Carbon isotopic fractionation in lipids from methanotrophic bacteria II: the effects of physiology and environmental parameters on the biosynthesis and isotopic signatures of biomarkers

Controls on the carbon isotopic signatures of methanotroph biomarkers have been further explored using cultured organisms. Growth under conditions which select for the membrane-bound particulate form of the methane monooxygenase enzyme (pMMO) leads to a significantly higher isotopic fractionation than does growth based on the soluble isozyme in both RuMP and serine pathway methanotrophs; in an RuMP type the delta delta 13Cbiomass equaled -23.9% for pMMO and -12.6% for sMMO. The distribution of biomarker lipids does not appear to be significantly affected by the dominance of one or the other MMO type and their isotopic compositions generally track those of the parent biomass. The 13C fractionation behaviour of serine pathway methanotrophs is very complex, reflecting the assimilation of both methane and carbon dioxide and concomitant dissimilation of methane-derived carbon. A limitation in CH4 availability leads to the production of biomass which is 13C-enriched with respect to both carbon substrates and this occurs irrespective of MMO type. This startling result indicates that there must be an additional fractionation step downstream from the MMO reaction which leads to incorporation of 13C-enriched carbon at the expense of dissimilation of 13C-depleted CO2. In these organisms, polyisoprenoid lipids are 13C-enriched compared to polymethylenic lipid which is the reverse of that found in the RuMP types. Serine cycle hopanoids, for example, can vary anywhere from 12% depleted to 10% enriched with respect to the CH4 substrate depending on its concentration. Decrease in growth temperature caused an overall increase in isotopic fractionation. In the total biomass, this effect tended to be masked by physiological factors associated with the type of organism and variation in the bulk composition. The effect was, however, clearly evident when monitoring the 13C signature of total lipid and individual biomarkers. Our results demonstrate that extreme carbon isotopic depletion in field samples and fossil biomarker lipids can be indicative of methanotrophy but the converse is not always true. For example, the hopanoids of a serine cycle methanotroph may be isotopically enriched by more than 10% compared to the substrate methane when the latter is limiting. In other words, hopanoids from some methanotrophs such as M. trichosporium would be indistinguishable from those of cyanobacteria or heterotrophic bacteria on the basis of either chemical structure or carbon isotopic signature.     

Carbon isotopic fractionation associated with lipid biosynthesis by a cyanobacterium: relevance for interpretation of biomarker records

For the cyanobacterium Synechocystis UTEX 2470, grown photoautotrophically to a logarithmic stage of growth, the total lipid extract is depleted in 13C by 4.8% relative to average biomass. Depletions observed for acetogenic (straight-chain) lipids range from 7.6 (hexadecanoic acid) to 9.9% (a C16 n-alkyl chain bound in a polar-lipid fraction), with a mass-weighted average of 9.1%. Polyisoprenoid lipids fall into two isotopic groups, with phytol, diplopterol, and diploptene depleted by 6.4-6.9% and bishomohopanol (produced from the extracts by the preparative degradation of bacteriohopanepolyol) depleted by 8.4%. Analysis of the pattern of depletions indicates that two carbon positions in each C5 biomonomer leading to polyisoprenoid products are probably depleted in 13C relative to average biomass. The depletion of bacteriohopanepolyol relative to other polyisoprenoids can be ascribed to changes that occur over the life of each cell: (1) the 13C content of carbon flowing to lipid biosynthesis decreases as the cell size increases and (2) a greater proportion of the bacteriohopanepolyol which, unlike other polyisoprenoids, is present mainly in the cytoplasm rather than in membranes and is synthesized when cells are larger. Chlorophyll a is depleted relative to average biomass by O.7%. Given the observed depletion of 13C in phytol, the heteroaromatic, chlorophyllide portion of chlorophyll must be enriched in 13C by 2.7%. This enrichment is large relative to that in chlorophyllides produced by eukaryotes and may be related to a parallel enrichment of 13C in cyanobacterial glutamic acid. As in many previous investigations of cyanobacterial lipids, long-chain n-alkanes (C22-C29) are found in the extracts. They are, however, enriched in 13C relative to biomass and have isotopic compositions suggesting that they are contaminants of petrochemical origin. Available results indicate that cyanobacterial lipids will be depleted relative to dissolved CO2 that has served as a carbon source by 22-30% and that a wider range of depletions will be characteristic of eukaryotic products. The absence of long-chain n-alkanes in cyanobacteria reduces the possibility that petroleum ever formed from pre-eukaryotic sedimentary debris.     

Hydrogen isotope fractionation in lipids of the methane-oxidizing bacterium Methylococcus capsulatus

Hydrogen isotopic compositions of individual lipids from Methylococcus capsulatus, an aerobic, methane-oxidizing bacterium, were analyzed by hydrogen isotope-ratio-monitoring gas chromatography-mass spectrometry (GC-MS). The purposes of the study were to measure isotopic fractionation factors between methane, water, and lipids and to examine the biochemical processes that determine the hydrogen isotopic composition of lipids. M. capsulatus was grown in six replicate cultures in which the δD values of methane and water were varied independently. Measurement of concomitant changes in δD values of lipids allowed estimation of the proportion of hydrogen derived from each source and the isotopic fractionation associated with the utilization of each source.All lipids examined, including fatty acids, sterols, and hopanols, derived 31.4 ± 1.7% of their hydrogen from methane. This was apparently true whether the cultures were harvested during exponential or stationary phase. Examination of the relevant biochemical pathways indicates that no hydrogen is transferred directly (with C-H bonds intact) from methane to lipids. Accordingly, we hypothesize that all methane H is oxidized to H₂O, which then serves as the H source for all biosynthesis, and that a balance between diffusion of oxygen and water across cell membranes controls the concentration of methane-derived H₂O at 31%. Values for α_l/w, the isotopic fractionation between lipids and water, were 0.95 for fatty acids and 0.85 for isoprenoid lipids. These fractionations are significantly smaller than those measured in higher plants and algae. Values for α_l/m, the isotopic fractionation between lipids and methane, were 0.94 for fatty acids and 0.79 for isoprenoid lipids. Based on these results, we predict that methanotrophs living in seawater and consuming methane with typical δD values will produce fatty acids with δD between −50 and −170‰, and sterols and hopanols with δD between −150 and −270‰.     

生物标志化合物碳同位素地球化学研究的几个相关问题

生物标志化合物稳定碳同位素地球化学是８０年代末期新兴的研究领域。单个生物标志化合物碳同位素组成的成因解释是该领域至关重要的问题。本文综合讨论了与此有关的生物合成过程中脂类化合物碳同位素变化、细菌生物合成过程中碳同位素分馏及其脂类化合物碳同位素组成特征，从而为我国的生物标志化合物碳同位素地球化学研究提供了新的理论依据     

Stable carbon-isotopic compositions of lipids isolated from the ammonia-oxidizing chemoautotroph Nitrosomonas europaea

For the ammonia-oxidizing bacterium Nitrosomonas europaea, grown autotrophically using semicontinuous culturing, average biomass was depleted in ¹³C relative to CO₂ dissolved in the medium by ca. 20‰ and the total-lipid extract was depleted in ¹³C relative to biomass by 3.7‰. The n-alkyl lipids (weighted average of fatty acids) and isoprenoid lipids (weighted average of hopanoids) were both depleted in ¹³C relative to biomass by about 9‰. The large depletion in the isoprenoid lipids seems to indicate that isotopic fractionations associated with the biosynthesis of methylerythritol phosphate (MEP) affected at least two carbon positions in each isoprene unit. Among the fatty acids, trans-9-hexadecenoic acid was most depleted (13.0‰ relative to biomass), followed by cis-9- hexadecenoic acid (9.6‰) and hexadecanoic acid (6.9‰). Isotopic relationships between the three acids suggest that significant isotope effects were associated with the desaturation and cis to trans isomerization of fatty acids. Given these observations, hopanoids produced by ammonia-oxidizing bacteria growing in natural waters are likely to be depleted in ¹³C by 26–30‰ relative to dissolved CO₂. Since CO₂ at aquatic oxyclines is often depleted in ¹³C, the range of δ values expected for hopanoids is ca. −34‰ to −55‰. The δ values of geohopanoids observed in numerous studies and attributed to unspecified chemoautotrophs fall within this range.     

An isotopic biogeochemical study of Neoproterozoic and Early Cambrian sediments from the Centralian Superbasin, Australia

Organic matter from Neoproterozoic and Early Cambrian sediments of the Amadeus and Officer basins of the Centralian Superbasin, Australia, has been studied for biomarker distributions and the carbon isotopic compositions of kerogen and individual hydrocarbons. These sediments represent both shallow and deep water marine facies in the older sections and marine and saline lacustrine carbonate deposits in the Cambrian. Hydrocarbon biomarker patterns were found to be quite consistent with the known sedimentary environments and provide valuable insights into the biogeochemical changes which accompanied the transition from a microbially-dominated ocean to the early stages of metazoan radiation. In particular, carbon isotopic data for n-alkyl and isoprenoid lipids presented here, and in earlier studies, showed a reversal in carbon isotopic ordering between the Proterozoic and Phanerozoic. By comparison with the delta 13C of kerogen, n-alkyl lipids from deep-water Proterozoic sediments were enriched in 13C and appear to be derived mainly from heterotrophs whilst open marine Phanerozoic counterparts are 13C depleted and evidently derived mainly from autotrophs. Data from the samples studied here are consistent with a model invoking a change in the redox structure of the ocean, possibly aided by the innovation of faecal pellets.     

Indication of methane movement from petroleum reservoir to surface, Löningen oilfield, NW-Germany

An existing method of detecting microgas seeps was applied in a free soil gas investigation above the Löningen oilfield, NW-Germany. Simple gas surveying was combined with sampling of soil gases for gas-chromatographic treatment and examination of the carbon isotopic composition. A zone of biological gas formation and an area of thermocatalytic methane were discovered, the latter probably caused by migration from the subsurface petroleum accumulation.The methane in the reservoir differs in isotopic composition depending on its origin: gas from gascap or petroleum. Methane dissolved from petroleum is depleted in ¹²C by about 5 ppt compared to methane from the gascap. In relation to the reservoir a general ¹³C-enrichment between 2.5 and 4.6 ppt is observed in the soil gas methanes which is supposedly due to isotopic fractionation during migration. The two species of reservoir methane are still distinguishable on the surface by their different composition. Gaseous hydrocarbons from C₂ to C₄ seem to be restricted by migration. Bacterial oxidation appears not to affect the isotopic composition of the thermocatalytic methane seriously.     

Carbon isotope fractionation in phospholipid fatty acid biomarkers of bacteria and fungi native to an acid mine drainage lake

This study identifies isotope signatures associated with autotrophic and heterotrophic microbial communities that may provide a means to determine carbon cycling relationships in situ for acid mine drainage (AMD) sites. Stable carbon isotope ratios (δ¹³C) of carbon sources, bulk cells, and membrane phospholipids (PLFA) were measured for autotrophic and heterotrophic microbial enrichment cultures from a mine tailings impoundment in northern Ontario, Canada, and for pure strains of the sulfur oxidizing bacteria Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. The autotrophic enrichments had indistinguishable PLFA distributions from the pure cultures, and the PLFA cyc-C_19:0 was determined to be a unique biomarker in this system for these sulfur oxidizing bacteria. The PLFA distributions produced by the heterotrophic enrichments were distinct from the autotrophic distributions and the C_18:2 PLFA was identified as a biomarker for these heterotrophic enrichments. Genetic analysis (16S, 18S rRNA) of the heterotrophic cultures indicated that these communities were primarily composed of Acremonium fungi.Stable carbon isotope analysis revealed that bulk cellular material in all autotrophic cultures was depleted in δ¹³C by 5.6–10.9‰ relative to their atmospheric CO₂ derived carbon source, suggesting that inorganic carbon fixation in these cultures is carbon limited. Individual PLFA from these autotrophs were further depleted by 8.2–14.6‰ compared to the bulk cell δ¹³C, which are among the largest biosynthetic isotope fractionation factors between bulk cell and PLFA reported in the literature. In contrast, the heterotrophic bulk cells were not significantly fractionated in δ¹³C relative to their carbon source and heterotrophic PLFA ranged from 3‰ enriched to 4‰ depleted relative to the isotopic composition of their total biomass. These distinct PLFA biomarkers and isotopic fractionations associated with autotrophic and heterotrophic activity in this laboratory study provide potential biomarkers for delineating autotrophic and heterotrophic carbon cycling in AMD environments.     

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.     

Formation of iron sulfide nodules during anaerobic oxidation of methane

The biomarker compositions of iron sulfide nodules (ISNs; upper Pliocene Valle Ricca section near Rome, Italy) that contain the ferrimagnetic mineral greigite (Fe₃S₄) were examined. In addition to the presence of specific terrestrial and marine biomarkers, consistent with formation in coastal marine sediments, these ISNs contain compounds thought to originate from sulfate reducing bacteria (SRB). These compounds include a variety of low-molecular-weight and branched alkanols and several non-isoprenoidal dialkyl glycerol diethers (DGDs). In addition, archaeal biomarkers, including archaeol, macrocyclic isoprenoidal DGDs and isoprenoidal glycerol dialkyl glycerol tetraethers are also present. Both SRB and archaeal lipid δ¹³C values are depleted in ¹³C (δ¹³C values are typically less than −50‰), which suggests that the SRB and archaea consumed ¹³C depleted methane. These biomarker and isotopic signatures are similar to those found in cold seeps and marine sediments where anaerobic oxidation of methane (AOM) occurs with sulfate serving as the terminal electron acceptor. Association of AOM with formation of greigite-containing ISNs could provide an explanation for documented remagnetization of the Valle Ricca sediments. Upward migration of methane, subsequent AOM and associated authigenic greigite formation are widespread processes in the geological record that have considerable potential to compromise paleomagnetic records.     

Aerobic methanotrophy at ancient marine methane seeps: A synthesis

The molecular fingerprints of the chemosynthesis based microbial communities at methane seeps tend to be extremely well preserved in authigenic carbonates. The key process at seeps is the anaerobic oxidation of methane (AOM), which is performed by consortia of methanotrophic archaea and sulphate reducing bacteria. Besides the occurrence of ¹³C depleted isoprenoids and n-alkyl chains derived from methanotrophic archaea and sulphate reducing bacteria, respectively, ¹³C depleted triterpenoids have been reported from a number of seep deposits. In order to evaluate the significance of these apparently non-AOM related molecular fossils, the biomarker inventories of one Campanian and two Miocene methane seep limestones are compared. These examples provide strong evidence that methane was not solely oxidized by an anaerobic process. Structural and carbon isotope data reveal that aerobic methanotrophy was common at some ancient methane seeps as well. The Miocene Marmorito limestone contains abundant 3β-methylated hopanoids (δ¹³C: −100‰). Most likely, 3β-methylated hopanepolyols, prevailing in aerobic methanotrophs, were the precursor lipids of these compounds. A series of isotopically depleted 4-methylated steranes (lanostanes; δ¹³C: −80‰ to −70‰) and similarly isotopically depleted 17β(H),21β(H)-32-hopanoic acid in the Miocene Pietralunga seep limestone also are derived probably from aerobic methanotrophs. Lanosterol, which is known to be produced by aerobic methanotrophs, is the most likely precursor of 4-methylated steranes. Less obvious is the origin of 8,14-secohexahydrobenzohopanes (δ¹³C: −110‰ to −107‰) in Late Cretaceous seep limestones. These hopanoids probably reflect early degradational products of precursor lipids locally produced by seep endemic aerobic methanotrophs.     

Biogeochemistry of the stable hydrogen isotopes

The fractionation of H isotopes between the water in the growth medium and the organically bonded H from microalgae cultured under conditions, where light intensity and wavelength, temperature, nutrient availability, and the H isotope ratio of the water were controlled, is reproducible and light dependant. All studies were based either on the H isotope ratios of the total organic H or on the lipids, where most of the H is firmly bonded to C. H bonded into other macromolecules, proteins, carbohydrates and nucleic acids, does not exchange with water, when algae are incubated in water enriched with deuterium. Only after the destruction of quaternary H bonds are labile hydrogens in macromolecules free to exchange with water. By growing algae (18 strains), including blue-green algae, green algae and diatoms, in continuous light, the isotope fractionations in photosynthesis were reproducibly ?93 to ?178 %. depending on the organism tested. This fractionation was not temperature dependent. Microalgae grown in total darkness with an organic substrate did not show the isotope fractionation seen in cells grown in light. In both light- and dark-grown algae, however, additional depletion of deuterium (?30 to ?60%.) in cellular organic matter occurs during the metabolism of carbohydrates to form lipids. Plants from several natural populations also fractionated isotopes during photosynthesis by an average of ?90 to ?110%. In addition, the organically bonded H in nonsaponifiable lipids was further fractionated by ?80%. from that in saponifiable lipids, isolated from two geographically distinct populations of marsh plants. This difference between H isotope ratios of these two groups of lipids provides an endogenous isotopic marker.     

Experimental investigation on the carbon isotope fractionation of methane during gas migration by diffusion through sedimentary rocks at elevated temperature and pressure

Molecular transport (diffusion) of methane in water-saturated sedimentary rocks results in carbon isotope fractionation. In order to quantify the diffusive isotope fractionation effect and its dependence on total organic carbon (TOC) content, experimental measurements have been performed on three natural shale samples with TOC values ranging from 0.3 to 5.74%. The experiments were conducted at 90°C and fluid pressures of 9 MPa (90 bar). Based on the instantaneous and cumulative composition of the diffused methane, effective diffusion coefficients of the ¹²CH₄ and ¹³CH₄ species, respectively, have been calculated.Compared with the carbon isotopic composition of the source methane (δ¹³C₁ = −39.1‰), a significant depletion of the heavier carbon isotope (¹³C) in the diffused methane was observed for all three shales. The degree of depletion is highest during the initial non-steady state of the diffusion process. It then gradually decreases and reaches a constant difference (Δ δ = δ¹³C_diff −δ¹³C_source) when approaching the steady-state. The degree of the isotopic fractionation of methane due to molecular diffusion increases with the TOC content of the shales. The carbon isotope fractionation of methane during molecular migration results practically exclusively from differences in molecular mobility (effective diffusion coefficients) of the ¹²CH₄ and ¹³CH₄ entities. No measurable solubility fractionation was observed.The experimental isotope-specific diffusion data were used in two hypothetical scenarios to illustrate the extent of isotopic fractionation to be expected as a result of molecular transport in geological systems with shales of different TOC contents. The first scenario considers the progression of a diffusion front from a constant source (gas reservoir) into a homogeneous “semi-infinite” shale caprock over a period of 10 Ma.In the second example, gas diffusion across a 100 m caprock sequence is analyzed in terms of absolute quantities and isotope fractionation effects. The examples demonstrate that methane losses by molecular diffusion are small in comparison with the contents of commercial size gas accumulations. The degree of isotopic fractionation is related inversely to the quantity of diffused gas so that strong fractionation effects are only observed for relatively small portions of gas.The experimental data can be readily used in numerical basin analysis to examine the effects of diffusion-related isotopic fractionation on the composition of natural gas reservoirs.     

Tracing H isotope effects in the dynamic metabolic network using multi-nuclear (1H, 2H and 13C) solid state NMR and GC–MS

Recent studies reveal substantial variability in biosynthetic ²H/¹H fractionation between lipids and water, which highlights the effect of central metabolic pathways on the H isotopic composition of the end products. Using multi-nuclear (¹H, ²H and ¹³C) solid state nuclear magnetic resonance (NMR) spectroscopy and gas chromatography–mass spectrometry (GC–MS), we were able to track the incorporation of ¹H and ²H metabolic fluxes into different cellular components of Escherichia coli during growth on two sets of media: (i) 10% deuterated water and non-deuterated glucose, and (ii) non-deuterated water and 10% deuterated glucose. In the 10% ²H water experiment, the ²H abundance of the bulk cell was 4.5%; the ²H uptake by membrane lipids, aliphatic H in proteins, and all the non-aliphatic H positions (including nucleic acids and sugars) was 6.2%, 2.3% and 6.2%, respectively. In the 10% ²H glucose experiment, the corresponding ²H uptake was 2.0%, 1.4% and 2.5%, respectively, and 1.9% for the bulk cell. The net fractionation of fatty acids (FAs) relative to water and glucose was consistent with that in studies employing natural ²H abundance, suggesting the 10% deuterated environment does not alter isotope fractionation during FA biosynthesis. Aliphatic H in proteins was significantly depleted in ²H relative to FAs by 290–640‰. This depletion is likely related to the dynamic regulation of central metabolic pathways that gradually builds up ²H in tricarboxylic acid (TCA) cycle intermediates though continuous interaction with water, leading to the rapid synthesis of isotopically light amino acids early in the cell cycle and the production of isotopically enriched FAs late in the cell cycle. Besides, enzyme malfunctioning caused by ²H substitution is likely to promote proteolysis, which could also contribute to the ²H depletion in proteins. Non-aliphatic H positions as a whole exhibited similar net fractionation factors to the lipids. The study provides an overview of H isotope distribution in the major molecular constituents of intact bacterial cells, offering insight into the mechanisms underlying the metabolic control on the H isotopic composition of biomarker precursors.     

Fractionation of carbon isotopes in biosynthesis of fatty acids by a piezophilic bacterium Moritella japonica strain DSK1

We examined stable carbon isotope fractionation in biosynthesis of fatty acids of a piezophilic bacterium Moritella japonica strain DSK1. The bacterium was grown to stationary phase at pressures of 0.1, 10, 20, and 50 MPa in media prepared using sterile-filtered natural seawater supplied with glucose as the sole carbon source. Strain DSK1 synthesized typical bacterial fatty acids (C_14-19 saturated, monounsaturated, and cyclopropane fatty acids) as well as long-chain polyunsaturated fatty acids (PUFA) (20:6ω3). Bacterial cell biomass and individual fatty acids exhibited consistent pressure-dependent carbon isotope fractionations relative to glucose. The observed Δδ_FA-glucose (−1.0‰ to −11.9‰) at 0.1 MPa was comparable to or slightly higher than fractionations reported in surface bacteria. However, bulk biomass and fatty acids became more depleted in ¹³C with pressure. Average carbon isotope fractionation (Δδ_FA-glucose) at high pressures was much higher than that for surface bacteria: −15.7‰, −15.3‰, and −18.3‰ at 10, 20, and 50 MPa, respectively. PUFA were more ¹³C depleted than saturated and monounsaturated fatty acids at all pressures. The observed isotope effects may be ascribed to the kinetics of enzymatic reactions that are affected by hydrostatic pressure and to biosynthetic pathways that are different for short-chain and long-chain fatty acids. A simple quantitative calculation suggests that in situ piezophilic bacterial contribution of polyunsaturated fatty acids to marine sediments is nearly two orders of magnitude higher than that of marine phytoplankton and that the carbon isotope imprint of piezophilic bacteria can override that of surface phytoplankton. Our results have important implications for marine biogeochemistry. Depleted fatty acids reported in marine sediments and the water column may be derived simply from piezophilic bacteria resynthesis of organic matter, not from bacterial utilization of a ¹³C-depleted carbon source (i.e., methane). The interpretation of carbon isotope signatures of marine lipids must be based on principles derived from piezophilic bacteria.     

Fractionation of carbon and hydrogen isotopes by methane-oxidizing bacteria

Carbon isotopic analysis of methane has become a popular technique in the exploration for oil and gas because it can be used to differentiate between thermogenic and microbial gas and can sometimes be used for gas-source rock correlations. Methane-oxidizing bacteria, however, can significantly change the carbon isotopic composition of methane; the origin of gas that has been partially oxidized by these bacteria could therefore be misinterpreted.We cultured methane-oxidizing bacteria at two different temperatures and monitored the carbon and hydrogen isotopic compositions of the residual methane. The residual methane was enriched in both ¹³C and D. For both isotopic species, the enrichment at equivalent levels of conversion was greater at 26°C than at 11.5°C. The change in δD relative to the change in δ¹³C was independent of temperature within the range studied. One culture exhibited a change in the fractionation pattern for carbon (but not for hydrogen) midway through the experiment, suggesting that bacterial oxidation of methane may occur via more than one pathway.The change in the δD value for the residual methane was from 8 to 14 times greater than the change in the δ¹³C value, indicating that combined carbon and hydrogen isotopic analysis may be an effective way of identifying methane which has been subjected to partial oxidation by bacteria.     

Mathematical simulation of the carbon isotopic fractionation between huminitic coals and related methane

In order to estimate the isotope fractionation effect between coals and methane during coalification a maturity-related fractionation model has been developed for coals and reservoir gases of NW Germany which is based on empirical data. Assuming that observed isotope shifts of the convertible carbon of coals of different maturities are related to a loss of methane during coalification and that this shift can be described by a Rayleigh distillation process, functions with preselected fractionation factors were fitted to measured isotope data of the convertible carbon of coals. The best approximation of theoretical and measured data was achieved with a low fractionation factor (α_c= 1.003). Using this model theoretical methane carbon isotope data were determined and compared to the isotopic composition of reservoir methanes of NW Germany. Although the methane isotope data of reservoir gases and the related maturity of the coals show a slight scatter, the theoretical data plot within the same range and follow the increase of the ¹³C concentration of reservoir gases with increasing maturity of the coals.     

The effect of aromatization on the isotopic compositions of hydrocarbons during early diagenesis

Polycyclic aromatic hydrocarbons with varying degrees of aromatization were isolated from the Eocene Messel Shale (Rheingraben, Germany). The high abundances of these compounds and their structural resemblances to cyclic triterpenoid lipids are consistent with derivation from microbial rather than thermal processes. Compounds structurally related to oleanane contain from five to nine double bonds; those within a series of aromatized hopanoids contain from three to nine. All are products of diagenetic reactions that remove hydrogen or methyl groups, and, in several cases, break carbon-carbon bonds to open rings. Aromatized products are on average depleted in 13C relative to possible precursors by l.2% (range: l.5% enrichment to 4% depletion, n = 9). The dependence of 13C content on the number of double bonds is not, however, statistically significant and it must be concluded that there is no strong evidence for isotopic fractionation accompanying diagenetic aromatization. Isotopic differences between series (structures related to ursane, des-A-ursane, des-A-lupane, des-A-arborane, and possibly, des-A-gammacerane are present) are much greater, indicating that 13C contents are controlled primarily by source effects. Fractionations due to chromatographic isotope effects during HPLC ranged from 0.1 to 2.8%.     

Controls on the stable carbon isotopic composition of biogenic methane produced in a tidal freshwater estaurine sediment

The δ¹³C value of methane in sediments from a tidal freshwater site in the White Oak River Estuary, North Carolina, exhibited a relatively small, but consistent, seasonal variation (∼3‰) with isotopically heavier values occurring during the warmer months (−66.1‰ summer, −69.2‰ winter). These isotopic shifts could have resulted from changes in: (1) isotopic compositions of precursor molecules; (2) kinetic isotope effects associated with methane production; or (3) pathways of methane production. Methane production rate and isotopic data from sediment incubation experiments and field measurements were used to determine the relative contributions of these factors to the observed seasonal variations. Although changes in δ¹³C values of biogenic methane are typically thought to result from changes in pathways of methane production, this study showed that a significant amount (36 ± 22%) of the seasonal variations between the δ¹³C value of methane produced in sediment incubation experiments could be attributed to changes in the δ¹³C value of the ΣCO₂ pool. This was due to increased methane production rates and removal of ¹²CO₂ with increasing temperature, a prevalent feature of methanogenic systems that may account for some of the frequently observed ¹³C enrichment in methane during warmer months. Combining the change in the δ¹³C value of the ΣCO₂ pool with temperature-controlled changes in fractionation (α) resulting from kinetic isotope effects accounted for (53 ± 22%) of the ¹³C enrichment observed during summer sediment incubation experiments. Although large pathway changes were not observed in sediment incubation experiments, the remaining differences in δ¹³C values could have resulted from smaller, undetectable changes in the percentage of methane production from acetate (∼14%) and/or a shift in the δ¹³C values of methane produced from acetate (∼4‰).