Stable carbon isotope discrimination in rice field soil during acetate turnover by syntrophic acetate oxidation or acetoclastic methanogenesis

Rice fields are an important source for the greenhouse gas methane. In Italian rice field soil CH₄ is produced either by hydrogenotrophic and acetoclastic methanogenesis, or by hydrogenotrophic methanogenesis and syntrophic acetate oxidation when temperatures are below and above about 40-45 °C, respectively. In order to see whether these acetate consumption pathways differently discriminate the stable carbon isotopes of acetate, we measured the δ¹³C of total acetate and acetate-methyl as well as the δ¹³C of CO₂ and CH₄ in rice field soil that had been pre-incubated at 45 °C and then shifted to different temperatures between 25 and 50 °C. Acetate transiently accumulated to about 6 mM, which is about one-third of the amount of CH₄ produced, irrespective of the incubation temperature and the CH₄ production pathway involved. However, the patterns of δ¹³C of the CH₄ and CO₂ produced were different at low (25, 30, 35 °C) versus high (40, 45, 50 °C) temperatures. These patterns were consistent with CH₄ being exclusively formed by hydrogenotrophic methanogenesis at high temperatures, and by a combination of acetoclastic and hydrogenotrophic methanogenesis at low temperatures. The patterns of δ¹³C of total acetate and acetate-methyl were also different at high versus low temperatures, indicating the involvement of different pathways of production and consumption of acetate at the two temperature regimes. Isotope fractionation during consumption of the methyl group of acetate was more pronounced at low (α = 1.010-1.025) than at high (α = 1.0-1.01) temperatures indicating that acetoclastic methanogenesis exhibits a stronger isotope effect than syntrophic acetate oxidation. Small amounts of propionate also transiently accumulated and were analyzed for δ¹³C. The δ¹³C values slightly increased (by about 10‰) during production and consumption of propionate, but were not affected by incubation temperature. Collectively, our results showed distinct isotope discrimination for different paths of acetate (and propionate) production and consumption, albeit differences were only small, and discrimination between methanogenic and syntrophic acetate consumption in nature may be difficult to detect.     

Stable isotopic evidence in support of active microbial methane cycling in low-temperature diffuse flow vents at 9°50′N East Pacific Rise

A unique dataset from paired low- and high-temperature vents at 9°50′N East Pacific Rise provides insight into the microbiological activity in low-temperature diffuse fluids. The stable carbon isotopic composition of CH₄ and CO₂ in 9°50′N hydrothermal fluids indicates microbial methane production, perhaps coupled with microbial methane consumption. Diffuse fluids are depleted in ¹³C by ∼10‰ in values of δ¹³C of CH₄, and by ∼0.55‰ in values of δ¹³C of CO₂, relative to the values of the high-temperature source fluid (δ¹³C of CH₄ =−20.1 ± 1.2‰, δ¹³C of CO₂ =−4.08 ± 0.15‰). Mixing of seawater or thermogenic sources cannot account for the depletions in ¹³C of both CH₄ and CO₂ at diffuse vents relative to adjacent high-temperature vents. The substrate utilization and ¹³C fractionation associated with the microbiological processes of methanogenesis and methane oxidation can explain observed steady-state CH₄ and CO₂ concentrations and carbon isotopic compositions. A mass-isotope numerical box model of these paired vent systems is consistent with the hypothesis that microbial methane cycling is active at diffuse vents at 9°50′N. The detectable ¹³C modification of fluid geochemistry by microbial metabolisms may provide a useful tool for detecting active methanogenesis.     

Stable carbon isotope composition and concentrations of CO2 and CH4 in the deep catotelm of a peat bog

Vertical profiles of concentration and C-isotopic composition of dissolved methane and carbon dioxide were observed over 26 months in the catotelm of a deep (6.5 m) peat bog in Switzerland. The dissolved concentrations of these gases increase with depth while CO₂ predominates over CH₄ (CO₂ ca. 5 times CH₄). This pattern can be reproduced by a reaction-advection-ebullition model, where CO₂ and CH₄ are formed in a ratio of 1:1. The less soluble methane is preferentially lost via outgassing (bubbles). The isotopic fractionation between CO₂ and CH₄ also increases with depth, with α_C values ranging from 1.045 to 1.075. The isotopic composition of the gases traces the passage of respiration-derived CO₂ (from the near surface) through a shallow zone with methanogenesis of low isotopic fractionation (splitting of fermentation-derived acetate). This solution then moves through the catotelm, where methanogenesis occurs by CO₂ reduction (large isotopic fractionation). In the upper part of the catotelm the C-13-depleted respiration-derived CO₂ pool buffers the isotopic composition of CO₂; the δ¹³C of CO₂ increases only slowly. At the same time strongly depleted CH₄ is formed as CO₂ reduction consumes the depleted CO₂. In the lower part of the catotelm, the respiration-derived CO₂ and shallow CH₄ become less important and CO₂ reduction is the dominant source of CO₂ and CH₄. Now, the δ¹³C values of both gases increase until equilibrium is reached with respect to the isotopic composition of the substrate. Thus, the δ¹³C values of methane reach a minimum at intermediate depth, and the deep methane has δ¹³C values comparable to shallow methane. A simple mixing model for the isotopic evolution is suggested. Only minor changes of the observed patterns of methanogenesis (in terms of concentration and isotopic composition) occur over the seasons. The most pronounced of these is a slightly higher rate of acetate splitting in spring.     

The relationship between total dissolved carbon dioxide and its stable carbon isotope ratio in aquatic sediments

A model is constructed to predict the stable carbon isotope ratio of the total dissolved CO₂ in aquatic sediments and laboratory reactors. The major parameters of the model are the fractionation between CO₂ and CH₄ as well as the intra-molecular fractionation of acetate, the relative production of CH₄ from CO₂ reduction versus acetate fermentation, the net production ratio of CO₂ to CH₄ and the stable carbon isotope ratio of the source organic carbon. The model is fitted to published data and to date from the littoral sediments of Lake Memphremagog, Quebec, Canada. The inclusion of the intra-molecular fractionation factor of acetate in the model provides a good fit to the data; without this factor, the values of the other parameters necessary for a good fit appear unreasonable.     

Carbon and hydrogen isotope fractionation by moderately thermophilic methanogens

A series of laboratory studies were conducted to increase understanding of stable carbon (¹³C/¹²C) and hydrogen (D/H) isotope fractionation arising from methanogenesis by moderately thermophilic acetate- and hydrogen-consuming methanogens. Studies of the aceticlastic reaction were conducted with two closely related strains of Methanosaeta thermophila. Results demonstrate a carbon isotope fractionation of only 7‰ (α = 1.007) between the methyl position of acetate and the resulting methane. Methane formed by this process is enriched in ¹³C when compared with other natural sources of methane; the magnitude of this isotope effect raises the possibility that methane produced at elevated temperature by the aceticlastic reaction could be mistaken for thermogenic methane based on carbon isotopic content. Studies of H₂/CO₂ methanogenesis were conducted with Methanothermobacter marburgensis. The fractionation of carbon isotopes between CO₂ and CH₄ was found to range from 22 to 58‰ (1.023 ≤ α ≤ 1.064). Greater fractionation was associated with low levels of molecular hydrogen and steady-state metabolism. The fractionation of hydrogen isotopes between source H₂O and CH₄ was found to range from 127 to 275‰ (1.16 ≤ α ≤ 1.43). Fractionation was dependent on growth phase with greater fractionation associated with later growth stages. The maximum observed fractionation factor was 1.43, independent of the δD-H₂ supplied to the culture. Fractionation was positively correlated with temperature and/or metabolic rate. Results demonstrate significant variability in both hydrogen and carbon isotope fractionation during methanogenesis from H₂/CO₂. The relatively small fractionation associated with deuterium during H₂/CO₂ methanogenesis provides an explanation for the relatively enriched deuterium content of biogenic natural gas originating from a variety of thermal environments. Results from these experiments are used to develop a hypothesis that differential reversibility in the enzymatic steps of the H₂/CO₂ pathway gives rise to variability in the observed carbon isotope fractionation. Results are further used to constrain the overall efficiency of electron consumption by way of the hydrogenase system in M. marburgensis, which is calculated to be less than 55%.     

Quantification of carbon flow from stable isotope fractionation in rice field soils with different organic matter content

Rice fields are an important source for the greenhouse gas methane produced by acetoclastic and hydrogenotrophic methanogenesis. Fractionation of ¹³C/¹²C can in principle be used to quantify the relative contribution of these pathways, but our knowledge of isotopic fractionation during reduction of CO₂ and turnover of acetate in different methanogenic environments is still scarce. We therefore measured δ¹³C signatures in two types of anoxic Italian rice field soils, one with high and one with low degradable organic matter (OM) content. Both soils were incubated in the presence and absence of methyl fluoride, a specific inhibitor of acetoclastic methanogenesis. Optimization of methyl fluoride concentration resulted in complete inhibition of acetoclastic methanogenesis. CH₄ was then exclusively produced by hydrogenotrophic methanogenesis, allowing determination of the isotopic signatures and fractionation factors specific for this methanogenic pathway. Acetate, which was then no longer consumed, accumulated and was used for determination of the isotopic signature of the fermentatively produced acetate (both total acetate and methyl carbon of acetate). Hence, all isotopic signatures, including fractionation factors were determined for the methanogenic soil. These data, were then used for computation of the relative contribution of the two methanogenic pathways. In the high OM soil, the contribution of acetoclastic methanogenesis to total CH₄ production increased simultaneously with decreasing acetate concentration. In the low OM soil, methanogenesis from H₂/CO₂ was clearly greater than theoretically expected. Furthermore, isotope fractionation of hydrogenotrophic methanogenesis indicated that the in situ energy status of methanogens strongly depended on the availability of organic carbon in the rice field soil system. Collectively, our data show that the study of isotopic fractionation in methanogenic environments allows a deeper insight into the ongoing processes, which may be quite different in the same ecosystem with different content of degradable OM.     

Fractionation of stable carbon isotopes during anaerobic production and degradation of propionate in defined microbial cultures

In many anoxic environments propionate is, after acetate, the second most important fermentation product, being degraded further to finally result in CH₄ production. In principle, isotope discrimination can be used to assess the path of organic matter degradation to acetate, CO₂ and CH₄. However, nothing is known about the isotope fractionation in primary and secondary fermentation steps involving propionate, although it is an important precursor of acetate. We therefore studied the degradation of propionate with a syntrophic coculture of Syntrophobacter fumaroxidans and Methanobacterium formicicum. The isotope enrichment factor for propionate degradation to acetate, CO₂ and CH₄ was almost negligible (ε_prop 0.9‰). The fermentative production of propionate was studied in cultures with Opitutus terrae growing on pectin, xylan and starch. These polysaccharides were fermented to acetate, succinate, propionate, H₂ and CO₂. While the δ¹³C value of the initially produced propionate was similar to that of the organic substrates (ca. −28 to −25‰), the δ¹³C value of the other fermentation products was higher. The δ¹³C values of all products generally decreased during the course of fermentation. Finally, a small depletion in ¹³C (ca. 6‰) with respect to the organic substrate was observed for propionate, while the other fermentation products where slightly enriched. Overall, stable carbon isotope discrimination was small during both fermentative production and consumption of propionate in the anaerobic microbial cultures, so that propionate turnover probably only marginally affects isotope fractionation during anaerobic degradation of organic matter.     

Potential of microbial methane formation in a high-temperature hydrocarbon seep

Hydrocarbon seepage is a surface expression where fluids mixed with sediments and hydrocarbons are expelled through fracture systems that potentially tap into gas–petroleum reservoirs. Hydrocarbons released from most seeps appear to be thermogenic on the basis of their relative abundance and isotopic composition. The potential for subsurface microbial processes modifying these geochemical fingerprints remains poorly constrained. In this study, microcosm incubations were conducted on mud slurries supplied with/without various methanogenic precursors at temperatures ranging from ambient conditions to 90 °C, in order to assess microbial CH₄ formation in the subsurface beneath hydrocarbon seeps. The analyses indicated that CH₄ production was positive at ?80 °C, regardless of whether or not or which precursors were added. However, the pattern of CH₄ production rates varied with the precursor and temperature. In general, the optimum CH₄ production from H₂/CO₂ and formate occurred over a wide range of temperatures (?40 °C), whereas that from acetate, methanol and methylamine was restricted to relatively lower temperatures (40–50 °C). The CH₄ recoveries, together with the C isotopic compositions of CH₄, further indicated that the quantities of CH₄ produced could not completely account for the quantities of precursor consumed, suggesting that a complex metabolic network was involved in the transformation of the added precursor and organic C inherited from inoculated sediments. Microbial CH₄ was estimated to constitute 7–61% of the CH₄ observed using experimentally-derived apparent isotope fractionations as the end member compositions. This illustrates the possibility that microbial CH₄ produced at shallower depths could quantitatively and isotopically alter deeply-sourced thermogenic CH₄ in hydrocarbon seep environments.     

An experimental study of oxygen isotope fractionation between inorganically precipitated aragonite and water at low temperatures

To determine oxygen isotope fractionation between aragonite and water, aragonite was slowly precipitated from Ca(HCO₃)₂ solution at 0 to 50°C in the presence of Mg²⁺ or SO₄²⁻. The phase compositions and morphologies of synthetic minerals were detected by X-ray diffraction (XRD) and scanning electron microscopy (SEM) techniques. The effects of aragonite precipitation rate and excess dissolved CO₂ gas in the initial Ca(HCO₃)₂ solution on oxygen isotope fractionation between aragonite and water were investigated. For the CaCO₃ minerals slowly precipitated by the CaCO₃ or NaHCO₃ dissolution method at 0 to 50°C, the XRD and SEM analyses show that the rate of aragonite precipitation increased with temperature. Correspondingly, oxygen isotope fractionations between aragonite and water deviated progressively farther from equilibrium. Additionally, an excess of dissolved CO₂ gas in the initial Ca(HCO₃)₂ solution results in an increase in apparent oxygen isotope fractionations. As a consequence, the experimentally determined oxygen isotope fractionations at 50°C indicate disequilibrium, whereas the relatively lower fractionation values obtained at 0 and 25°C from the solution with less dissolved CO₂ gas and low precipitation rates indicate a closer approach to equilibrium. Combining the lower values at 0 and 25°C with previous data derived from a two-step overgrowth technique at 50 and 70°C, a fractionation equation for the aragonite-water system at 0 to 70°C is obtained as follows:

     

Chemical and isotopic equilibrium between CO2 and CH4 in fumarolic gas discharges: Generation of CH4 in arc magmatic-hydrothermal systems

The chemical and isotopic composition of fumarolic gases emitted from Nisyros Volcano, Greece, and of a single gas sample from Vesuvio, Italy, was investigated in order to determine the origin of methane (CH₄) within two subduction-related magmatic-hydrothermal environments.Apparent temperatures derived from carbon isotope partitioning between CH₄ and CO₂ of around 340°C for Nisyros and 470°C for Vesuvio correlate well with aquifer temperatures as measured directly and/or inferred from compositional data using the H₂O-H₂-CO₂-CO-CH₄ geothermometer. Thermodynamic modeling reveals chemical equilibrium between CH₄, CO₂ and H₂O implying that carbon isotope partitioning between CO₂ and CH₄ in both systems is controlled by aquifer temperature.N₂/³He and CH₄/³He ratios of Nisyros fumarolic gases are unusually low for subduction zone gases and correspond to those of midoceanic ridge environments. Accordingly, CH₄ may have been primarily generated through the reduction of CO₂ by H₂ in the absence of any organic matter following a Fischer-Tropsch-type reaction. However, primary occurrence of minor amounts of thermogenic CH₄ and subsequent re-equilibration with co-existing CO₂ cannot be ruled out entirely. CO₂/³He ratios and δ¹³C_CO2 values imply that the evolved CO₂ either derives from a metasomatized mantle or is a mixture between two components, one outgassing from an unaltered mantle and the other released by thermal breakdown of marine carbonates. The latter may contain traces of organic matter possibly decomposing to CH₄ during thermometamorphism.     

Carbon and hydrogen isotopic compositions of products of open-system catalytic hydrogenation of CO2: Implications for abiogenic hydrocarbons in Earth’s crust

This paper reports the isotope effects in an open-system Fischer-Tropsch type (FTT) synthesis, with implications for the origin of natural abiogenic hydrocarbons. The starting form of carbon was CO₂, with carbon and hydrogen isotopic compositions measured for products of catalytic hydrogenation of CO₂ on iron and cobalt catalysts (FTCO₂-Fe and FTCO₂-Co) at 350 and 245 °C, respectively, and 10 MPa. The carbon isotopic composition of the resulting saturated hydrocarbons (alkanes) as a function of carbon number shows a positive trend for both FTCO₂-Fe and FTCO₂-Co, with a fractionation of 2-4‰ and 3-6‰ between CH₄ and C₂H₆ over the Fe and Co catalysts, respectively. The unsaturated hydrocarbons (alkenes) do not show any trend. A strong kinetic isotope fractionation (>40‰) occurred between CO₂ and CH₄ in both experiments. The hydrogen isotope fractionation between alkanes appeared to be similar to that found in natural (thermogenic and biogenic) gases, with enrichment in deuterium of longer hydrocarbon chains; the dominant H/D fractionation occurred between CH₄ and C₂H₆. Alkenes in the products of the FTCO₂-Fe reaction are enriched in deuterium (∼50‰) and do not show any trend versus carbon number. We suggest that other than FTT reactions or a simple mixing are responsible for the occurrence of the inverse isotopic trends in both δ¹³C and δD found in light hydrocarbons in some terrestrial environments and meteorites.     

Carbon isotopic fractionation during a mesocosm bloom experiment dominated by Emiliania huxleyi: Effects of CO2 concentration and primary production

We investigated the effect of CO₂ and primary production on the carbon isotopic fractionation of alkenones and particulate organic matter (POC) during a natural phytoplankton bloom dominated by the coccolithophore Emiliania huxleyi. In nine semi-closed mesocosms (∼11 m³ each), three different CO₂ partial pressures (pCO₂) in triplicate represented glacial (∼180 ppmv CO₂), present (∼380 ppmv CO₂), and year 2100 (∼710 ppmv CO₂) CO₂ conditions. The largest shift in alkenone isotopic composition (4-5‰) occurred during the exponential growth phase, regardless of the CO₂ concentration in the respective treatment. Despite the difference of ∼500 ppmv, the influence of pCO₂ on isotopic fractionation was marginal (1-2‰). During the stationary phase, E. huxleyi continued to produce alkenones, accumulating cellular concentrations almost four times higher than those of exponentially dividing cells. Our isotope data indicate that, while alkenone production was maintained, the interaction of carbon source and cellular uptake dynamics by E. huxleyi reached a steady state. During stationary phase, we further observed a remarkable increase in the difference between δ¹³C of bulk organic matter and of alkenones spanning 7-12‰. We suggest that this phenomenon is caused mainly by a combination of extracellular release of ¹³C-enriched polysaccharides and subsequent particle aggregation induced by the production of transparent exopolymer particles (TEP).     

Carbon isotopic fractionation between CO2 vapour,silicate and carbonate melts: an experimental study to 30 kbar

The carbon isotopic fractionation between CO₂ vapour and sodamelilite (NaCaAlSi₂O₇) melt over a range of pressures and temperatures has been investigated using solid-media piston-cylinder high pressure apparatus. Ag₂C₂O₄ was the source of CO₂ and experimental oxygen fugacity was buffered at hematite-magnetite by the double capsule technique. The abundance and isotopic composition of carbon dissolved in sodamelilite (SM) glass were determined by stepped heating and the ¹³C of coexisting vapour was determined directly by capsule piercing. CO₂ solubility in SM displays a complex behavior with temperature. At pressures up to 10 kbars CO₂ dissolves in SM to form carbonate ion complexes and the solubility data suggest slight negative temperature dependence. Above 20 kbars CO₂ reacts with SM to form immiscible Na-rich silicate and Ca-rich carbonate melts and CO₂ solubility in Na-enriched silicate melt rises with increasing temperature above the liquidus. Measured values for carbon isotopic fractionation between CO₂ vapour and carbonate ions dissoived in sodamelilite melt at 1200°–1400° C and 5–30 kbars average 2.4±0.2, favouring¹³C enrichment in CO₂ vapour. The results are maxima and are independent of pressure and temperature. Similar values of 2 are obtained for the carbon isotopic fractionation between CO₂ vapour and carbonate melts at 1300°–1400° C and 20–30 kbars.     

The influence of carbon source on abiotic organic synthesis and carbon isotope fractionation under hydrothermal conditions

A series of laboratory experiments were performed to investigate the relative contributions of CO and other single-carbon compounds to abiotic synthesis of organic compounds in hydrothermal environments. Experiments were conducted by heating aqueous solutions of CO, CO₂, HCOOH, or CH₄ at 250 °C under reducing conditions, and observing production of CH₄ and other hydrocarbons. Native Fe was included in the experiments as a source of H₂ through reaction with water and as a potential catalyst. Experiments with CO or HCOOH as the carbon source resulted in rapid generation of CH₄ and other hydrocarbons that closely resembled typical products of Fischer-Tropsch organic synthesis. In contrast, experiments using CO₂ or CH₄ as the carbon source yielded no detectable hydrocarbon products. Carbon isotope measurements of reaction products from the CO experiments indicate that the CH₄ and other hydrocarbons were substantially depleted in ¹³C, with CH₄ δ¹³C values 30 to 34‰ lighter than the initial CO. Most of the fractionation apparently occurs during attachment of CO to the catalyst surface and subsequent reduction to surface-bound methylene. The initial step in polymerization of these methylene units to form hydrocarbons involves a small, positive fractionation, so that ethane and ethene are slightly enriched in ¹³C relative to CH₄. However, subsequent addition of carbon molecules to the growing hydrocarbon chain proceeds with no net observable fractionation, so that the isotopic compositions of the C₃₊ light hydrocarbons are controlled by isotopic mass balance. This result is consistent with a previously proposed model for carbon isotopic patterns of light hydrocarbons in natural samples. The abundance and isotopic composition of light hydrocarbons produced with HCOOH as the carbon source were similar to those generated with CO, but the isotopic compositions of non-volatile hydrocarbons diverged, suggesting that the higher hydrocarbons were formed by different mechanisms in the CO and HCOOH experiments. The experiments indicate that CO, and possibly HCOOH, may be critical intermediates in the abiotic formation of organic compounds in geologic environments, and suggest that the low levels of these compounds present in most hydrothermal systems could represent a bottleneck restricting the extent of abiotic organic synthesis in some circumstances.     

Phosphoric acid fractionation factors for smithsonite and cerussite between 25 and 72°C

The intramolecular kinetic oxygen isotope fractionation between CO₂ and CO₃²⁻ during reaction of phosphoric acid with natural smithsonite (ZnCO₃) and cerussite (PbCO₃) has been determined between 25 and 72°C. While cerussite decomposes in phosphoric acid within a few hours at 25°C, smithsonite reacts very slowly with the acid at 25°C providing yields of CO₂ < 25% after 2 weeks. The low yields result in a low precision for oxygen isotope measurements of the acid-liberated CO₂ (±1.65‰, 1σ, n = 9). The yield and reproducibility of oxygen isotope values of the acid-liberated CO₂ from smithsonite can be improved, the latter to ∼±0.15‰, by increasing the reaction temperature to 50°C for 12 h or to 72°C for 1 h. Our new phosphoric acid fractionation factor for natural cerussite at 25°C deviates significantly from a previously published value on synthetic material. The temperature dependence of the oxygen isotope factionation factor, α between acid-liberated CO₂ and carbonate at 25 to 72°C is given by the following equations

     

Thermal decarboxylation of acetic acid: Implications for origin of natural gas

Laboratory experiments on the thermal decarboxylation of solutions of acetic acid at 200°C and 300°C were carried out in hydrothermal equipment allowing for on-line sampling of both the gas and liquid phases for chemical and stable-carbon-isotope analyses. The solutions had ambient pH values between 2.5 and 7.1; pH values and the concentrations of the various acetate species at the conditions of the experiments were computed using a chemical model.Results show that the concentrations of acetic acid, and not total acetate in solution, control the reaction rates which follow a first order equation based on decreasing concentrations of acetic acid with time. The decarboxylation rates at 200°C (1.81 × 10^?8 per second) and 300°C (8.17 × 10^?8 per second) and the extrapolated rates at lower temperatures are relatively high. The activation energy of decarboxylation is only 8.1 kcal/mole. These high decarboxylation rates, together with the distribution of short-chained aliphatic acid anions in formation waters, support the hypothesis that acid anions are precursors for an important portion of natural gas.Results of the δ¹³C values of CO₂, CH₄, and total acetate show a reasonably constant fractionation factor of about 20 permil between CO₂ and CH₄ at 300°C. The δ¹³C values of CO₂ and CH₄ are initially low and become higher as decarboxylation increases.     

Carbon isotope effects associated with mixed-acid fermentation of saccharides by Clostridium papyrosolvens

In anoxic environments, microbial fermentation is the first metabolic process in the path of organic matter degradation. Since little is known about carbon isotope fractionation during microbial fermentation, we studied mixed-acid fermentation of different saccharides (glucose, cellobiose, and cellulose) in Clostridium papyrosolvens. The bacterium was grown anaerobically in batch under different growth conditions, both in pure culture and in co-culture with Methanobacterium bryantii utilizing H₂/CO₂ or Methanospirillum hungatei utilizing both H₂/CO₂ and formate. Fermentation products were acetate, lactate, ethanol, formate, H₂, and CO₂ (and CH₄ in methanogenic co-culture), with acetate becoming dominant at low H₂ partial pressures. After complete conversion of the saccharides, acetate was ¹³C-enriched (α_sacc/ac = 0.991-0.997), whereas lactate (α_sacc/lac = 1.001-1.006), ethanol (α_sacc/etoh = 1.007-1.013), and formate (α_sacc/form = 1.007-1.011) were ¹³C-depleted. The total inorganic carbon produced was only slightly enriched in ¹³C, but was more enriched, when formate was produced in large amounts, as ¹²CO₂ was preferentially converted with H₂ to formate. During biomass formation, ¹²C was slightly preferred (α_sacc/biom ≈ 1.002). The observations in batch culture were confirmed in glucose-limited chemostat culture at growth rates of 0.02-0.15 h⁻¹ at both low and high hydrogen partial pressures. Our experiments showed that the carbon flow at metabolic branch points in the fermentation path governed carbon isotope fractionation to the accumulated products. During production of pyruvate, C isotopes were not fractionated when using cellulose, but were fractionated to different extents depending on growth conditions when using cellobiose or glucose. At the first catabolic branch point (pyruvate), the produced lactate was depleted in ¹³C, whereas the alternative product acetyl-CoA was ¹³C enriched. At the second branch point (acetyl-CoA), the ethanol formed was 15.6-18.6‰ depleted in ¹³C compared to the alternative product acetate. At low hydrogen partial pressures, as normally observed under environmental conditions, fermentation of saccharides should mainly result in the production of acetate that is only slightly enriched in ¹³C (<3‰).     

Variable carbon isotope fractionation expressed by aerobic CH4-oxidizing bacteria

Carbon isotope fractionation factors reported for aerobic bacterial oxidation of CH₄(α_CH4-CO2) range from 1.003 to 1.039. In a series of experiments designed to monitor changes in the carbon isotopic fractionation of CH₄ by Type I and Type II methanotrophic bacteria, we found that the magnitude of fractionation was largely due to the first oxidation step catalyzed by methane monooxygenase (MMO). The most important factor that modulates the (α_CH4-CH3OH) is the fraction of the total CH₄ oxidized per unit time, which strongly correlates to the cell density of the growth cultures under constant flow conditions. At cell densities of less than 0.1 g/L, fractionation factors greater than 1.03 were observed, whereas at cell densities greater than 0.5 g/L the fractionation factors decreased to as low as 1.002. At low cell densities, low concentrations of MMO limit the amount of CH₄ oxidized, while at higher cell densities, the overall rates of CH₄ oxidation increase sufficiently that diffusion of CH₄ from the gaseous to dissolved state and into the cells is likely the rate-determining step. Thus, the residual CH₄ is more fractionated at low cell densities, when only a small fraction of the total CH₄ has been oxidized, than at high cell densities, when up to 40% of the influent CH₄ has been utilized. Therefore, since Rayleigh distillation behavior is not observed, δ¹³C values of the residual CH₄ cannot be used to infer the amount oxidized in either laboratory or field-studies. The measured (α_CH4-CH3OH) was the same for both Type I and Type II methanotrophs expressing particulate or soluble MMO. However, large differences in the δ¹³C values of biomass produced by the two types of methanotrophs were observed. Methylosinus trichosporium OB3b (Type II) produced biomass with δ¹³C values about 15‰ higher than the dissimilated CO₂, whereas Methylomonas methanica (Type I) produced biomass with δ¹³C values only about 6‰ higher than the CO₂. These effects were independent of the magnitude of the initial carbon isotope fractionation caused by MMO and were relatively constant despite changing ratios of assimilatory to dissimilatory carbon transformation by the organisms. This suggests that the difference in biomass carbon isotopes is primarily due to differences in the fractionation effect at the formaldehyde branch point in the metabolic pathway, rather than assimilation of CO₂ by Type II methanotrophs.     

Sulfides,oxides and sphene in high-pressure schists from New Caledonia

In the New Caledonia high-pressure schists pyrite, pyrrhotite, chalcopyrite, rutile and sphene are common phases while hematite and ilmenite are rare and magnetite is absent. The parageneses of these minerals were clarified from their occurrence as inclusions in garnet, from phase relations in the Cu-Fe-S and Fe-Ti-O-S systems, and from phase rule considerations for the multi-component system. The sulfur fugacity estimated for pelites and basites containing pyrrhotite, pyrite and rutile increased with increasing metamorphic grade; the oxygen fugacity in these schists was less than 10^–27.6 bars at 400° C, 10 kb and 10^–22.3 bars at 500° C, 11 kb. Among the other components of the metamorphic fluid in pelites, H₂O was major, CH₄, CO₂ and H₂S minor, and H₂, CO, COS and SO₂ rare. The fluid composition altered with advancing metamorphic grade, such that H₂O decreased while CO₂, CH₄ and H₂S increased, and this change was linked to concurrent massive decarbonization in the rock matrices.     

Preliminary reservoir model of enhanced coalbed methane (ECBM) in a subbituminous coal seam,Huntly Coalfield,New Zealand

The Huntly coalfield has significant coal deposits that contain biogenically-sourced methane. The coals are subbituminous in rank and Eocene in age and have been previously characterised with relatively low to moderate measured gas (CH₄) contents (2–4 m³/ton). The CO₂ holding capacity is relatively high (18.0 m³/ton) compared with that of CH₄ (2.6 m³/ton) and N₂ (0.7 m³/ton) at the same pressure (4 MPa; all as received basis). The geothermal gradient is also quite high at 55 °C/km.A study has been conducted which simulates enhancement of methane recovery (ECBM) from these deposits using a new version of the TOUGH2 (version 2) reservoir simulator (ECBM-TOUGH2) that can handle non-isothermal, multi-phase flows of mixtures of water, CH₄, CO₂ and N₂. The initial phase of the simulation is CH₄ production for the first 5 years of the field history. The model indicates that methane production can be significantly improved (from less than 80% recovery to nearly 90%) through injection of CO₂. However, although an increase in the rate of CO₂ injection increases the amount of CO₂ sequestered, the methane recovery (because of earlier breakthrough with increasing injection rate) decreases. Modeling of pure N₂ injection produced little enhanced CH₄ production. The injection of a hypothetical flue gas mixture (CO₂ and N₂) also produced little increase in CH₄ production. This is related to the low adsorption capacity of the Huntly coal to N₂ which results in almost instantaneous breakthrough into the production well.