Direct mass spectrometric measurement of gases in peat cores

Abstract Dissolved gas concentrations (O₂, CH₄, CO₂) in peat cores were monitored simultaneously using a fine (1.56 mm diameter) membrane inlet probe connected to a quadrupole mass spectrometer. This technique allows direct measurements at specific locations within the sample with minimal disturbance. Detailed gas profiles in completely waterlogged peat samples (hollows) and samples in which the water table was several cm below the vegetation surface (hummocks) were compared. The depth of the water table played a central role in the distribution of gases. In a hollow, oxygen was present (90 μM) at the surface but was not detectable (<0.5 μM) at depths greater than 2 cm. Concentrations of CH₄ and CO₂ increased from 6 and 300 μM respectively at the surface to maxima of 450 and 3900 μM at 13 cm depth. At a hummock, O₂ and CO₂ were present above the water table but CH₄ was not detectable. CH₄ was measurable 2 cm below the water table. Both CH₄ and CO₂ concentrations increased with depth but maxima were not attained in the sampled cores.     

Temporal change of gas metabolism by hydrogen-syntrophic methanogenic bacterial associations in anoxic paddy soil

Abstract Interspecies H₂ transfer within methanogenic bacterial associations (MBA) accounted for 95–97% of the conversion of ¹⁴CO₂ to ¹⁴CH₄ in anoxic paddy soil. Only 3–5% of the ¹⁴CH₄ were produced from the turnover of dissolved H₂. The H₂-syntrophic MBA developed within 5 days after the paddy soil had been submerged and placed under anoxic atmosphere. Afterwards, both the contribution of MBA to H₂-dependent methanogenesis and the turnover of dissolved H₂ did not change significantly for up to 7 months of incubation. However, while the rates of H₂-dependent methanogenesis stayed relatively constant, the rates of total methanogenesis decreased. The contribution of MBA to H₂-dependent methanogenesis was further enhanced to 99% when the temperature was shifted from 30°C to 17°C, or when the soil had been planted with rice. This enhancement was partially due to an increased utilization of dissolved H₂ by chloroform-insensitive non-methanogenic bacteria, most probably homoacetogens, so that CH₄ production was almost completely restricted to H₂-syntrophic MBA. The activity of MBA, as measured by the conversion of ¹⁴CO₂ to ¹⁴CH₄, was stimulated by glucose, lactate, and ethanol to a similar or greater extent than by exogenous H₂. Propionate and acetate had no effect.     

Thymidine incorporation into lake water bacterioplankton and pure cultures of chemolithotrophic (CO, H2) and methanotrophic bacteria

Abstract Incorporation of [³H]methyl thymidine into bacterial DNA was measured using samples of bacterioplankton from Lake Constance and pure cultures of CO, H₂ and CH₄-oxidizing bacteria. Thymidine was incorporated by Pseudomonas carboxydovorans, Paracoccus denitrificans, Methylosinus trichosporium, Methylomonas agile , and by various chemolithotropic or methylotrophic isolates from Lake Constance. Thymidine incorporation by bacterial cultures was stimulated by increasing concentrations of CO or H₂. Increased CH₄ concentrations stimulated thymidine incorporation by Ms. trichosporium only if the cells had been starved. In contrast to bacterial cultures, thymidine incorporation by bacterioplankton samples was not stimulated by increasing     

Links between methanotroph community composition and CH4 oxidation in a pine forest soil

The main gap in our knowledge about what determines the rate of CH₄ oxidation in forest soils is the biology of the microorganisms involved, the identity of which remains unclear. In this study, we used stable-isotope probing (SIP) following ¹³CH₄ incorporation into phospholipid fatty acids (PLFAs) and DNA/RNA, and sequencing of methane mono-oxygenase ( pmoA ) genes, to identify the influence of variation in community composition on CH₄ oxidation rates. The rates of ¹³C incorporation into PLFAs differed between horizons, with low ¹³C incorporation in the organic soil and relatively high ¹³C incorporation into the two mineral horizons. The microbial community composition of the methanotrophs incorporating the ¹³C label also differed between horizons, and statistical analyses suggested that the methanotroph community composition was a major cause of variation in CH₄ oxidation rates. Both PLFA and pmoA -based data indicated that CH₄ oxidizers in this soil belong to the uncultivated 'upland soil cluster α'. CH₄ oxidation potential exhibited the opposite pattern to ¹³C incorporation, suggesting that CH₄ oxidation potential assays may correlate poorly with in situ oxidation rates. The DNA/RNA-SIP assay was not successful, most likely due to insufficient ¹³C-incorporation into DNA/RNA. The limitations of the technique are briefly discussed.     

Sulphate reduction in oxic and sub-oxic North-East Atlantic sediments

Abstract Oxic and sub-oxic N.-E. Atlantic sediments were examined for sulphate-reducing activity. Oxygen and/or nitrate reduction are probably the dominant mineralisation processes in the abyssal plain sediment studied. A low rate of sulphate reduction (0.1 nmol SO²⁻₄/ml/day) was recorded in the surface 5 cm of the continental slope sediment, together with the presence of a range of sulphate-reducing bacteria (SRB). A higher activity of sulphate reduction (2.2 nmol SO²⁻₄/ml/day) occurred in the continental shelf sediment which led to a small decrease in pore water sulphate and an increase in titration alkalinity. This sediment contained approx. 10²–10³ acetate, lactate and propionate oxidising SRB/ml. No low- M _r organic acids were detected in these sediments. However, amendment with 75 μM acetate stimulated sulphate-reducing activity in the shelf sediment.     

Development of a gas diffusion probe for the determination of methane concentrations and diffusion characteristics in flooded paddy soil

Abstract A probe for the measurement of dissolved CH₄ in anoxic methanogenic environments was developed. The probe was based on the diffusion of dissolved CH₄ through a silicone membrane into a gas space at the end of the probe. This gas space was flushed with N₂ and analyzed gas-chromatographically for CH₄. The probe had a spatial resolution of < 1.3 mm, the detection limit was about 20 μM CH₄, the precision of the measurement was 9%, and consecutive measurements could be made every 4 min. Memory effects after analysis of high CH₄ concentrations could be avoided by flushing the probe with N₂ between each measurement. The probe was sensitive for water movement and, therefore, was calibrated in an artificial sediment of glass beads (100 μm diam.) immersed by aqueous solutions of known CH₄ concentrations. Sensitivity of the probe for changes in the sediment's porosity could not presently be excluded. The probe was used to measure vertical profiles of dissolved CH₄ in microcosms of anoxic paddy soil. The vertical CH₄ profiles measured with the probe compared fairly well with those measured after an extraction procedure. The profiles clearly showed that CH₄ was produced in deeper layers and diffused upwards to be consumed in the oxic top 2 mm soil layers. The probe was also used to determine the diffusion coefficient of CH₄ in an inactivated paddy soil microcosm using a set-up which allowed modelling of a measured CH₄ concentration profile using Fick's 2nd law.     

Methanogenesis and methanotrophy within a Sphagnum peatland

Abstract: Methane production and consumption activities were examined in a Massachusetts peatland. Peat from depths of 5–35 cm incubated under anaerobic conditions, produced an average of 2 nmol CH₄ g⁻¹ h⁻¹ with highest rates for peat fractions between 25–30 cm depth. Extracted microbial nucleic acids showed the strongest relative hybridization with a 16S rRNA oligonucleotide probe specific for Archaea with samples from the 25–30 cm depth. In aerobic laboratory incubations, the peat consumed methane with a maximum velocity of 67 nmol CH₄ g⁻¹ h⁻¹ and a K _s of 1.6 μM. Methane consumption activity was concentrated 4–9 cm below the peat surface, which corresponds to the aerobic, partially decomposed region in this peatland. Phospholipid fatty acid analysis of peat fractions demonstrated an abundance of methanotrophic bacteria within the region of methane consumption activity. Increases in temperature up to 30°C produced an increase in methane consumption rates for shallow samples, but not for samples taken from depths greater than 9 cm. Nitrogen fixation experiments were carried out using ¹⁵N₂ uptake in order to avoid problems associated with inhibition of methanotrophy. These experiments demonstrated that methane in peat samples did not stimulate nitrogen fixation activity, nor could activity be correlated with the presence of methanotrophic bacteria in peat fractions.     

Confirmation of operative acetate, H2/CO2 and methanol methanogenic pathways in the solid-state refuse fermentation

By use of the radiolabelled substrates sodium [1–¹⁴C] acetate, sodium [2–¹⁴C] acetate, NaH¹⁴CO₃ and ¹⁴CH₃OH, three of the possible methanogenic pathways in fermenting refuse were confirmed. Due to the absence of a methanol pool, however, the relative contribution of each could not be determined. Circumstantial evidence for an operative trimethylamine pathway was gained but not confirmed whilst preliminary attempts to stimulate methanogenesis in refuse by supplementation with mono-and dimethylamine proved unsuccessful.     

Acute impact of agriculture on high-affinity methanotrophic bacterial populations

Exposure of mineral soils to atmospherically relevant concentrations of ¹³CH₄ (2 ppmv) followed by ¹³C-phospholipid fatty acid stable isotope probing allows assessment of the high-affinity methanotrophic bacterial sink in hitherto unattainable detail. Utilizing this approach, inorganic fertilizer-treated soils from a long-term agricultural experiment were shown to display dramatic reduction, by > 70%, of the methanotrophic bacterial cell numbers. Reduction in the methane sink capacity of the soils was slightly lower than the directly observed reduction in methanotrophic bacterial counts, indicating that the inhibitory effects on high-affinity methanotrophic bacteria are not fully expressed through CH₄ oxidation rates. The results emphasize the need to rigorously assess commonly applied agricultural practices with respect to their unseen negative impacts on soil microbial diversity in relation to terrestrial sinks for atmospheric trace gases.     

Metabolism of position-labelled glucose in anoxic methanogenic paddy soil and lake sediment

Abstract Turnover times of radioactive glucose were shorter in paddy soil (4–16 min) than in Lake Constance sediment (18–62 min). In the paddy soil, 65–75% of the radioactive glucose was converted to soluble metabolites. In the sediment, only about 25% of the radioactive glucose was converted to soluble metabolites, the rest to particulate material. In anoxic paddy soil, the degradation pattern of position-labelled glucose was largely consistent with glucose degradation via the Embden-Meyerhof-Parnas (EMP) pathway followed by methanogenic acetate cleavage: CO₂ mainly originated from C-3,4, whereas CH₄ mainly originated from C-1 and C-6 of glucose. Acetate-carbon originated from C-1, C-2 and C-6 rather than from C-3,4 of glucose. In both paddy soil and Lake Constance sediment acetate and CO₂ were the most important early metabolites of radioactive glucose. Other early products included propionate, ethanol/butyrate, succinate, and lactate, but accounted each for less than 1–8% of the glucose utilized. The labelling of propionate by [3,4-¹⁴C]glucose suggests that it was mainly produced from glucose or lactate rather than from ethanol. Isopropanol and caproate were also detectable in paddy soil, but were not produced from radioactive glucose. Chloroform inhibited methanogenesis, inhibited the further degradation of radioactive acetate and resulted in the accumulation of H₂, however, did not inhibit glucose degradation. Since acetate was the main soluble fermentation product of glucose and was produced at a relatively high molar acetate: CO₂ ratio (2.5:1), homoacetogenesis appeared to be the most important glucose fermentation pathway.     

N 5, N10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri

N ⁵ N ¹⁰-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri . The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin ( K _m=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F₄₂₀ ( K _m=12 μM) at a specific rate ( V _max) of 2200 μmol min⁻¹· mg protein⁻¹ ( K _cat=1320 s⁻¹). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH₂=H₄MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri .     

Measurement of denitrification in estuarine sediment using membrane inlet mass spectrometry

Abstract Denitrification was measured in intact sediment cores and in homogenised slurries using membrane inlet mass spectrometry. Dissolved concentrations of O₂, N₂, N₂O and CO₂ were simultaneously monitored. Using a 0.8 mm diameter needle probe, a comparison was made of the gas profiles of intact cores obtained under different conditions, i.e. with air or argon as the headspace gas and after the addition of nitrate and/or a carbon source to the sediment surface. O₂ was detectable to a depth of 1 cm under a headspace of air and the depth at which the maxima of denitrification products occurred was 1.5–2 cm. Denitrification products (N₂O, N₂) occurred in the surface layers where O₂ was above the minimum level of detectability (> 0.25 μM): diffusion of N₂ and N₂O upwards from the anoxic zone, local anaerobic microenvironments or aerobic denitrification are alternative explanations for this observation. The addition of nitrate and/or acetate increased the concentrations of N₂, N₂O and CO₂ in the sediment core. In sediment slurries, the pH, nitrate concentration, carbon source and the depth from which the sample was taken affected the rate of denitrification. Nitrogen was the sole detectable end product. Maximum denitrification occurred at pH 7.5 and at 20 mM nitrate. Denitrification was at a maximum in those slurries prepared from sections of core at 1–2 cm depth.     

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.     

Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acids

Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH₄ into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH₄ flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using ¹³C-labelled CH₄ directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to ¹³CH₄ for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.     

Hydrogen turnover by psychrotrophic homoacetogenic and mesophilic methanogenic bacteria in anoxic paddy soil and lake sediment

Abstract The effect of temperature on CH₄ production, turnover of dissolved H₂, and enrichment of H₂-utilizing anaerobic bacteria was studied in anoxic paddy soil and sediment of Lake Constance. When anoxic paddy soil was incubated under an atmosphere of H₂/CO₂, rates of CH₄ production increased 25°C, but decreased at temperatures lower than 20°C. Chloroform completely inhibited methano-genesis in anoxic paddy soil and lake sediment, but did not or only partially inhibit the turnover of dissolved H₂, especially at low incubation temperatures. Cultures with H₂ as energy source resulted in the enrichment of chemolithotrophic homoacetogenic bacteria whenever incubation temperatures were lower than 20°C. Hydrogenotrophic methanogens could only be enriched at 30°C from anoxic paddy soil. A homoacetogen     

Sulphate-reducing bacteria in deep aquifer sediments of the London Basin: their role in anaerobic mineralization of organic matter

Sulphate-reducing potential was measured in sandy aquifer sediments of the London Basin. Sulphate reduction could be stimulated in the laboratory by saturating the sands with groundwater, and creating an anaerobic environment. The stimulation of vigorous sulphate reduction through the addition of an external substrate was associated with an increase in Fe_T concentration. Molybdate and selenate were added to sediment/groundwater slurries as specific inhibitors of sulphate-reducing bacteria. Under sulphate-reducing conditions acetate accumulated, but was inhibited by molybdate and selenate. ¹⁴C-acetate was used to measure the rate of acetate metabolism in the sediments.     

Hydrogen Production by Rumen Holotrich Protozoa: Effects of Oxygen and Implications for Metabolic Control by In Situ Conditions

Experiments with washed suspensions of holotrich protozoa (Isotricha spp. and Dasytricha ruminantium ) showed that both organisms have an efficient 0,-scavenging capability (apparent K_m values 2.3 and 0.3 μM, respectively). Reversible inhibition of H₂, production increased almost linearly with increasing O₂ up to 1.5 μM; higher levels of O₂ gave irreversible inhibition. In situ determinations of H, CH₄, O₂, and CO₂, in ovine rumen liquor, using a membrane inlet mass spectrometer probe, indicated that O₂, was present before feeding at 1-1.5 μM and decreased to undetectable levels (<0.25 μM) within 25 min after feeding. A transient increase in O₂. concentration after feeding occurred only in defaunated animals and resulted in suppression of CH₄ and CO₂ production. The presence of washed holotrich protozoa decreases the O₂ sensitivity of CH₄ production by suspensions of a cultured methanogenic bacterium Methanosarcina barkeri . It is concluded that holotrich protozoa play a role in ruminal O₂ utilization as well as in the production of fermentation end products (especially short-chain volatile fatty acids) utilized by the ruminant and H, utilized by methanogenic bacteria. These hydrogenosome-containing protozoa thus both control patterns of fermentation by influencing O₂ levels, and are themselves regulated by the low ambient O₂ concentrations they experience in the rumen.     

The Uptake of Carnitine by Slices of Rat Cerebral Cortex

Abstract: The properties of carnitine transport were studied in rat brain slices. A rapid uptake system for carnitine was observed, with tissue-medium gradients of 38 ± 3 for L-[¹⁴CH₃]carnitine and 27 ± 3 for D-[¹⁴CH₃]carnitine after 180 min incubation at 37°C in 0.64 mM substrate. Uptake of L- and D-carnitine showed saturability. The estimated values of K _m for L- and D-carnitine were 2.85 mM and 10.0 mM, respectively; but values of V _max (1 μmol/min/ml in-tracellular fluid) were the same for the two isomers. The transport system showed stereospecificity for L-carnitine. Carnitine uptake was inhibited by structurally related compounds with a four-carbon backbone containing a terminal carboxyl group. L-Carnitine uptake was competitively inhibited by γ-butyrobetaine ( K _i= 3.22 mM), acetylcarnitine ( K _i= 6.36 mM), and γ-aminobutyric acid ( K _i= 0.63 mM). The data suggest that carnitine and γ-aminobutyric acid interact at a common carrier site. Transport was not significantly reduced by choline or lysine. Carnitine uptake was inhibited by an N₂ atmosphere, 2,4-dinitrophenol, carbonylcyanide- N -chlorophenylhydrazone, potassium cyanide, n-ethylmaleimide, and ouabain. Transport was abolished by low temperature (4°C) and absence of glucose from the medium. Carnitine uptake was Na⁺-dependent, but did not require K⁺ or Ca²⁺.     

Bicarbonate-dependent production and methanogenic consumption of acetate in anoxic paddy soil

Abstract Acetate turnover was measured in slurries of anoxic methanogenic paddy soil after addition of carrier-free [2-¹⁴C]-acetate. Acetate concentrations stayed fairly constant for about 1–2 days indicating steady state between production and consumption reactions. Depending on the experiment, acetate concentrations were between 100 and 3000 μM. Turnover rates were determined from the logarithmic decrease of [2-¹⁴C]-acetate or from the accumulation of acetate in the presence of chloroform resulting in similar values, i.e. 12–13 nmol h⁻¹g⁻¹d.w. soil at 17°C and 36–88 nmol h⁻¹g⁻¹d.w. at 30°C. Acetate consumption was completely inhibited by chloroform. The respiratory index (RI) was < 0.27. Hence, acetate was apparently consumed by methanogenic bacteria. About 80–90% of the CH₄ produced originated from the methyl group of acetate. The role of homoacetogenesis for acetate production was studied by measuring the incorporation of radioactive bicarbonate into acetate. In different experiments, CO₂ incorporation accounted for fractions of 1–60% of the acetate produced, about 10% being the most likely value for steady-state conditions. The fraction increased at high H₂ concentrations and decreased at high acetate concentrations. The rate of H₂ production that was required for chemolithotrophic acetate production from CO₂ was two orders of magnitude higher than the actually measured rate. Hence, most of the CO₂ incorporation into acetate was caused by electron donors other than H₂ (e.g., carbohydrates) and/or by exchange reactions.