Influence of recent vegetation on labile and recalcitrant carbon soil pools in central Queensland, Australia: evidence from thermal analysis-quadrupole mass spectrometry-isotope ratio mass spectrometry

The effect of a recent vegetation change (<100 years) from C(4) grassland to C(3) woodland in central Queensland, Australia, on soil organic matter (SOM) composition and SOM dynamics has been investigated using a novel coupled thermogravimetry-differential scanning calorimetry-quadrupole.mass spectrometry-isotope ratio mass spectrometry (TG-DSC-QMS-IRMS) system. TG-DSC-QMS-IRMS distinguishes the C isotope composition of discrete SOM pools, showing changes in labile, recalcitrant and refractory carbon in the bulk soil and particle size fractions which track the vegetation changes. Analysis of evolved gases (by QMS) from thermal decomposition, rather than observed weight loss, proved essential in determining the temperature at which SOM decomposes, because smectite and kaolinite clays contribute to observed weight losses. The delta(13)C analyses of the CO(2) evolved at different temperatures for bulk soil and particle size-separates showed that most of the labile SOM under the more recent woody vegetation was C(3)-derived carbon whereas the delta(13)C values in the recalcitrant SOM showed greater C(4) contributions. This indicated a shift from grass (C(4))- to tree (C(3))-derived carbon in the woodland, which was also supported by the two-phase (13)C enrichment with depth, i.e. C(3) vegetation dominated the top soil (0-10 cm), but the C(4) contribution increased with depth (more gradual). This is perturbed by the inclusion of charcoal from forest fires ((14)C age incursions) and by the deep incorporation of C(3) carbon due to root penetration.     

Evaluating high time-resolved changes in carbon isotope ratio of respired CO2 by a rapid in-tube incubation technique

Recent insights into fractionation during dark respiration and rapid dynamics in isotope signatures of leaf- and ecosystem-respired CO(2) indicate the need for new methods for high time-resolved measurements of the isotopic signature of respired CO(2) (delta(13)C(res)). We present a rapid and simple method to analyse delta(13)C(res) using an in-tube incubation technique and an autosampler for small septum-capped vials. The effect of storage on the delta(18)O and delta(13)C ratios of ambient CO(2) concentrations was tested with different humidity and temperatures. delta(13)C ratios remained stable over 72 h, whereas delta(18)O ratios decreased after 24 h. Storage at 4 degrees C improved the storage time for delta(18)O. Leaves or leaf discs were incubated in the vials, flushed with CO(2)-free air and respired CO(2) was automatically sampled within 5 min on a microGas autosampler interfaced to a GV-Isoprime isotope ratio mass spectrometer. Results were validated by simultaneous on-line gas-exchange measurements of delta(13)C(res) of attached leaves. This method was used to evaluate the short-term (5-60 min) and diurnal dynamics of delta(13)C(res) in an evergreen oak (Quercus ilex) and a herb (Tolpis barbata). An immediate depletion of 2-4 per thousand from the initial delta(13)C(res) value occurred during the first 30 min of darkening. Q. ilex exhibited further a substantial diurnal enrichment in delta(13)C(res) of 8 per thousand, followed by a progressive depletion during the night. In contrast, T. barbata did not exhibit a distinct diurnal pattern. This is in accordance with recent theory on fractionation in metabolic pathways and may be related to the different utilisation of the respiratory substrate in the fast-growing herb and the evergreen oak. These data indicate substantial and rapid dynamics (within minutes to hours) in delta(13)C(res), which differed between species and probably the growth status of the plant. The in-tube incubation method enables both high time-resolved analysis and extensive sampling across different organs, species and functional types.     

Measuring the 13C content of soil-respired CO2 using a novel open chamber system

Carbon dioxide respired by soils comes from both autotrophic and heterotrophic respiration. 13C has proved useful in differentiating between these two sources, but requires the collection and analysis of CO2 efflux from the soil. We have developed a novel, open chamber system which allows for the accurate and precise quantification of the delta13C of soil-respired CO2. The chamber was tested using online analyses, by configuring a GasBench II and continuous flow isotope ratio mass spectrometer, to measure the delta13C of the chamber air every 120 s. CO2 of known delta13C value was passed through a column of sand and, using the chamber, the CO2 concentration stabilized rapidly, but 60 min was required before the delta13C value was stable and identical to the cylinder gas (-33.3 per thousand). Changing the chamber CO2 concentration between 200 and 900 micromol.mol(-1) did not affect the measured delta13C of the efflux. Measuring the delta13C of the CO2 efflux from soil cores in the laboratory gave a spread of +/-2 per thousand, attributed to heterogeneity in the soil organic matter and roots. Lateral air movement through dry sand led to a change in the delta13C of the surface efflux of up to 8 per thousand. The chamber was used to measure small transient changes (+/-2 per thousand) in the delta13C of soil-respired CO2 from a peaty podzol after gradual heating from 12 to 35 degrees C over 12 h. Finally, soil-respired CO2 was partitioned in a labelling study and the contribution of autotrophic and heterotrophic respiration to the total efflux determined. Potential applications for the chamber in the study of soil respiration are discussed.     

(13)C/(12)C isotope labelling to study leaf carbon respiration and allocation in twigs of field-grown beech trees

In situ (13)C/(12)C isotopic labelling was conducted in field-grown beech (Fagus sylvatica) twigs to study carbon respiration and allocation. This was achieved with a portable gas-exchange open system coupled to an external chamber. This method allowed us to subject leafy twigs to CO(2) with a constant carbon isotope composition (delta(13)C of -51.2 per thousand) in an open system in the field. The labelling was done during the whole light period at two different dates (in June 2002 and October 2003). The delta(13)C values of respiratory metabolites and CO(2) that is subsequently respired during the night were measured. It was found that night-respired CO(2) is not completely labelled (only ca. 58% and 27% of new carbon is found in respired CO(2) immediately after the labelling in June 2002 and October 2003, respectively) and the labelling level progressively disappeared during the next day. It is concluded that the carbon respired by beech leaves after illumination was supplied by a mixture of carbon sources in which current carbohydrates were not the only contributors. In addition, as has been found in herbaceous plants, isotopic data before labelling showed that carbon isotope discrimination favoring the (13)C isotope occurred during the night respiration of beech leaves.     

Isotopic composition of inorganic carbon as an indicator of benzoate degradation by pseudomonas putida: temperature, growth rate and pH effects

Degradation experiments of benzoate by Pseudomonas putida resulted in enzymatic carbon isotope fractionations. However, isotopic temperature effects between experiments at 20 and 30 degrees C were minor. Averages of the last three values of the CO(2) isotopic composition (delta(13)C(CO2(g))) were more negative than the initial benzoate delta(13)C value (-26.2 per thousand Vienna Pee Dee Belenite (VPDB)) by 3.8, 3.4 and 3.2 per thousand at 20, 25 and 30 degrees C, respectively. Although the maximum isotopic temperature difference found was only 0.6 per thousand, more extreme temperature variations may cause larger isotope effects. In order to understand the isotope effects on the total inorganic carbon (TIC), a better measure is to calculate the proportions of the inorganic carbon species (CO(2)(g), CO(2)(aq) and HCO(3)(-)) and to determine their cumulative delta(13)C(TIC). In all three experiments delta(13)C(TIC) was more positive than the initial isotopic composition of the benzoate at a pH of 7. This suggests an uptake of (12)C in the biomass in order to match the carbon balance of these closed system experiments.     

Assessing the use of delta(13)C natural abundance in separation of root and microbial respiration in a Danish beech (Fagus sylvatica L.) forest

Our understanding of forest biosphere-atmosphere interactions is fundamental for predicting forest ecosystem responses to climatic changes. Currently, however, our knowledge is incomplete partly due to inability to separate the major components of soil CO(2) effluxes, viz. root respiration, microbial decomposition of soil organic matter and microbial decomposition of litter material. In this study we examined whether the delta(13)C characteristics of solid organic matter and respired CO(2) from different soil-C components and root respiration in a Danish beech forest were useful to provide information on the root respiration contribution to total CO(2) effluxes. The delta(13)C isotopic analyses of CO(2) were performed using a FinniganMAT Delta(PLUS) isotope-ratio mass spectrometer coupled in continuous flow mode to a trace gas preparation-concentration unit (PreCon). Gas samples in 2-mL crimp seal vials were analysed in a fully automatic mode with an experimental standard error +/-0.11 per thousand. We observed that the CO(2) derived from root-free mineral soil horizons (A, B(W)) was more enriched in (13)C (delta(13)C range -21.6 to -21.2 per thousand ) compared with CO(2) derived from root-free humus layers (delta(13)C range -23.6 to -23.4 per thousand ). The CO(2) evolved from root respiration in isolated young beech plants revealed a value intermediate between those for the soil humus and mineral horizons, delta(13)C(root) = -22.2 per thousand, but was associated with great variability (SE +/- 1.0 per thousand ) due to plant-specific differences. delta(13)C of CO(2) from in situ below-ground respiration averaged -22.8 per thousand, intermediate between the values for the humus layer and root respiration, but variability was great (SE +/- 0.4 per thousand ) due to pronounced spatial patterns. Overall, we were unable to statistically separate the CO(2) of root respiration vs. soil organic matter decomposition based solely on delta(13)C signatures, yet the trend in the data suggests that root respiration contributed approximately 43% to total respiration. The vertical gradient in delta(13)C, however, might be a useful tool in partitioning respiration in different soil layers. The experiment also showed an unexpected (13)C-enrichment of CO(2) (>3.5 per thousand ) compared with the total-C signatures in the individual soil-C components. This may suggest that analyses of bulk samples are not representative for the C-pools actively undergoing decomposition.     

Evolution of the delta13C signature related to total carbon contents and carbon decomposition rate constants in a soil profile under grassland

Evolution of the total carbon (C) content and the (13)C enrichment (delta(13)C signature) of soil organic matter (SOM) with increasing depth in a soil profile under permanent grassland (C(3) vegetation) were investigated. The relationship between the total C content and the delta(13)C signature at different depths in the upper 30 cm of the soil profile could be well fitted by the Rayleigh equation (y = -29.8 - 2.3x, R(2) = 0.95, p < 0.001), describing the enrichment in (13)C as resulting from isotopic fractionation associated with C mineralization (isotope enrichment factor epsilon = -2.3 per thousand). Potential C dynamics of SOM in four depth intervals of the profile (0-10, 10-20, 20-30 and 30-40 cm depth) were investigated through an incubation study. The C decomposition rate constants decreased with increasing sampling depth from 0.0479 yr(-1) (0-10 cm sampling depth) to 0.0256 yr(-1) (30-40 cm sampling depth) and were highly correlated (y = 0.02 + 0.13x, R(2) = 0.93, p < 0.05) with the corresponding deltadelta(13)C values (average change of the delta(13)C signature per depth increment). These results suggest that changes of the delta(13)C signature of SOM in undisturbed soil profiles under continuous C(3) vegetation may serve as an indicator of the variation of SOM quality with increasing depth.     

Carbon-isotopic composition of soil-respired carbon dioxide in static closed chambers at equilibrium

The carbon-isotopic composition (delta13C) of soil-respired CO2 has been employed to evaluate soil carbon-cycling processes and the contribution of soil CO2 emissions to canopy and tropospheric air. These evaluations can be successful only when accurate isotope values of soil-respired CO2 are available. Here, we tested the robustness of delta13C values of soil-respired CO2 obtained after long incubations in static closed chambers that were initially flushed with soil air. The rationale of this approach is that the equilibrium carbon-isotope values of chamber-headspace CO2 are theoretically equal to those of CO2 produced within the soil. Static closed chambers were installed in replicated grass monocultures, and measurements of headspace CO2 concentrations and delta13C values were performed at regular time intervals for 24 h in July 2005. The results revealed no significant effects of grass species on headspace CO2 concentrations or delta13C values (repeated measures analysis of variance (ANOVA), P>0.1). As predicted by theory, isotope values asymptotically approached equilibrium conditions, which in our experimental setting occurred after 10 h. This good match between model predictions and our results suggests that an accurate determination of delta13C values of CO2 produced within soils is obtained through the isotopic measurement of chamber-headspace CO2 once equilibrium conditions have been reached with the underlying soils. An additional advantage of this approach is that only one sample per chamber is required, which, combined with the low uncertainties of these measurements, facilitates the investigation of the spatial (landscape) variability of soil-respired CO2.     

Use of 13C to monitor soil organic matter transformations caused by a simulated forest fire

Soil organic matter (SOM) transformations caused by heating were analyzed using the stable carbon isotope (13)C as a tracer to follow C mineralization dynamics and C transfers between different organic compartments. A (13)C-labelled soil, obtained by incorporation of (13)C-enriched Lolium perenne phytomass into a pine forest soil, was heated for 10 min at 385 degrees C to reproduce conditions typical of a forest fire and changes in total C content, potential C mineralization activity and C distribution between the different soil organic fractions were determined. Changes caused by heating on the potential soil C mineralization, determined by laboratory aerobic incubation, reveal alterations to the SOM biodegradability; some stabilized SOM showed an increase in biodegradability, whereas less stabilized SOM became more resistant to microorganisms. Chemical fractionations of SOM allowed us to monitor changes in its composition. As a consequence of heating, the less polymerized humic fractions were the most strongly affected, with the total disappearance of fulvic acids. A significant increase in the quantity and degree of polymerization of the humic acids at the expense of other more (13)C-enriched substances was also found. Finally, a large decrease in humin was observed, its solubilizable part disappearing completely, probably as a consequence of the incorporation of the byproducts into the free organic matter fraction.     

A rapid and precise technique for measuring delta(13)C-CO(2) and delta(18)O-CO(2) ratios at ambient CO(2) concentrations for biological applications and the influence of container type and storage time on the sample isotope ratios

A simple modification to a commercially available gas chromatograph isotope ratio mass spectrometer (GC/IRMS) allows rapid and precise determination of the stable isotopes ((13)C and (18)O) of CO(2) at ambient CO(2) concentrations. A sample loop was inserted downstream of the GC injection port and used to introduce small volumes of air samples into the GC/IRMS. This procedure does not require a cryofocusing step and significantly reduces the analysis time. The precisions for delta(13)C and delta(18)O of CO(2) at ambient concentration were +/-0.164 and +/-0.247 per thousand, respectively. This modified GC/IRMS was used to test the effects of storage on the (18)O and (13)C isotopic ratios of CO(2) at ambient concentrations in four container types. On average, the change in the (13)C-CO(2) and (18)O-CO(2) ratios of samples after one week of storage in glass vials equipped with butyl rubber stoppers (Bellco Glass Inc.) were depleted by 0.12 and by 0.20 per thousand, respectively. The (13)C ratios in aluminum canisters (Scotty II and IV, Scott Specialty Gasses) after one month of storage were depleted, on average, by 0.73 and 2.04 per thousand, respectively, while the (18)O ratios were depleted by 0.38 and 1.20 per thousand for the Scotty II and IV, respectively. After a month of storage in electropolished containers (Summa canisters, Biospheric Research Corporation), the (13)C-CO(2) and (18)O-CO(2) ratios were depleted, on average, by 0.26 and enriched by 0.30 per thousand, respectively, close to the precision of measurements. Samples were collected at a mature hardwood forest for CO(2) concentration determination and isotopic analysis. A comparison of CO(2) concentrations determined with an infrared gas analyzer and from sample voltages, determined on the GC/IRMS concurrent with the isotopic analysis, indicated that CO(2) concentrations can be determined reliably with the GC/IRMS technique. The (13)C and (18)O ratios of nighttime ecosystem-respired CO(2), determined from the intercept of Keeling plots, were -26.11 per thousand (V-PDB) and -8.81 per thousand (V-PDB-CO(2)), respectively.     

Characterization of soil organic matter fractions from grassland and cultivated soils via C content and delta13C signature

Variations in (13)C natural abundance and distribution of total C among five size and density fractions of soil organic matter, water soluble organic C (WSOC) and microbial biomass C (MBC) were investigated in the upper layer (0-20 cm) of a continuous grassland soil (CG, C(3) vegetation), a C(3)-humus soil converted to continuous maize cultivation (CM, C(4) vegetation) and a C(3)-humus soil converted to a rotation of maize cultivation and grassland (R). The amounts of WSOC and MBC were both significantly larger in the CG than in the CM and the R. In the three soils, WSOC was depleted while MBC was enriched in (13)C as compared with whole soil C. The relative contributions to the total C content of C stored in the macro-organic matter and in the size fraction 50-150 microm decreased with decreasing total C contents in the order CG > R > CM, while the relative contribution of C associated with the clay- and silt-sized fraction <50 microm increased. This reflects a greater stability and physical protection against microbial degradation associated with soil disruption (tillage) of the clay- and silt-associated organic C, in relation to the organic C in larger size fractions. The size and density fractions from the CG soil showed significant differences in (13)C enrichment, indicating different degrees of microbial degradation and stability of soil organic C associated with physically different soil organic matter (SOM) fractions. Delta(13)C analysis of the size and density fractions from CM and R soils reflected a decreasing turnover rate of soil organic C with increasing density among the macro-organic matter fractions and with decreasing particle size.     

Site-specific carbon isotope analysis of aromatic carboxylic acids by elemental analysis/pyrolysis/isotope ratio mass spectrometry

Site-specific carbon isotope composition of organic compounds can provide useful information on their origin and history in natural environments. Site-specific isotope analyses of small amounts of organic compounds (sub-nanomolar level), such as short-chain carboxylic acids and amino acid analogues, have been performed using gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/pyrolysis/IRMS). These analyses were previously limited to volatile compounds. In this study, site-specific carbon isotope analysis has been developed for non-volatile aromatic carboxylic acids at sub-micromolar level by decarboxylation using a continuous flow elemental analysis (EA)/pyrolysis/IRMS technique. Benzoic acid, 2-naphthylacetic acid and 1-pyrenecarboxylic acid were pyrolyzed at 500-1000 degrees C by EA/pyrolysis/IRMS to produce CO2 for delta13C measurement of the carboxyl group. These three aromatic acids were most efficiently pyrolyzed at 750 degrees C. Conventional sealed-tube pyrolysis was also conducted for comparison. The delta13C values of CO2 generated by the continuous flow technique were within 1.0 per thousand of those performed by the conventional technique, indicating that the new continuous flow technique can accurately analyze the carbon isotopic composition of the carboxyl group in aromatic carboxylic acids. The new continuous flow technique is simple, rapid and uses small sample sizes, so this technique will be useful for characterizing the isotopic signature of carboxyl groups in organic compounds.     

Automated analysis of 13C/12C ratios in CO2 and dissolved inorganic carbon for ecological and environmental applications

Stable carbon isotope ratios (13C/12C) are a valuable tool for studying a wide range of environmental processes, including carbon cycling and subsurface microbial activity. Recent advances in automated analysis provide the opportunity to increase greatly the ease and consistency of isotopic analysis. This study evaluated an automated headspace sampler linked to a commercially available CO2 preconcentration system and continuous flow isotope ratio mass spectrometer. Field sampling and analysis methods are illustrated for delta13C of soil respired CO2, from both tracer and natural abundance experiments, and dissolved inorganic carbon from contaminated groundwater. The automated system demonstrated accuracy, precision, and linearity, with standard errors below 0.1 per thousand for replicate gas standards run at concentrations varying five-fold. It measured 40 samples per 10-hour run, with concentrations ranging from ppb to percentage levels. In the field, gas samples were injected into nitrogen-filled autosampler vials, thereby allowing use of small sample volumes, control of analyte concentration, and direct analysis by the automated system with no further preparation. A significant linear relationship between standard concentrations and peak area allows for accurate estimates of sample CO2 concentration from the mass spectrometric data. The ability to analyze multiple small-volume samples with minimal off-line preparation should enhance the application of isotopes to well-replicated field experiments for process-level studies and spatial and temporal scaling.     

Quantification of priming and CO2 respiration sources following slurry-C incorporation into two grassland soils with different C content

The fate of incorporated slurry-C was examined in a laboratory experiment using two UK grassland soils, i.e. a Pelostagnogley (5.1 %C) and a Brown Earth (2.3 %C). C3 and C4 slurries were incorporated into these two wet-sieved (C3) soils (from 4-10 cm depth). Gas samples were collected 0.2, 1, 2, 3, 4, 6, 9, 20, 30 and 40 days after slurry application and analyzed for CO2 concentration and delta13C content. Slurry incorporation into the soil strongly increased soil CO2 respiration compared with the unamended soil. Total (40 day) cumulative CO2 flux was higher for the Pelostagnogley than the Brown Earth. The 13C natural abundance tracer technique enabled quantification of the sources of respired CO2 and priming effects (days 0-9). Proportionally more slurry-derived C was respired from the Pelostagnogley (46%) than the Brown Earth (36%). The incorporated slurry-C was lost twice as fast as the native soil C in both soils. Slurry incorporation induced a priming effect, i.e. additional release of soil-derived C, most pronounced in the Pelostagnogley (highest C content). The majority of respired soil-derived C (>70%) was primed C. The study indicated that potential reductions in ammonia volatilisation following slurry injection to grasslands might be negated by enhanced loss of primed soil C (i.e. pollution swapping).     

Post-photosynthetic fractionation of stable carbon isotopes between plant organs--a widespread phenomenon

Discrimination against 13C during photosynthesis is a well-characterised phenomenon. It leaves behind distinct signatures in organic matter of plants and in the atmosphere. The former is depleted in 13C, the latter is enriched during periods of preponderant photosynthetic activity of terrestrial ecosystems. The intra-annual cycle and latitudinal gradient in atmospheric 13C resulting from photosynthetic and respiratory activities of terrestrial plants have been exploited for the reconstruction of sources and sinks through deconvolution by inverse modelling. Here, we compile evidence for widespread post-photosynthetic fractionation that further modifies the isotopic signatures of individual plant organs and consequently leads to consistent differences in delta13C between plant organs. Leaves were on average 0.96 per thousand and 1.91 per thousand more depleted than roots and woody stems, respectively. This phenomenon is relevant if the isotopic signature of CO2-exchange fluxes at the ecosystem level is used for the reconstruction of individual sources and sinks. It may also modify the parameterization of inverse modelling approaches if it leads to different isotopic signatures of organic matter with different residence times within the ecosystems and to a respiratory contribution to the average difference between the isotopic composition of plant organic matter and the atmosphere. We discuss the main hypotheses that can explain the observed inter-organ differences in delta13C.     

Collection and storage of CO2 for 13C analysis: An application to separate soil CO2 efflux into root- and soil-derived components

Soil surface CO2 efflux is comprised of CO2 from (i) root respiration and rhizosphere microbes and (ii) heterotrophic respiration from the breakdown of soil organic matter (SOM). This efflux may be partitioned between these sources using delta13C measurements. To achieve this, continuous flow isotope ratio mass spectrometry can be used and, in conjunction with 10 mL septum-capped vials, large numbers of samples may be analysed using a Finnigan MAT Delta(plus)XP interfaced to a Gas Bench II. Here we describe a number of advances to facilitate such work, including: (i) a technique for monitoring mass spectrometer performance, (ii) improvements to sample storage, and (iii) a gas-handling system for incubating and sampling the CO2 derived from roots and soils. Mass spectrometer performance was monitored using an automated refillable vial. Compressed air analysed with this system had mean delta13C of -9.61 +/- 0.16 per thousand (+/- 1sigma, n = 28) collected over four runs. Heating the butyl rubber septa used to seal the vials at 105 degrees C for 12 h improved the sample storage. After air transportation over 12 days, the isotope composition of the CO2 at ambient concentrations was unchanged (before: -35.2 +/- 0.10 per thousand, n = 4; after: -35.3 +/- 0.10 per thousand, n = 15); without heat treatment of the septa the CO2 became slightly enriched (-35.0 +/- 0.14 per thousand, n = 15). The linearity of the Gas Bench II was found to decline above 8000 micromol CO2 mol(-1). To stay within a linear range and to allow the incubation of soil and root material we describe a gas-handling system based around a peristaltic pump. Finally, we demonstrate these methods by growing a C-4 grass (Guinea grass, Panicum maximum Jacq.) in a C-3 soil. Root respiration was found to contribute between 5 and 22% to the soil surface CO2 efflux. These methodologies will facilitate experiments aimed at measuring the isotopic composition of soil-derived CO2 across a range of ecological applications.     

Discrimination against 13C during degradation of simple and complex substrates by two white rot fungi

Changes in isotopic 13C signatures of CO2-C evolved during decomposition of a sugar (glucose), a fatty acid (palmitic acid), a protein (albumin), a structural biopolymer (lignin) and bulk plant tissue (aerial shoots from Lolium perenne) were monitored over a period of 76 days. All materials were sterilized and inoculated with either of two different species of white rot fungi, Phanerochaete chrysosporium or Coriolus versicolor, and incubated in sealed bottles at 28 degrees C. The CO2 concentration in the jars was periodically determined using an infrared gas analyzer and its isotopic (13C) signature was assessed using a trace gas (ANCA TGII) module coupled to an isotope ratio mass spectrometer (IRMS, Europa 20-20). L. perenne material inoculated with C. versicolor showed the highest C mineralization activity with approximately 70% of total C evolved as CO2 after 76 days of incubation, followed by glucose. Substrates inoculated with C. versicolor generally decomposed faster than when degraded by P. chrysosporium, except for lignin, where no significant differences between the two fungi types were found and CO2-C released was less than 2% of the initial C. Considerable 13C isotopic fractionation during the degradation of plant tissue and of pure biochemical compounds was revealed as well as progressive shifts in cumulative CO2-13C isotopic signatures over time. During the first stages of decomposition, the CO2-C released was usually depleted in 13C as compared with the initial solid substrate, but with ongoing decomposition the CO2-C evolved became progressively more enriched in 13C. P. chrysosporium usually showed a slightly higher 13C fractionation than C. versicolor during the first decomposition phase. At posterior decomposition stages isotopic discrimination was often stronger by C. versicolor. These findings on isotopic 13C discrimination during microbial degradation both of simple biochemical compounds and of complex vegetal tissue confirmed not only the existence of significant 13C isotopic fractionation during plant residue decomposition, but also the existence of non-random isotopic distribution within substrates. They also demonstrated the ability of microorganisms to selectively discriminate against 13C even when degrading an isolated simple substrate.     

Effects of temperature on isotopic enrichment in Daphnia magna: implications for aquatic food-web studies

Laboratory experiments were conducted with Daphnia magna and Hyalella sp. grown on a single food source of known isotopic composition at a range of temperatures spanning the physiological optima for each species. Daphnia raised at 26.5 degrees C were enriched in delta(13)C and delta(15)N by 3.1 and 2.8 per thousand, respectively, relative to diet. Daphnia raised at 12.8 degrees C were enriched 1.7 and 5.0 per thousand in delta(13)C and delta(15)N, respectively. Results imply a significant negative relationship between the delta(13)C and delta(15)N of primary consumers when a temperature gradient exists. Similar responses were observed for Hyalella. Results indicate a general increase in delta(13)C enrichment and decrease in delta(15)N enrichment as temperature rises. Deviations from the commonly applied isotopic enrichment values used in aquatic ecology were attributed to changes in temperature-mediated physiological rates. Field data from a variety of sources also showed a general trend toward delta(13)C enrichment with increasing temperature in marine and lacustrine zooplankton. Multivariate regression models demonstrated that, in oligotrophic and mesotrophic lakes, zooplankton delta(13)C was related to lake-specific POM delta(13)C, lake surface temperature and latitude. Temperature-dependent isotopic separation (enrichment) between predator and prey should be taken into consideration when interpreting the significance of isotopic differences within and among aquatic organisms and ecosystems, and when assigning organisms to food-web positions on the basis of observed isotope values.     

Temperature dependencies of high-temperature reduction on conversion products and their isotopic signatures

High-temperature reduction (HTR) is widely used for oxygen and hydrogen isotope determination. Decomposition of cellulose, sucrose and polyethylene foil by HTR is quantitative for temperatures around 1450 degrees C. For lower reaction temperature production of CO(2), water and the deposition of carbon inside the reactor are significant and thus the element of interest for isotopic analysis is split into different pools, leading to isotope fractionation. After reduction of cellulose or sucrose at 1125 degrees C less than 60% of the oxygen is found as CO, which is monitored with the isotope ratio mass spectrometer to determine the delta(18)O value. The remaining oxygen is unevenly distributed between CO(2) and H(2)O, preferentially as CO(2). Raising the reaction temperature to 1425 degrees C yields almost quantitative conversion of oxygen into CO and results in a 3 per thousand more positive delta(18)O value. Similarly, only 40-50% of the carbon of cellulose and sucrose is transformed into CO in the HTR reactor at 1125 degrees C. This is far from the stoichiometric expected value of 83% for quantitative carbon transfer for cellulose and 92% for sucrose. Of the carbon 40-50% is deposited in the reactor and the remainder can be found as CO(2). Based on the comparison of carbon isotope results from HTR and those obtained from combustion, we hypothesize that CO produced during the HTR originates partly from sample carbon and glassy carbon. A combined combustion and HTR carbon isotope determination may provide an insight into the intramolecular carbon distribution of organic substances. These results suggest that HTR should be carried out at temperatures above 1450 degrees C to make sure that fractionations associated with the reduction process are minimal. If this is not possible frequent calibration is required using reference materials of the same structure as the sample.     

Three sources of CO2 efflux from soil partitioned by 13C natural abundance in an incubation study

This study describes a novel approach to separate three soil carbon (C) sources by one tracer method (here 13C natural abundance). The approach is based on the combination of C3 and C4 sources in different treatments, identical decomposition of C3 and C4 substances in soil, and subsequent calculation of their contribution to the total CO2 efflux. We used the temporal dynamics of the CO2 efflux from a C3 grassland soil amended with added C3 or C4 slurry and/or C3 or C4 sugar to estimate contributions of three separate C sources: native soil organic matter (SOM), slurry and sugar, to CO2 efflux. Soil with slurry and/or sugar was incubated under controlled conditions, and concentration and delta13C values of evolved CO2 were measured over a 2-week period. The main assumption needed for separation of three C sources in CO2 efflux, i.e. identical decomposition of applied C3 and C4 sugars in soil, was investigated and proven. The relative contribution to the CO2 efflux increased, but its duration decreased with an increased microbial availability of the C source, i.e. sugar > slurry > SOM. The microorganisms used the C sources according to their availability. The contribution of sugar to the CO2 efflux was finished after 2-4 days. Separation of three CO2 sources and comparison of CO2 from different treatments tracing the changes of SOM and slurry decomposition induced by addition of sugar were investigated. During the sugar decomposition (the first 2-4 days), the SOM decomposition strongly decreased. At the same time the contribution of slurry-C to CO2 increased. The shortcomings and limitations as well as possible future applications of the suggested method including FACE (Free Air Carbon dioxide Enrichments) and continuous labelling experiments are discussed.