration depth, oxygen fluxes of 8–34 mmol m−2 d−1 were estimated.
Permeable sediments are common across continental shelves and are critical contributors to marine biogeochemical cycling. Organic matter in permeable sediments is dominated by microalgae, which as eukaryotes have different anaerobic metabolic pathways to bacteria and archaea. Here we present analyses of flow-through reactor experiments showing that dissolved inorganic carbon is produced predominantly as a result of anaerobic eukaryotic metabolic activity. In our experiments, anaerobic production of dissolved inorganic carbon was consistently accompanied by large dissolved H2 production rates, suggesting the presence of fermentation. The production of both dissolved inorganic carbon and H2 persisted following administration of broad spectrum bactericidal antibiotics, but ceased following treatment with metronidazole. Metronidazole inhibits the ferredoxin/hydrogenase pathway of fermentative eukaryotic H2 production, suggesting that pathway as the source of H2 and dissolved inorganic carbon production. Metabolomic analysis showed large increases in lipid production at the onset of anoxia, consistent with documented pathways of anoxic dark fermentation in microalgae. Cell counts revealed a predominance of microalgae in the sediments. H2 production was observed in dark anoxic cultures of diatoms (Fragilariopsis sp.) and a chlorophyte (Pyramimonas) isolated from the study site, substantiating the hypothesis that microalgae undertake fermentation. We conclude that microalgal dark fermentation could be an important energy-conserving pathway in permeable sediments.
Commentary: http://www.nature.com/ngeo/journal/vaop/ncurrent/full/ngeo2855.html
Continental shelves are predominately (~70%) covered with permeable, sandy sediments. While identified as critical sites for intense oxygen, carbon and nutrient turnover, constituent exchange across permeable sediments remains poorly quantified. The central North Sea largely consists of permeable sediments and has been identified as increasingly at risk for developing hypoxia. Therefore, we investigate the benthic O2 exchange across the permeable North Sea sediments using a combination of in situ microprofiles, a benthic chamber and aquatic eddy correlation. Tidal bottom currents drive the variable sediment O2 penetration depth (from ~3 to 8 mm) and the concurrent turbulence-driven 25-fold variation in the benthic sediment O2 uptake. The O2 flux and variability were reproduced using a simple 1-D model linking the benthic turbulence to the sediment porewater exchange. The high O2 flux variability results from deeper sediment O2 penetration depths and increased O2 storage during high velocities, which is then utilized during low flow periods. The study reveals that the benthic hydrodynamics, sediment permeability and porewater redox oscillations are all intimately linked and crucial parameters determining the oxygen availability in permeable sediments. These parameters must all be considered when evaluating mineralization pathways of organic matter and nutrients in permeable sediments.
Marine H2 and CO cycles in the sea adjacent to Honshu Island in Japan were investigated using vertical and diurnal seawater sampling. The vertical profiles of the H2 concentration differed among three stations that were located near the Kuroshio Current, off Suruga Bay, and in the center of Sagami Bay. Surface H2 enrichment was found near the Kuroshio Current, whereas subsurface H2 maxima within the pycnocline appeared at the Kuroshio and Suruga Bay stations. Biological N2 fixation likely accounts for the surface and subsurface H2 enrichment while the fermentative H2 production remains as the other possible process. In addition, twenty-four-hour observation at the Sagami Bay station revealed nearly constant H2 levels through depth and time, whereas a noon-high surface-high pattern was observed in the CO concentrations.
We measured denitrification in permeable sediments in a sealed flume tank with environmentally representative fluid flow and solute transport behavior using novel measurements. Numerical flow and reactive transport models representing the flume experiments were implemented to provide mechanistic insight into the coupled hydrodynamic and biogeochemical processes. There was broad agreement between the model results and experimental data. The model showed that the coupling between nitrification and denitrification was relatively weak in comparison to that in cohesive sediments. This was due to the direct advective transport between anoxic pore water and the overlying water column, and little interaction between the mostly oxic advective region and the underlying anoxic region. Denitrification was therefore mainly fueled by nitrate supplied from the water column. This suggests that the capacity of permeable sediments with nonmigratory ripples to remove bioavailable nitrogen from coastal ecosystems is lower than that of cohesive sediments. We conclude that while experimental measurements provide a good starting point for constraining key parameters, reactive transport models with realistic kinetic and transport parameters provide critical insight into biogeochemical processes in permeable sediment that are difficult to experimentally evaluate.
Hydrogen concentrations have been measured in sulfate‐reducing sediments of a British Columbian fjord and in Skan Bay, Alaska, as well as in the hemipelagic sediments of the eastern north tropical Pacific off the Mexican coast. In the sediments of both the Mexican shelf and Canadian fjord, hydrogen levels were lowest near the surface, increased with depth and reached a maximum pore‐water concentration of about 25–35 nM before sulfate was totally depleted. Deeper in the sediments, H 2 levels decreased again. In Skan Bay sediments, concentrations of pore‐water hydrogen increased almost linearly with depth to a maximum level of 60 nM. Measured rates of hydrogen production in Skan Bay sediments varied little with depth (about 200 nM d ⁻¹ ), and in Skan Bay the hydrogen pool had a turnover time of, at most, several hours. Model‐predicted net rates of hydrogen production in Skan Bay are not significantly different than zero. These results are consistent with the hypothesis of interspecies hydrogen transfer between hydrogen‐producing and ‐consuming bacterial populations, which predicts low ambient hydrogen concentrations and tight coupling between production and consumption.
Molecular hydrogen plays a central role in bacterially mediated anoxic sediment chemistry, as both an important electron transfer agent and a key thermodynamic control. We studied the response of hydrogen concentrations to changes in temperature, specific electron acceptor, sulfate concentration, and pH in a series of laboratory experiments using sediments from Cape Lookout Bight, North Carolina. Hydrogen concentrations were found to depend significantly on each of these factors in a fashion that suggests thermodynamic control. In general, the change in hydrogen concentrations was apparently driven by a necessity to maintain a constant ΔG for the predominant terminal electron-accepting process. We hypothesize this situation derives from highly competetive conditions that force terminal metabolic bacteria to operate right at their thermodynamic limits. The response of hydrogen to individual controls in the laboratory experiments was reflected in relatively quantitiative fashion in down-core, seasonal, and inter-environmental variations observed in sediment cores from Cape Lookout Bight and the White Oak River, NC.
Hydrogen, methane, and relevant microbiological and hydrographic observations were made to study the processes responsible for the observed distributions of the gases in Saanich Inlet, a seasonally anoxic fjord. Concentrations of surface waters were up to 22 (H2) and 13 (CH4) times atmospheric saturation. Below the surface hydrogen fell to undersaturation, rising to above saturation in the anoxic layer. Methane increased to a maximum at 30 m, and after a minimum at 125 m, increased greatly in the anoxic layer. Microbes cultured from inlet surface water produced hydrogen under experimental conditions, not by nitrogen fixation but while apparently denitrifying. Large numbers of protozoa present may provide anaerobic microniches for hydrogen and methane production, as might the intestinal tracts of the large populations of higher organisms in the inlet. Methane profiles in the upper 50 m are typical of waters outside the inlet, indicating regional sources and sinks. Hydrogen production and consumption rates, inferred to be rapid in the anoxic waters, nearly balance so that only slight increases in hydrogen occur there.
We present the first reported net loss-rate constants of molecular hydrogen, H2, in seawater. Net loss rates and depth profiles of hydrogen were measured in coastal seawater at two mid-latitude sites in eastern Canada: the St. Lawrence Estuary and Halifax Harbour, between November 2005 and July 2006. Net loss-rate constants ranged between 0.29 d− 1 and 6.07 d− 1 in the St. Lawrence Estuary, and from 0.14 d− 1 to 8.67 d− 1 in Halifax Harbour. The 0.2 μm−5 μm particle size fraction was associated with H2 loss, implying bacterial consumption. There was a correlation between net loss-rate constants for H2 and CO suggesting that both substrates shared a common sink. Dissolved hydrogen profiles measured in the St. Lawrence Estuary and Bedford Basin during winter and spring generally showed H2 levels below atmospheric equilibrium in surface waters and declining with increasing depth. Bedford Basin surface water was supersaturated (125–257%) on sunny days in June and July suggesting that net H2 production was linked to irradiance, although the exact nature of the source is yet to be determined. A series of light and dark incubation experiments showed lower net H2 uptake rates in irradiated samples suggesting either H2 photo production or light inhibition of H2 uptake.
A convenient method is described for analyzing the deuterium/hydrogen (D/H) ratio of atmospheric molecular hydrogen (H(2)) based on mass spectrometric isotope-ratio monitoring. The method requires small amounts of air ( approximately 300 mL STP), is operated on-line, and comprises four steps: (1). the condensation of the air matrix at approximately 40 K; (2). the collection of the non-condensed components of the air sample (H(2), Ne, He, and traces of N(2)) in a 5 A molecular sieves pre-concentration trap at approximately 63 K; (3). gas chromatographic purification of H(2) in a flow of He; and (4) quantification of the D/H ratio in an isotope-ratio mass spectrometer. The precision of the determination of the D/H ratio is better than 2 per thousand, which is comparable to, or better than, that obtained by conventional duel-inlet off-line analysis. There are, however, discrepancies relative to the D/H ratios determined by conventional duel-inlet analysis. This is due to differences in peak shape between reference and sample air, depending on the amount of H(2) injected. Consequently, calibration runs are required. After the calibration of the system, we obtained an accuracy of 1.5 per thousand, so that the accumulated uncertainty is estimated to be less than 4 per thousand. The method also allows determination of the H(2) concentration, with an uncertainty estimated to be 2%.
