Bioenergetic control of soil carbon dynamics across depth.

Soil carbon dynamics is strongly controlled by depth globally, with increasingly slow dynamics found at depth. The mechanistic basis remains however controversial, limiting our ability to predict carbon cycle-climate feedbacks. Here we combine radiocarbon and thermal analyses with long-term incubations in absence/presence of continuously 13C/14C-labelled plants to show that bioenergetic constraints of decomposers consistently drive the depth-dependency of soil carbon dynamics over a range of mineral reactivity contexts. The slow dynamics of subsoil carbon is tightly related to both its low energy density and high activation energy of decomposition, leading to an unfavourable 'return-on-energy-investment' for decomposers. We also observe strong acceleration of millennia-old subsoil carbon decomposition induced by roots ('rhizosphere priming'), showing that sufficient supply of energy by roots is able to alleviate the strong energy limitation of decomposition. These findings demonstrate that subsoil carbon persistence results from its poor energy quality together with the lack of energy supply by roots due to their low density at depth.

Can stable isotopes quantify soil carbon build-up from organic fertilizers?

The application of organic fertilizers (OF) can supply carbon (C) to the soil in crop fields. OF-derived C (OF-C) is often estimated using the differential method that can be biased due to indirect effects of OF on soil C. This study tested three methods to quantify OF-C: (i) the widespread differential method, (ii) the synchronic isotope method comparing plots with and without OF and (iii) the asynchronic isotope method mimicking a trial without a control plot. These methods were implemented on an Arenosol and an Andosol supplied during 13 years with slurry or compost. The results highlighted the relevance of using the synchronic isotope method, which focuses on the direct effect of OFs on the soil organic matter (without bias of vegetation change) and considers control soil's evolution. The higher the isotopic difference between soil and OF, the shorter the method implementation time needed: for an initial difference of 7.5 ‰ and 3.5 ‰, quantification is suitable after 4 and 9 years of fertilization respectively. Attention should be paid to OF-δ13C variability to guarantee the method validity. The method proved to be suitable to study the factors controlling the OF-C fate in tropical soils.

Can N2 O emissions offset the benefits from soil organic carbon storage?

To respect the Paris agreement targeting a limitation of global warming below 2°C by 2100, and possibly below 1.5°C, drastic reductions of greenhouse gas emissions are mandatory but not sufficient. Large-scale deployment of other climate mitigation strategies is also necessary. Among these, increasing soil organic carbon (SOC) stocks is an important lever because carbon in soils can be stored for long periods and land management options to achieve this already exist and have been widely tested. However, agricultural soils are also an important source of nitrous oxide (N2 O), a powerful greenhouse gas, and increasing SOC may influence N2 O emissions, likely causing an increase in many cases, thus tending to offset the climate change benefit from increased SOC storage. Here we review the main agricultural management options for increasing SOC stocks. We evaluate the amount of SOC that can be stored as well as resulting changes in N2 O emissions to better estimate the climate benefits of these management options. Based on quantitative data obtained from published meta-analyses and from our current level of understanding, we conclude that the climate mitigation induced by increased SOC storage is generally overestimated if associated N2 O emissions are not considered but, with the exception of reduced tillage, is never fully offset. Some options (e.g. biochar or non-pyrogenic C amendment application) may even decrease N2 O emissions.

Liquid chromatographic isolation of individual carbohydrates from environmental matrices for stable carbon analysis and radiocarbon dating.

Carbohydrates are among the most abundant organic molecules in both aquatic and terrestrial ecosystems; however, very few studies have addressed their isotopic signature using compound-specific isotope analysis, which provides additional information on their origin (δ13C) and fate (Δ14C). In this study, semi-preparative liquid chromatography with refractive index detection (HPLC-RI) was employed to produce pure carbohydrate targets for subsequent offline δ13C and Δ14C isotopic analysis. δ13C analysis was performed by elemental analyzer-isotope ratio mass spectrometer (EA-IRMS) whereas Δ14C analysis was performed by an innovative measurement procedure based on the direct combustion of the isolated fractions using an elemental analyzer coupled to the gas source of a mini carbon dating system (AixMICADAS). In general, four successive purifications with Na+, Ca2+, Pb2+, and Ca2+ cation-exchange columns were sufficient to produce pure carbohydrates. These carbohydrates were subsequently identified using mass spectrometry by comparing their mass spectra with those of authentic standards. The applicability of the proposed method was tested on two different environmental samples comprising marine particulate organic matter (POM) and total suspended atmospheric particles (TSP). The obtained results revealed that for the marine POM sample, the δ13C values of the individual carbohydrates ranged from -18.5 to -16.8‰, except for levoglucosan and mannosan, which presented values of -27.2 and -26.2‰, respectively. For the TSP sample, the δ13C values ranged from -26.4 to -25.0‰. The galactose and glucose Δ14C values were 19 and 43‰, respectively, for the POM sample. On the other hand, the levoglucosan radiocarbon value was 33‰ for the TSP sample. These results suggest that these carbohydrates exhibit a modern age in both of these samples. Radiocarbon HPLC collection window blanks, measured after the addition of phthalic acid (14C free blank), ranged from -988 to -986‰ for the abovementioned compounds, indicating a very small background isotopic influence from the whole purification procedure. Overall, the proposed method does not require derivatization steps, produces extremely low blanks, and may be applied to different types of environmental samples.

Including Stable Carbon Isotopes to Evaluate the Dynamics of Soil Carbon in the Land-Surface Model ORCHIDEE.

Soil organic carbon (SOC) is a crucial component of the terrestrial carbon cycle and its turnover time in models is a key source of uncertainty. Studies have highlighted the utility of δ13C measurements for benchmarking SOC turnover in global models. We used 13C as a tracer within a vertically discretized soil module of a land-surface model, Organising Carbon and Hydrology In Dynamic Ecosystems- Soil Organic Matter (ORCHIDEE-SOM). Our new module represents some of the processes that have been hypothesized to lead to a 13C enrichment with soil depth as follows: 1) the Suess effect and CO2 fertilization, 2) the relative 13C enrichment of roots compared to leaves, and 3) 13C discrimination associated with microbial activity. We tested if the upgraded soil module was able to reproduce the vertical profile of δ13C within the soil column at two temperate sites and the short-term change in the isotopic signal of soil after a shift in C3/C4 vegetation. We ran the model over Europe to test its performance at larger scale. The model was able to simulate a shift in the isotopic signal due to short-term changes in vegetation cover from C3 to C4; however, it was not able to reproduce the overall vertical profile in soil δ13C, which arises as a combination of short and long-term processes. At the European scale, the model ably reproduced soil CO2 fluxes and total SOC stock. These findings stress the importance of the long-term history of land cover for simulating vertical profiles of δ13C. This new soil module is an emerging tool for the diagnosis and improvement of global SOC models.

Atmosphere-soil carbon transfer as a function of soil depth.

The exchange of carbon between soil organic carbon (SOC) and the atmosphere affects the climate1,2 and-because of the importance of organic matter to soil fertility-agricultural productivity3. The dynamics of topsoil carbon has been relatively well quantified4, but half of the soil carbon is located in deeper soil layers (below 30 centimetres)5-7, and many questions remain regarding the exchange of this deep carbon with the atmosphere8. This knowledge gap restricts soil carbon management policies and limits global carbon models1,9,10. Here we quantify the recent incorporation of atmosphere-derived carbon atoms into whole-soil profiles, through a meta-analysis of changes in stable carbon isotope signatures at 112 grassland, forest and cropland sites, across different climatic zones, from 1965 to 2015. We find, in agreement with previous work5,6, that soil at a depth of 30-100 centimetres beneath the surface (the subsoil) contains on average 47 per cent of the topmost metre's SOC stocks. However, we show that this subsoil accounts for just 19 per cent of the SOC that has been recently incorporated (within the past 50 years) into the topmost metre. Globally, the median depth of recent carbon incorporation into mineral soil is 10 centimetres. Variations in the relative allocation of carbon to deep soil layers are better explained by the aridity index than by mean annual temperature. Land use for crops reduces the incorporation of carbon into the soil surface layer, but not into deeper layers. Our results suggest that SOC dynamics and its responses to climatic control or land use are strongly dependent on soil depth. We propose that using multilayer soil modules in global carbon models, tested with our data, could help to improve our understanding of soil-atmosphere carbon exchange.

Labelled microbial culture as a calibration medium for (13) C-isotope measurement of derivatized compounds: application to tert-butyldimethylsilyl amino acids.

RATIONALE: Compound-specific stable carbon isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is widely used in studies of environmental or biological functioning. In the case of derivatized molecules, a calibration might be required due to added non-analyte carbon and in some cases non-stoichiometric recovery by the mass spectrometer. METHODS: Two biological materials of known isotopic composition were produced by microbial cell cultures on either (13) C-labelled glucose or non-labelled glucose as sole source of carbon. Subsequent hydrolyzed amino acids were derivatized as tert-butyldimethylsilyl (tBDMSi) derivatives and analyzed by GC/C/IRMS. The (13) C-enrichment measurements were used as a direct calibration to calculate the original (13) C/(12) C ratios of individual amino acids. We tested this calibration on both known and unknown samples. RESULTS: For the main proteinogenic amino acids we could determine the number of non-analyte added carbon atoms and assess the non-stoichiometrical recovery of tBDMSi carbon atoms, due to their incomplete oxidation in the combustion step of GC/C/IRMS. The calibration enabled the determination of the natural abundances (δ(13) C values) of amino acids with an average accuracy of ±1.1 ‰. We illustrate the application of the calibration to determine the (13) C/(12) C ratios of amino acids, and the associated uncertainty, in biological and plant materials. CONCLUSIONS: The analysis of a labelled microbial cell culture offers a straightforward, rapid and reliable estimate of non-analyte carbon contribution to stable isotope composition. We recommend this method as a calibration or a control in artificial or natural (13) C-tracing experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Deep soil carbon dynamics are driven more by soil type than by climate: a worldwide meta-analysis of radiocarbon profiles.

The response of soil carbon dynamics to climate and land-use change will affect both the future climate and the quality of ecosystems. Deep soil carbon (>20 cm) is the primary component of the soil carbon pool, but the dynamics of deep soil carbon remain poorly understood. Therefore, radiocarbon activity (Δ14C), which is a function of the age of carbon, may help to understand the rates of soil carbon biodegradation and stabilization. We analyzed the published 14C contents in 122 profiles of mineral soil that were well distributed in most of the large world biomes, except for the boreal zone. With a multivariate extension of a linear mixed-effects model whose inference was based on the parallel combination of two algorithms, the expectation-maximization (EM) and the Metropolis-Hasting algorithms, we expressed soil Δ14C profiles as a four-parameter function of depth. The four-parameter model produced insightful predictions of soil Δ14C as dependent on depth, soil type, climate, vegetation, land-use and date of sampling (R2=0.68). Further analysis with the model showed that the age of topsoil carbon was primarily affected by climate and cultivation. By contrast, the age of deep soil carbon was affected more by soil taxa than by climate and thus illustrated the strong dependence of soil carbon dynamics on other pedologic traits such as clay content and mineralogy.

Measurement of δ13C values of soil amino acids by GC-C-IRMS using trimethylsilylation: a critical assessment.

In this study, we evaluated trimethylsilyl (TMS) derivatives as derivatization reagents for the compound-specific stable carbon isotope analysis of soil amino acids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). We used non-proteinogenic amino acids to show that the extraction-derivatization-analysis procedure provides a reliable method to measure δ(13)C values of amino acids extracted from soil. However, we found a number of drawbacks that significantly increase the final total uncertainty. These include the following: production of multiple peaks for each amino acid, identified as di-, tri- and tetra-TMS derivatives; a number of TMS-carbon (TMS-C) atoms added lower than the stoichiometric one, possibly due to incomplete combustion; different TMS-C δ(13)C for di-, tri- and tetra-TMS derivatives. For soil samples, only four amino acids (leucine, valine, threonine and serine) provide reliable δ(13)C values with a total average uncertainty of 1.3 ‰. We conclude that trimethylsilyl derivatives are only suitable for determining the (13)C incorporation in amino acids within experiments using (13)C-labelled tracers but cannot be applied for amino acids with natural carbon isotope abundance until the drawbacks described here are overcome and the measured total uncertainty significantly decreased.

Molecular turnover time of soil organic matter in particle-size fractions of an arable soil.

The composition and molecular residence time of soil organic matter (SOM) in four particle-size fractions (POM >200 microm, POM 63-200 microm, silt and clay) were determined using Curie-point pyrolysis/gas chromatography coupled on-line to mass spectrometry. The fractions were isolated from soils, either continuously with a C(3) wheat (soil (13)C value = -26.4 per thousand), or transferred to a C(4) maize (soil (13)C value = -20.2 per thousand) cropping system 23 years ago. Pyrograms contained up to 45 different pyrolysis peaks; 37 (ca. 85%) were identifiable compounds. Lignins and carbohydrates dominated the POM fractions, proteins were abundant, but lignin was (nearly) absent in the silt and clay fractions. The mean turnover time (MRT) for the pyrolysis products in particulate organic matter (POM) was generally <15 years (fast C pool) and 20-300 years (medium or slow C pools) in silt and clay fractions. Methylcyclopentenone (carbohydrate) in the clay fraction and benzene (mixed source) in the silt fraction exhibited the longest MRTs, 297 and 159 years, respectively. Plant-derived organic matter was not stored in soils, but was transformed to microbial remains, mainly in the form of carbohydrates and proteins and held in soil by organo-mineral interactions. Selective preservation of plant-derived OM (i.e. lignin) based on chemical recalcitrance was not observed in these arable soils. Association/presence of C with silt or clays in soils clearly increased MRT values, but in an as yet unresolved manner (i.e. 'truly' stabilized, or potentially still 'labile' but just not accessible C).

Plant host habitat and root exudates shape soil bacterial community structure.

The rhizosphere is active and dynamic in which newly generated carbon, derived from root exudates, and ancient carbon, in soil organic matter (SOM), are available for microbial growth. Stable isotope probing (SIP) was used to determine bacterial communities assimilating each carbon source in the rhizosphere of four plant species. Wheat, maize, rape and barrel clover (Medicago truncatula) were grown separately in the same soil under (13)CO(2) (99% of atom (13)C) and DNA extracted from rhizosphere soil was fractionated by isopycnic centrifugation. Bacteria-assimilating root exudates were characterized by denaturing gradient gel electrophoresis (DGGE) analysis of (13)C-DNA and root DNA, whereas those assimilating SOM were identified from (12)C-DNA. Plant species root exudates significantly shaped rhizosphere bacterial community structure. Bacteria related to Sphingobacteriales and Myxococcus assimilated root exudates in colonizing roots of all four plants, whwereas bacteria related to Sphingomonadales utilized both carbon sources, and were identified in light, heavy and root compartment DNA. Sphingomonadales were specific to monocotyledons, whereas bacteria related to Enterobacter and Rhizobiales colonized all compartments of all four plants, used both fresh and ancient carbon and were considered as generalists. There was also evidence for an indirect important impact of root exudates, through stimulation of SOM assimilation by a diverse bacterial community.

Identification of cellulolytic bacteria in soil by stable isotope probing.

Plant residues, mainly made up of cellulose, are the largest fraction of organic carbon material in terrestrial ecosystems. Soil microorganisms are mainly responsible for the transfer of this carbon to the atmosphere, but their contribution is not accurately known. The aim of the present study was to identify bacterial populations that are actively involved in cellulose degradation, using the DNA-stable isotope probing (DNA-SIP) technique. (13)C-cellulose was produced by Acetobacter xylinus and incubated in soil for 7, 14, 30 and 90 days. Total DNA was extracted from the soil, the (13)C-labelled (heavy) and unlabelled (light) DNA fractions were separated by ultracentrifugation, and the structure of active bacterial communities was analysed by bacterial-automated ribosomal intergenic spacer analysis (B-ARISA) and characterized with denaturing gradient gel electrophoresis (DGGE). Cellulose degradation was associated with significant changes in bacterial community structure issued from heavy DNA, leading to the appearance of new bands and increase in relative intensities of other bands until day 30. The majority of bands decreased in relative intensity at day 90. Sequencing and phylogenetic analysis of 10 of these bands in DGGE profiles indicated that most sequences were closely related to sequences from organisms known for their ability to degrade cellulose or to uncultured soil bacteria.

Dynamics and identification of soil microbial populations actively assimilating carbon from 13C-labelled wheat residue as estimated by DNA- and RNA-SIP techniques.

This work is the first report on the use of DNA-, RNA-SIP approaches to elucidate the dynamics and the diversity of bacterial populations actively assimilating C derived from plant residues labelled at more than 90% (13)C. Wheat-residues, were incorporated and incubated into soil microcosms for 28 days. At the end of the incubation time, no more than 55% of the total CO(2) released was (13)C-labelled, suggesting the occurrence of an important priming effect process. After 7 days, more than 30% of the whole DNA extracted were labelled, allowing an efficient separation of labelled from unlabelled DNA using density gradient centrifugation. The genetic structure of bacterial community, assessed by Automated Ribosomal Intergenic Spacer Analysis technique, was deduced from the (13)C- and (12)C-fractions of control and enriched conditions, over the time course of the experiment. Dynamics showed that wheat residues directly induced a rapid and durable stimulation of fresh organic matter (FOM) degrading populations ((13)C), while specific soil organic matter (SOM) degrading populations ((12)C) seemed to be indirectly stimulated only at the early time point (t7d). After 14 days of incubations, 16S rRNA clone libraries were elaborated on (12)C- and (13)C-RNA extracted from enriched microcosms, as well as (12)C-RNA extracted from control condition. Stimulation of the beta- and gamma-subgroups of proteobacteria, where numerous populations were previously described as r-strategists or copiotrophic organisms, was recorded in the (13)C-fraction. In the mean time, several phyla like Actinobacteria, Cyanobacteria, Candidate, Gemmatimonadetes and Planctomycetes were only present in (12)C fractions. Surprisingly, several sequences affiliated to species characterized as oligotrophic organisms were retrieved in both types of fraction. Trophic relationships between soil bacteria involved in FOM and SOM degradation were discussed on the basis of different hypotheses of Fontaine and colleagues (2003) concerning the mechanisms of the priming effect induction.

Long term changes in the distribution and delta(15)N values of individual soil amino acids in the absence of plant and fertiliser inputs.

The long-term 'biodegradation' on soil amino acids was examined in the control plots of '42 parcelles' experiment, established in 1928 at INRA, Versailles (France). None of the plots is cultivated, but is kept free of weeds, and mixed to a depth of 25 cm twice yearly. Topsoil (0-10 cm depth) samples collected in 1929, 1963 and 1997 were subjected to acid hydrolysis (6 N HCl) for comparison. The distribution and delta(15)N natural abundance of 20 individual amino acids in the soils were determined, using ion chromatography (IC) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The total N and amino acid-N (AA-N), respectively, decreased by 54 % and 73 % in the period from 1929 to 1997. The average N loss was comparable for 1929-1963 (period 1) and 1963-1997 (period 2), but AA-N loss was three times faster in the former period. This significant reduction in total AA-N content was mirrored in the individual amino acids, which decreased by 74 % +/- 1 % (ranging 58-89 %) between 1929 and 1997. The bulk delta(15)N values generally increased from 1929 to 1997, mainly associated with comparable or even higher increase of delta(15)N of the non-AA-N in the soil. The residence time (t(1/2), time in which half of N was lost from a specific soil pool) was ca. 65 +/- 5 years for the bulk soil, and comparable for periods 1 and 2. However, between periods 1 and 2 it decreased from 128 to 41 years in the non-AA pool, but increased from 59 to 92 years in the AA-N pool. Proline and amino acids that appear early in soil microbial metabolic pathways (e.g. glutamic acid, alanine, aspartic acid and valine) had relatively high delta(15)N values. Phenylalanine, threonine, glycine and leucine had relatively depleted delta(15)N values. The average delta(15)N value of the individual amino acids (IAAs) increased by 1delta unit from 1929 to 1997, associated with a similar rise from 1929 to 1963, and no change thereafter till 1997. However, the delta(15)N values of phenylalanine decreased by more than 7delta(15)N units between 1929 and 1997. The delta(15)N shift of IAAs from 1929 to 1963 and from 1929 to 1997 was not influenced by the relative amount of N remaining compared with the 1929 soil concentrations. The only exception was phenylalanine which showed decreasing delta(15)N associated with its decreasing concentration in the soil. We conclude therefore that in the absence of plant and fertiliser inputs, no change in the delta(15)N value of individual soil amino acids occurs, hence the original delta(15)N values are preserved and diagnostic information on past soil N (cycling) is retained. The exception was phenylalanine, its delta(15)N decreased with decreasing concentration from 1929 to 1997, hence it acted as a 'potential' marker for the land use changes (i.e. arable cropping to a fallow). The long term biological processing and reworking of residual amino acids resulted in a (partial) stabilisation in the soil, evidenced by reduced N loss and increased residence time of amino acid N during the period 1963-1997.

Zn speciation in the organic horizon of a contaminated soil by micro-X-ray fluorescence, micro- and powder-EXAFS spectroscopy, and isotopic dilution.

Soils that have been acutely contaminated by heavy metals show distinct characteristics, such as colonization by metal-tolerant plant species and topsoil enrichment in weakly degraded plant debris, because biodegradation processes are strongly inhibited by contamination. Such an organic topsoil, located downwind of an active zinc smelter and extremely rich in Zn (approximately 2%, dry weight), was investigated by X-ray diffraction, synchrotron-based X-ray microfluorescence, and powder- and micro-extended X-ray absorption fine structure (EXAFS) spectroscopy for Zn speciation and by isotopic dilution for Zn lability. EXAFS spectra recorded on size fractions and on selected spots of thin sections were analyzed by principal component analysis and linear combination fits. Although Zn primary minerals (franklinite, sphalerite, and willemite) are still present (approximately 15% of total Zn) in the bulk soil, Zn was found to be predominantly speciated as Zn-organic matter complexes (approximately 45%), outer-sphere complexes (approximately 20%), Zn-sorbed phosphate (approximately 10%), and Zn-sorbed iron oxyhydroxides (approximately 10%). The bioaccumulated Zn fraction is likely complexed to soil organic matter after the plants' death. The proportion of labile Zn ranges from 54 to 92%, depending on the soil fraction, in agreement with the high proportion of organically bound Zn. Despite its marked lability, Zn seems to be retained in the topsoil thanks to the huge content of organic matter, which confers to this horizon a high sorption capacity. The speciation of Zn in this organic soil horizon is compared with that found in other types of soils.

Measurement of the 13C/12C ratio of soil-plant individual sugars by gas chromatography/combustion/isotope-ratio mass spectrometry of silylated derivatives.

Carbohydrate is an important pool in the terrestrial carbon cycle. The potential offered by natural and artificial 13C-labelling techniques should therefore be applied to the investigation of the dynamics of individual sugars in soils. For this reason, we evaluated the method of 13C sugar analysis by gas chromatography/combustion/isotope-ratio mass spectrometry (GC/C/IRMS) after hydrolysis and direct trimethylsilylation. Trimethylsilylation involved the addition of several carbon atoms per sugar. These atoms have to be taken into account in the estimation of the carbon isotope ratio. The analysis of standard and natural pentoses and hexoses of known 13C enrichments revealed that the number of analysed added carbon atoms was less than expected from stoichiometry. This was attributed to incomplete derivatization and/or incomplete oxidation of methylsilyl carbon before IRMS. Using a calibration of the number of analysed added carbon atoms, the isotope excess of enriched samples could be determined with a relative error close to 5%. Concerning the determination of natural abundances by GC/C/IRMS, we could measure the delta 13C of standard C3- and C4-derived sugars with an accuracy of +/-1.5 per thousand using the previous calibration. We were able to apply this technique to plant-soil systems labelled by pulse-chase of 13CO2, revealing the nature and dynamics of sugars in the plant rhizosphere.

Earthworm diet related to soil organic matter dynamics through 13C measurements.

The ability of earthworms to assimilate soil organic pools of different ages was investigated in field conditions through natural 13C labelling. Provided that 13C natural abundance of earthworm tissues is determined by their diet, the assimilation of soil organic matter by earthworms was estimated by measuring 13C/12C ratio of tissues of earthworms sampled in soils containing organic pools with differing 13C/12C ratios. Earthworms were collected in two locations where the vegetation shifted from a C3 to a C4 type (France), or from a C4 to a C3 type (Ivory Coast) 3 to 20 years ago. The results show that irrespective of their ecology (litter or soil-feeding), earthworms mainly feed on recent soil organic pools and assimilate soil organic matter with the same age distribution as the overall decomposers in the soil.