Fractionation of Carbon Isotopes during Biogenesis of Atmospheric Isoprene

The stable carbon isotope composition of isoprene emitted from leaves of red oak (Quercus rubra L.) was measured. Isoprene was depleted in ¹³C relative to carbon recently fixed by photosynthesis. The difference in isotope composition between recently fixed carbon and emitted isoprene was independent of the isotopic composition of the source CO₂. β-Carotene, an isoprenoid plant constituent, was depleted in ¹³C relative to whole leaf carbon to the same degree as isoprene, but fatty acids were more depleted. Isoprene emitted from leaves fed abscisic acid was much less depleted in ¹³C than was isoprene emitted from unstressed leaves. We conclude that isoprene is made from an isoprenoid precursor that is derived from acetyl-CoA made from recent photosynthate. The carbon isotope composition of isoprene in the atmosphere is likely to be slightly more negative (less ¹³C) than C₃ plant material but when plants are stressed the isotopic composition could vary.     

Carbon Isotope Ratios (13C/12C) in Fractions of Field-Grown Grape

The carbon isotope ratio (δ¹³C%) in leaves and berries of Vitis vinifera L. was determined during the fruiting season. The δ¹³C was from ?24.6 to ?26.3 in total leaf tissue, while it is from ?22.7 to ?24.3 in the aqueous extract of leaves. The δ¹³C in the berries was more stable, around ?24, with no change during ripening. Malic and tartaric acid, isolated from the berries at the onset of ripening, showed a δ¹³C of ?23.5 and ?23.0 respectively. It was concluded that malic acid accumulated in the berries at the onset of ripening is not formed directly through β-carboxylation.     

Methanogenesis in Eocene Arctic soils inferred from δC of tree fossil carbonates

We report on the carbon and oxygen stable isotope composition of fossil tree material collected at the White Mountain locality of the Buchanan Lake Formation on Axel Heiberg Island in the High Arctic of Canada. The fossils are Middle Eocene in age and have been permineralized with carbonate. Microscopic examination of fossils revealed them to be the remains of Metasequoia stems, composed of secondary carbonate (calcite) and original wood intermingled at the cellular level. Because the specimens show little compression, crushing, or tissue degradation, we believe that carbonate permineralization occurred soon after burial, and therefore provides insight into Eocene carbon cycling at the locality. The carbon isotope signature of the carbonate suggests that methanogenesis resulted in a ¹³C-enriched CO₂ pool that equilibrated with soil water and gave rise to unusually ¹³C-enriched CaCO₃. Tree fossil carbonate exhibited strikingly high δ¹³C values (+4.0 to +7.4‰) compared to published Phanerozoic pedogenic carbonate δ¹³C values. These δ¹³C values, in conjunction with fractionation factors (α) previously determined for carbonate precipitation and methanogenic pathways, indicate an Eocene soil CO₂ pool containing 80-95% CO₂ produced as a by-product of acetate-fermentation methanogenesis. Because methane in the atmosphere is a powerful greenhouse gas, we suggest that methane emissions from Axel Heiberg soils contributed to the relatively warm Arctic climate during the Middle Eocene.     

Effects of temperature and food quality on isotopic turnover and discrimination in a cladoceran

Our experimental study was designed to assess the effects of temperature on nitrogen isotope turnover and to measure the effects of temperature and food quality on the stable carbon and nitrogen isotope discrimination factors (Δ¹³C and Δ¹⁵N) in a cladoceran. For the first part of our study, Daphnia were fed with non-enriched or ¹⁵N-enriched Scenedesmus obliquus at 12, 15, 20, and 25 °C. For the second part, Daphnia were reared at 15, 20, and 25 °C on Scenedesmus or Cryptomonas sp. There were no clear effects of temperature on turnover rates of the nitrogen isotope of cladocerans. However, a general increase in Δ¹³C with increasing temperature was measured, regardless of the food source. Δ¹⁵N was also affected by temperature, but contrasting results were measured depending on the food source used. There were significant effects of food quality on Δ¹³C and Δ¹⁵N in Daphnia, as values obtained for Daphnia fed Scenedesmus were always higher than those obtained for Daphnia fed Cryptomonas. Our experiments produced discrimination factors that were very different from those usually considered in isotope studies and showed that the values used for isotope model implementation to analyze field data need to be adapted to environmental conditions.     

Comparison of the exploitation of methane-derived carbon by tubicolous and non-tubicolous chironomid larvae in a temperate eutrophic lake

Carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) in three sympatric species of larval chironomids were analyzed in a temperate eutrophic shallow lake in Japan. Markedly lower δ¹³C values were reported in Chironomus plumosus (?51.2 ‰) and Tanypus sp. (?43.5 ‰) than those in photoautotrophic carbon sources [particulate organic matter (POM) and sediment]. There were positive correlations between δ¹³C and δ¹⁵N in the two chironomid species. These results indicated that they assimilated carbon derived from biogenic methane by exploiting methane-oxidizing bacteria (MOB). In contrast, Propsilocerus akamusi exhibited similar δ¹³C values to those of POM or sediment. A δ¹³C-based isotope mixing model was used to estimate the dietary contributions of MOB to each chironomid species. The mean contributions ranged from 11 to 15 % in C. plumosus, 13 to 19 % in Tanypus sp., but only up to 5 % in P. akamusi. In an aquarium, we observed that individuals of C. plumosus and Tanypus sp., which exhibited low δ¹³C values, built U-shaped larval tubes in the sediment, and an oxidized layer developed around these tubes. Propsilocerus akamusi did not exhibit this behavior. These results suggest that tube building may provide larval chironomids with greater access to methane-derived carbon through increased opportunities to feed on MOB.     

Evidence for novel and specialized mycorrhizal parasitism: the orchid Gastrodia confusa gains carbon from saprotrophic Mycena

We investigated the physiological ecology of the Asian non-photosynthetic orchid Gastrodia confusa. We revealed its mycorrhizal partners by using molecular identification and identified its ultimate nutritional source by analysing carbon and nitrogen natural stable isotope abundances. Molecular identification using internal transcribed spacer and large subunit nrDNA sequences showed that G. confusa associates with several species of litter- and wood-decomposer Mycena fungi. The carbon and nitrogen isotope signatures of G. confusa were analysed together with photosynthetic plant reference samples and samples of the ectomycorrhizal epiparasite Monotropa uniflora. We found that G. confusa was highly enriched in ¹³C but not greatly in ¹⁵N, while M. uniflora was highly enriched in both ¹³C and ¹⁵N. The ¹³C and ¹⁵N signatures of G. confusa were the closest to those of the fruit bodies of saprotrophic fungi. Our results demonstrate for the first time using molecular and mass-spectrometric approaches that myco-heterotrophic plants gain carbon through parasitism of wood or litter decaying fungi. Furthermore, we demonstrate that, several otherwise free-living non-mycorrhizal, Mycena can be mycorrhizal partners of orchids.     

Evidence of Substantial Carbon Isotope Fractionation among Substrate, Inorganic Carbon, and Biomass during Aerobic Mineralization of 1,2-Dichloroethane by Xanthobacter autotrophicus

Carbon isotope fractionation during aerobic mineralization of 1,2-dichloroethane (1,2-DCA) by Xanthobacter autotrophicus GJ10 was investigated. A strong enrichment of ¹³C in residual 1,2-DCA was observed, with a mean fractionation factor α ± standard deviation of 0.968 ± 0.0013 to 0.973 ± 0.0015. In addition, a large carbon isotope fractionation between biomass and inorganic carbon occurred. A mechanistic model that links the fractionation factor α to the rate constants of the first catabolic enzyme was developed. Based on the model, it was concluded that the strong enrichment of ¹³C in 1,2-DCA arises because the first irreversible step of the initial enzymatic transformation of 1,2-DCA consists of an S_N2 nucleophilic substitution. S_N2 reactions are accompanied by a large kinetic isotope effect. The substantial carbon isotope fractionation between biomass and inorganic carbon could be explained by the kinetic isotope effect associated with the initial 1,2-DCA transformation and by the metabolic pathway of 1,2-DCA degradation. Carbon isotope fractionation during 1,2-DCA mineralization leads to 1,2-DCA, inorganic carbon, and biomass with characteristic carbon isotope compositions, which may be used to trace the process in contaminated environments.     

Terrestrial and marine trophic pathways support young-of-year growth in a nearshore Arctic fish

River discharge supplies nearshore communities with a terrestrial carbon source that is often reflected in invertebrate and fish consumers. Recent studies in the Beaufort Sea have documented widespread terrestrial carbon use among invertebrates, but only limited use among nearshore fish consumers. Here, we examine the carbon source and diet of rapidly growing young-of-year Arctic cisco (Coregonus autumnalis) using stable isotope values (δ¹³C and δ¹⁵N) from muscle and diet analysis (stomach contents) during a critical and previously unsampled life stage. Stable isotope values (δ¹⁵N and δ¹³C) may differentiate between terrestrial and marine sources and integrate over longer time frames (weeks). Diet analysis provides species-specific information, but only from recent foraging (days). Average δ¹³C for all individuals was ?25.7 ‰, with the smallest individuals possessing significantly depleted δ¹³C values indicative of a stronger reliance of terrestrial carbon sources as compared to larger individuals. Average δ¹⁵N for all individuals was 10.4 ‰, with little variation among individuals. As fish length increased, the proportion of offshore Calanus prey and neritic Mysis prey increased. Rapid young-of-year growth in Arctic cisco appears to use terrestrial carbon sources obtained by consuming a mixture of neritic and offshore zooplankton. Shifts in the magnitude or phenology of river discharge and the delivery of terrestrial carbon may alter the ecology of nearshore fish consumers.     

Leaf carbon isotope ratio and water use efficiency of urban roadside trees in summer in Kyoto city

The effect of summer climate on leaf carbon isotope composition (δ¹³C) of the major roadside tree species Prunus × yedoensis (P. yedoensis) was investigated in Kyoto city, Japan, to explore the implications for alterations in urban environments. Temperature and the vapor pressure deficit were higher at sites of higher traffic volumes, possibly affected by a heat island effect. The leaf δ¹³C of P. yedoensis trees was affected strongly by leaf carbon isotope discrimination (Δ), with much less effect of δ¹³C on atmospheric CO₂. Leaf Δ values in the summer were smaller at sites of higher traffic volumes with high atmospheric temperatures, suggesting a higher long-term water use efficiency (WUE) at these sites. Gas exchange measurements of P. yedoensis leaves indeed suggested a higher intrinsic WUE at sites of higher traffic volumes with high atmospheric temperatures. These results suggest that leaf Δ is related to the response of WUE to summer climates, and that leaf δ¹³C in urban areas is a useful tracer for understanding the influences of urban environments on plant photosynthetic processes.     

Use of Carbon Isotopes to Estimate Incorporation of Added CO(2) by Greenhouse-Grown Tomato Plants

A method is presented which uses the ¹³C and ¹⁴C isotope abundance in CO₂-enriched greenhouse crops to determine the percentage of plant organic carbon derived from artificially added CO₂. In a greenhouse experiment with CO₂ concentrations elevated to 1100 ± 100 microliters per liter during part of the daylight hours and maintained at normal atmospheric concentrations (340 microliters per liter) during the rest of the time, it was shown by ¹⁴C analysis that between 41% and 42% of the carbon in tomato plants (Lycopersicon esculentum var 4884) came from the artificially added CO₂. Similar results were obtained from ¹³C analyses when the CO₂ pressure-dependent isotope separation was taken into account.     

The effect of acidification and the combined effects of acidification/lipid extraction on carbon stable isotope ratios for sub-arctic and arctic marine zooplankton species

Stable isotope ratios of carbon (δ ¹³C) in zooplankton are widely applied in ecosystem-level studies of energy flux and trophic interactions. Carbonate (CaCO₃) and lipid content are highly variable both among and within zooplankton species. Such variability can arise from the δ ¹³C-depleted nature of lipids as well as differences in carbon incorporation among tissues (e.g., relative amount of carbonate). Critically, the impact of the common lipid- and carbonate-normalization steps of extraction and acidification is poorly understood and applied in an inconsistent manner. Here, we investigated the effect of lipid extraction and sample acidification (CaCO₃ removal) on δ ¹³C in sub-arctic and arctic marine zooplankton species. Our results indicate that, with the exception of the shelled mollusc Limacina helicina, acidification of samples can be omitted for all other marine zooplankton considered in this study. In the case of L. helicina, δ ¹³C can be corrected for carbonate content using the linear equation developed in this study. In contrast, the δ ¹³C for all species was significantly enriched by a combination of lipid extraction and acidification (up to +4.9 ‰) prior to stable isotope analysis. Our data were used to develop simple, predictive species-specific correction models for lipid-acid-normalized δ ¹³C using C:N and/or untreated δ ¹³C values. Our results indicate that the δ ¹³C value for all species, including those with lower C:N ratios (~3–4), should be corrected for lipid content. We recommend lipid extraction whenever possible, or else the use of species/taxon-specific δ ¹³C lipid-normalization models for accurate determination of carbon sources and dynamics for arctic and sub-arctic marine zooplankton.     

Diet of Japanese eels Anguilla japonica in the Kojima Bay-Asahi River system, Japan

The diet of Japanese eels, Anguilla japonica, was investigated using stomach content and stable isotope analyses. Stable isotope enrichment of carbon and nitrogen (Δδ¹³C and Δδ¹⁵N) was first estimated for A. japonica by comparing the isotopic signatures (δ¹³C and δ¹⁵N) of reared eels to that of their food. The estimated isotope enrichment was then applied to the diet estimation of A. japonica in the Kojima Bay-Asahi River system, Japan, combined with conventional stomach content analysis. Stable isotope enrichment varied among tissues, from 0.2‰ to 0.8‰ for carbon and from 1.3‰ to 2.1‰ for nitrogen. Nitrogen isotope enrichment of A. japonica muscle estimated in this study was 2.1‰, which was different from the previously reported mean δ¹⁵N enrichment of several animals of 3.4‰. These results indicate that isotope-based diet estimations for A. japonica need to use species- and tissue-specific values of isotope enrichment. In the diet analysis, stomach contents and stable isotopes revealed that (1) A. japonica appear to usually feed on a single type of prey species in each feeding session, (2) principal prey species were mud shrimp, Upogebia major, in brackish Kojima Bay and crayfish, Procambarus clarkia, in the Asahi River, (3) A. japonica in Kojima Bay primarily depend on the pelagic food web as a carbon source due to mud shrimp being filter feeders and eels in the Asahi River primarily depend on the littoral food web. Based on these results and the recently reported eel movements between Kojima Bay and the Asahi River, it appears that A. japonica can adapt to various feeding environments as opportunists, but also utilize the food resources by targeting a single type of prey species during a single feeding session.     

Mechanistic studies of sesquiterpene cyclases based on their carbon isotope ratios at natural abundance

During the process of terpene biosynthesis, C–C bond breaking and forming steps are subjected to kinetic carbon isotope effects, leading to distinct carbon isotopic signatures of the products. Accordingly, carbon isotopic signatures could be used to reveal the ‘biosynthetic history’ of the produced terpenoids. Five known sesquiterpene cyclases, regulating three different pathways, representing simple to complex biosynthetic sequences, were heterologously expressed and used for in vitro assays with farnesyl diphosphate as substrate. Compound specific isotope ratio mass spectrometry measurements of the enzyme substrate farnesyl diphosphate (FDP) and the products of all the five cyclases were performed. The calculated δ¹³C value for FDP, based on δ¹³C values and relative amounts of the products, was identical with its measured δ¹³C value, confirming the reliability of the approach and the precision of measurements. The different carbon isotope ratios of the products reflect the complexity of their structure and are correlated with the frequency of carbon–carbon bond forming and breaking steps on their individual biosynthetic pathways. Thus, the analysis of carbon isotopic signatures of terpenes at natural abundance can be used as a powerful tool in elucidation of associated biosynthetic mechanisms of terpene synthases and in future in vivo studies even without ‘touching’ the plant.     

Scope for use of stable carbon isotopes in discerning the incorporation of forest detritus into aquatic foodwebs

Stable isotope analysis of carbon has been proposed as a means for discerning the incorporation of terrestrial forest detritus into aquatic foodwebs, and as such, has the potential to be used as a biomonitor of the aquatic effects of riparian deforestation. A synthesis of ¹³C/¹²C data from the literature indicates, however, that the scope for successful use of carbon isotope analysis in separating allochthonous and autochthonous food provenance is much more limited than was once thought. This occurs due the overlap in carbon isotope ratios between terrestrial forest detritus and those of both lotic attached algae and lentic filamentous attached algae. Only within rockyshored, oligotrophic lakes without macrophytes, and forest-fringed estuaries and lagoons, where the carbon isotope ratios for attached algae and forest detritus are significantly different, is there any likelihood of discerning the incorporation of allochthonous carbon into aquatic foodwebs using ¹³C/¹²C values alone.     

Carbon isotope ratios of amylose,amylopectin and mutant starches

Carbon isotope ratios (expressed as δ¹³C values) were determined for various sources of starch and the starch fractions amylose and amylopectin. The δ¹³C values of amylose were consistently less negative, 0.4–2.3 ‰, than those of amylopectin in kernal starch from maize (Zea mays) and barley (Hordeum vulgare) and in tuber starch from potato (Solanum tuberosum). Kernel starch isolated from the maize mutants wx1 and ae1, with known genetic lesions in the starch biosynthetic pathway, also showed significant differences in δ¹³C values. Collectively, these results suggest that variation in carbon isotope ratios in the amylose and amylopectin components of starch may be attributed to isotopic discrimination by the enzymes involved in starch biosynthesis.     

Late Cretaceous marine arthropods relied on terrestrial organic matter as a food source: Geochemical evidence from the Coon Creek Lagerstätte in the Mississippi Embayment

The Upper Cretaceous Coon Creek Lagerstätte of Tennessee, USA, is known for its extremely well‐preserved mollusks and decapod crustaceans. However, the depositional environment of this unit, particularly its distance to the shoreline, has long been equivocal. To better constrain the coastal proximity of the Coon Creek Formation, we carried out a multiproxy geochemical analysis of fossil decapod (crab, mud shrimp) cuticle and associated sediment from the type section. Elemental analysis and Raman spectroscopy confirmed the presence of kerogenized carbon in the crabs and mud shrimp; carbon isotope (δ¹³C) analysis of bulk decapod cuticle yielded similar mean δ¹³C values for both taxa (?25.1‰ and ?26‰, respectively). Sedimentary biomarkers were composed of n‐alkanes from C₁₆ to C₃₆, with the short‐chain n‐alkanes dominating, as well as other biomarkers (pristane, phytane, hopanes). Raman spectra and biomarker thermal maturity indices suggest that the Coon Creek Formation sediments are immature, which supports retention of unaltered, biogenic isotopic signals in the fossil organic carbon remains. Using our isotopic results and published calcium carbonate δ¹³C values, we modeled carbon isotope values of carbon sources in the Coon Creek Formation, including potential marine (phytoplankton) and terrestrial (plant) dietary sources. Coon Creek Formation decapod δ¹³C values fall closer to those estimated for terrigenous plants than marine phytoplankton, indicating that these organisms were feeding primarily on terrigenous organic matter. From this model, we infer that the Coon Creek Formation experienced significant terrigenous organic matter input via a freshwater source and thus was deposited in a shallow, nearshore marine environment proximal to the shoreline. This study helps refine the paleoecology of nearshore settings in the Mississippi Embayment during the global climatic shift in the late Campanian–early Maastrichtian and demonstrates for the first time that organic δ¹³C signatures in exceptionally preserved fossil marine arthropods are a viable proxy for use in paleoenvironmental reconstructions.     

A new biosynthetic pathway of porphyrins from isopropanol

A new biosynthetic pathway, which can produce both vitamin B₁₂ and large amounts of porphyrins from isopropanol, was identified in Arthrobacter hyalinus using carbon-13 stable isotope tracer techniques and carbon-13 nuclear magnetic resonance (¹³C-NMR) spectroscopy. Studies on the incorporation of [2-¹³C]isopropanol, [1- or 2-¹³C]sodium acetate, l-[1-¹³C]glutamate, and [1-, 2-, 3-, 4-, 5-¹³C]5-aminolevulinic acid into uroporphyrinogen III showed that isopropanol was metabolized into uroporphyrinogen III through acetyl CoA and that 5-aminolevulinic acid was produced from l-glutamic acid and not via Shemin's pathway.     

Targeted distribution of photo-assimilate in Striga hermonthica (Del.) Benth parasitic on Sorghum bicolor L.

Autotrophic photosynthesis in developing plants of Striga hermonthica, a C3 plant, parasitizing Sorghum bicolor, a C4 plant, has been quantified by measuring the ¹³C/¹²C isotope ratio (δ¹³C). This parameter has been determined in cellulose and chlorophyll extracted from plants 8 weeks after emergence. It is shown that these components are both proportionately high in autotrophic carbon in the leaf material, thus confirming the previous hypothesis that St. hermonthica photo-assimilate is directed primarily towards the synthesis of new photosynthetic capacity.     

Stable Isotope Labeled n-Alkanes to Assess Digesta Passage Kinetics through the Digestive Tract of Ruminants

We describe the use of carbon stable isotope (¹³C) labeled n-alkanes as a potential internal tracer to assess passage kinetics of ingested nutrients in ruminants. Plant cuticular n-alkanes originating from intrinsically ¹³C labeled ryegrass plants were pulse dosed intraruminally in four rumen-cannulated lactating dairy cows receiving four contrasting ryegrass silage treatments that differed in nitrogen fertilization level (45 or 90 kg nitrogen ha⁻¹) and maturity (early or late). Passage kinetics through the gastrointestinal tract were derived from the δ¹³C (i.e. the ratio ¹³C:¹²C) in apparently undigested fecal material. Isotopic enrichment was observed in a wide range of long-chain n-alkanes (C₂₇–C₃₆) and passage kinetics were determined for the most abundant C₂₉, C₃₁ and C₃₃ n-alkanes, for which a sufficiently high response signal was detected by combustion isotope ratio mass spectrometry. Basal diet treatment and carbon chain length of n-alkanes did not affect fractional passage rates from the rumen (K ₁) among individual n-alkanes (3.71–3.95%/h). Peak concentration time and transit time showed a quantitatively small, significant (p≤0.002) increase with carbon chain length. K ₁ estimates were comparable to those of the ¹³C labeled digestible dry matter fraction (3.38%/h; r = 0.61 to 0.71; p≤0.012). A literature review has shown that n-alkanes are not fermented by microorganisms in the rumen and affirms no preferential depletion of ¹³C versus ¹²C. Our results suggest that ¹³C labeled n-alkanes can be used as nutrient passage tracers and support the reliability of the δ¹³C signature of digestible feed nutrients as a tool to measure nutrient-specific passage kinetics.     

Stable nitrogen and carbon isotope ratios in wild native honeybees: the influence of land use and climate

The eastern hive bee Apis cerana is a major honeybee species in Asia providing numerous ecosystem services. Understanding how much the honeybees depend on natural and human-influenced plants and landscapes in different climates is important could contribute to evaluate how wild honeybees use food resources and to measure the ecosystem services. We investigated the effects of land use and climate changes on stable nitrogen and carbon isotope ratios in wild populations of A. cerana. In populations from 139 individual sites throughout Japan, we measured nitrogen (δ¹⁵N) and carbon (δ¹³C) stable isotope ratios and analyzed the effects of land use and climate. Our results showed that forested areas and annual precipitation had significant effects on δ¹⁵N, and that paddy fields and urban areas had significant effects on δ¹³C. These results suggest that A. cerana sensibly uses available food resources in the various environments and that stable nitrogen and carbon isotope ratios clearly reflect the effects of land use and climate changes on the populations of A. cerana. Thus, stable nitrogen and carbon isotope ratios in A. cerana, which widely distributes in Asia, can be used as indicators of the environments, such as land use and climate, of an area within its foraging range.