Evidence of old soil carbon in grass biosilica particles – Evidência inequívoca de carbono antigo do solo em partículas de biossílica de gramíneas

Evidência inequívoca de carbono antigo do solo em partículas de biossílica de gramíneas

Resumo: Partículas de biossílica vegetal (fitólitos) contêm pequenas quantidades de carbono chamadas phytC. Com base nas suposições de que o phytC é de origem fotossintética e um sistema fechado, foram feitas recentemente alegações de que os fitólitos de várias espécies monocotiledôneas importantes para a agricultura desempenham um papel significativo no sequestro de CO2 atmosférico. No entanto, datas anômalas de radiocarbono phytC (14C) sugeriram contribuições de uma fonte não fotossintética para o phytC. Aqui abordamos esta hipótese de fonte não fotossintética usando medições isotópicas comparativas (14C e δ13C) de fitC, tecidos vegetais, CO2 atmosférico e matéria orgânica do solo. Métodos de última geração garantiram a pureza do fitólito, enquanto a combustão sequencial passo a passo revelou propriedades complexas de decomposição químico-térmica do phytC. Embora a fotossíntese seja a principal fonte de carbono no tecido vegetal, descobriu-se que o phytC é parcialmente derivado do carbono do solo, que pode ter vários milhares de anos. O fato de o phytC não ser constituído exclusivamente de C fotossintético limita a utilidade do phytC como ferramenta de datação ou como um sumidouro significativo de CO2 atmosférico. Além disso, são necessários mais experimentos para investigar como o C derivado da SOM é acessível às raízes e se acumula na biossílica vegetal, para uma melhor compreensão dos processos mecanísticos subjacentes ao processo de biomineralização do silício em plantas superiores.

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Plant biosilica particles (phytoliths) contain small amounts of carbon called phytC. Based on the assumptions that phytC is of photosynthetic origin and a closed system, claims were recently made that phytoliths from several agriculturally important monocotyledonous species play a significant role in atmospheric CO2 sequestration. However, anomalous phytC radiocarbon (14C) dates suggested contributions from a non-photosynthetic source to phytC. Here we address this non-photosynthetic source hypothesis using comparative isotopic measurements (14C and δ13C) of phytC, plant tissues, atmospheric CO2, and soil organic matter. State-of-the-art methods assured phytolith purity, while sequential stepwise-combustion revealed complex chemical-thermal decomposability properties of phytC. Although photosynthesis is the main source of carbon in plant tissue, it was found that phytC is partially derived from soil carbon that can be several thousand years old. The fact that phytC is not uniquely constituted of photosynthetic C limits the usefulness of phytC either as a dating tool or as a significant sink of atmospheric CO2. It additionally calls for further experiments to investigate how SOM-derived C is accessible to roots and accumulates in plant biosilica, for a better understanding of the mechanistic processes underlying the silicon biomineralization process in higher plants.

$Figure 2. Above-ground C manipulation procedure. (a) Averaged Fm 14 C values versus averaged phytC yields (or concentration in % of phytoliths). Constant solid lines correspond to the averaged Fm 14 C values obtained for stems and leaves (SL) of origin and the oldest extracted SOM fraction. (b) Oldest SOM-derived C contribution to phytC calculated using the mixing equation (Eq. 1) presented in the text expressing the 14 C signature of phytC as the result of mixing between the C derived from plant photosynthesis and the C derived from the oldest extracted SOM fraction. Phytolith samples are labeled according to the extraction protocol (1a, 1b, 2a, 2b described in caption and in the text) used and the laboratory of extraction (UCI, CEREGE, LacCore and SSAL).$

$Figure 3. Above-ground C manipulation experiment. δ 13 C values of stems and leaves, phytC, and soil SOM fractions obtained for (a) sorghum and (b) durum wheat experiments. To facilitate comparisons between groups, samples from ambient and enriched CO 2 plots are plotted next to each other. Values are reported as per mil (‰) related to PDB. Results of the bulk and refractory SOM fractions were averaged; consequently results and uncertainties indicate multiple data points. Individual results are shown in Tables S1 and S2.$

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Biogeosciences, 13, 1269–1286, 2016www.biogeosciences.net/13/1269/2016/doi:10.5194/bg-13-1269-2016© Author(s) 2016. CC Attribution 3.0 License.Unambiguous evidence of old soil carbon in grass biosilica particlesPaul E. Reyerson1,2,a, Anne Alexandre3, Araks Harutyunyan1, Remi Corbineau3, Hector A. Martinez De La Torre1,Franz Badeck4, Luigi Cattivelli4, and Guaciara M. Santos11Earth System Science, University of California, Irvine, USA2Department of Botany, University of Wisconsin-Madison, USA3Aix Marseille Université, CNRS, IRD, CEREGE UM34, 13545 Aix-en-Provence CEDEX 4, France4Consiglio per la Ricerca in Agricoltura e l’analisi dell’economis agraria – Genomics Research Centre,Fiorenzuola d’Arda, Italyacurrent address: Department of Geography and Earth Science, University of Wisconsin-La Crosse, USACorrespondence to: Guaciara M. Santos (gdossant@uci.edu)Received: 19 June 2015 – Published in Biogeosciences Discuss.: 17 September 2015Revised: 27 January 2016 – Accepted: 8 February 2016 – Published: 1 March 2016Abstract. Plant biosilica particles (phytoliths) contain smallamounts of carbon called phytC. Based on the assumptionsthat phytC is of photosynthetic origin and a closed system,claims were recently made that phytoliths from several agri-culturally important monocotyledonous species play a sig-nificant role in atmospheric CO2 sequestration. However,anomalous phytC radiocarbon (14C) dates suggested contri-butions from a non-photosynthetic source to phytC. Herewe address this non-photosynthetic source hypothesis us-ing comparative isotopic measurements (14C and δ13C) ofphytC, plant tissues, atmospheric CO2, and soil organic mat-ter. State-of-the-art methods assured phytolith purity, whilesequential stepwise-combustion revealed complex chemical-thermal decomposability properties of phytC. Although pho-tosynthesis is the main source of carbon in plant tissue, itwas found that phytC is partially derived from soil carbonthat can be several thousand years old. The fact that phytC isnot uniquely constituted of photosynthetic C limits the use-fulness of phytC either as a dating tool or as a significantsink of atmospheric CO2. It additionally calls for further ex-periments to investigate how SOM-derived C is accessible toroots and accumulates in plant biosilica, for a better under-standing of the mechanistic processes underlying the siliconbiomineralization process in higher plants.1 IntroductionSilicon (Si) is the most abundant element in the Earth’s crustand is widely recycled by higher plants. Si is acquired byroots from soils and precipitated in or between the cellsas micrometric hydrous amorphous biosilica particles calledphytoliths. Phytolith abundances range from < 1 % of dryweight (d wt) in many plants to several % d wt in grassesthat are Si-accumulators (Geis, 1973; Runge, 1999; Webband Longstaffe, 2000; Raven, 2003). Phytoliths contain smallamounts of carbon (C) occluded during silica precipitation(Alexandre et al., 2015), commonly termed as phytC or phy-tOC and assumed to be of photosynthetic origin (Carter2009; Piperno, 2006) (Fig. 1a). Thus, phytC isotopic signa-tures (δ13C and 14C) obtained from buried soils and sedimen-tary archives have been interpreted in terms of paleoenviron-mental changes (Kelly et al., 1991; Carter, 2009; McInerneyet al., 2011), or used as a dating tool (McClaran and Umlauf,2000; Piperno and Stothert, 2003; Parr and Sullivan, 2005;Piperno, 2006).Motivated by anthropogenic emissions of carbon diox-ide (CO2) (Mauna Loa Observatory; NOAA-ESRL data athttp://www.esrl.noaa.gov/) and their direct association withclimate change, a set of recent studies has advanced the ideathat many monocotyledonous crop species (bamboo, sug-arcane, maize, rice, etc.) as well as grasslands in general(among the largest ecosystems in the world – Suttie et al.,2005) may play a significant role in C sequestration through anewly evidenced mechanism: CO2 biosequestration in grassPublished by Copernicus Publications on behalf of the European Geosciences Union.

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1270 P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particlesFigure 1. Sketch of (a) the conventional hypothesis of plant C oc-clusion during silica precipitation based solely on atmospheric CO2as a source, and (b) the emerging hypothesis of a dual origin (atmo-spheric CO2 and SOM) for plant C (and phytC). Young and old soilC distributed in leaf epidermis (green tissue) and phytoliths (illus-trated by the bilobate type shape outlined in black) are representedby black and orange dots, respectively, in the microscope diagram.biosilica particles (Parr and Sullivan, 2005, 2011; Parr et al.,2009, 2010; Song et al., 2013, 2014; Toma et al., 2013). Ifcorrect, encapsulated atmospheric CO2 can be slowly andsteadily accumulated in soils, with turnover times of the or-der of several hundreds to thousands of years (Parr and Sul-livan, 2005). Selective use of silica accumulator crops couldfurther enhance this sequestration mechanism (Song et al.,2013).However, the validity of these interpretations has recentlybeen challenged. First, attempts to properly calibrate the geo-chemical signals borne by phytC were inconclusive (Wild-ing, 1967; Kelly et al., 1991; McClaran and Umlauf, 2000;Smith and White, 2004; Webb and Longstaffe, 2010). Sec-ond, differences in the efficiency of phytolith extraction pro-tocols may have contributed to inconsistencies and overes-timations in phytC quantification (from 0.1 to 20 % of phy-tolith d wt) (Corbineau et al., 2013 and references therein,Song et al., 2014 and references therein). Third, systematicoffsets of phytC 14C ages relative to the 14C ages of theplant tissues from which phytoliths originate have been pub-lished (Santos et al., 2010, 2012a, b, 2016; Sullivan and Parr,2013; Yin et al., 2014; Piperno, 2016). These offsets can beas large as hundreds to several thousands of years, regard-less of the chemical protocol used for phytolith extractions,indicating the presence of a secondary contributor of C tophytC. Together, these observations led to the hypothesis thata whole or a fraction of phytC may come from old soil C(Santos et al., 2012a) (Fig. 1b). Previous analyses of macro-molecules embedded in phytoliths suggested a variety of or-ganic molecules (Bauer et al., 2011 and references therein),but there is no direct evidence that they are solely synthe-sized by the plant. Moreover, a recent Nano Secondary IonMass Spectrometry (NanoSIMS) investigation of phytC dis-tribution in the silica structure suggests that a significant partof phytC can be lost at the very first stage of phytolith disso-lution (Alexandre et al., 2015), thus dissociating the conceptof phytC protection from phytolith stability.Therefore, if the soil C to phytC hypothesis is definitivelyconfirmed, it casts doubt on the efficiency of paleoenviron-mental reconstructions based on phytC as a proxy of plantC, and raises questions regarding the present estimates ofcrop and grasslands phytolith efficiency in sequestering at-mospheric CO2, as well as its assessment of long-term stabi-lization in soils based on fossil phytolith 14C dating (decadesversus hundreds, or thousands of years, as suggested by Parrand Sullivan, 2005). Additionally, confirmation of a dual ori-gin (soil organic matter (SOM) and photosynthetic) of phytCwould open new questions regarding plant–soil interactionsand SOM recycling, relevant for our understanding of therole of terrestrial ecosystems in the C cycle.To unequivocally establish that a fraction of phytC is in-deed from soils, a robust data set is produced here by con-sidering and ruling out all other factors that can possiblybias the isotopic signatures of phytC. We reassess the oldsoil C contribution to phytC hypothesis (Santos et al., 2012a)on the basis of > 200 isotopic results (δ13C and/or 14C) ofphytoliths and associated materials (grass tissues, SOM frac-tions, amendments and hydroponic solutions, CO2 respiredfrom substrates or extracted from air). Pure phytolith concen-trates were acquired from sets of above- and below-ground Cmanipulation experiments. Phytolith concentrates were ex-tracted using several protocols with different degrees of ag-gressiveness (Corbineau et al., 2013) in four different lab-oratories. Cutting-edge techniques assured phytolith purity,and multiple analyses of carbon isotope reference materialsassured high quality and reproducibility of the isotopic re-sults. Furthermore, to establish a link between phytC hetero-geneity in the sense of molecular complexity and resistanceto oxidation (labile vs. recalcitrant), we subjected duplicatesof pure phytolith extracts to thermal treatments. The multi-methodology approach used in this study allows us to com-pletely address: (a) the anomalous 14C results associated withphytC in the literature, (b) the implications of a soil C con-tribution to phytC for 14C geochronology dates, and (c) theshortcomings of using phytC as an atmospheric CO2 sink.2 Material and methods2.1 SamplesOur experimental design is based on a two-step process.First, in order to evidence whether the 14C signatures ofBiogeosciences, 13, 1269–1286, 2016 www.biogeosciences.net/13/1269/2016/

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P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particles 1271phytC are solely of photosynthetic origin, we select sam-ples from known-year specimens, and compare plant mate-rial grown under normal atmospheric CO2 conditions to theartificially altered plant C isotope content of photosyntheti-cally assimilated depleted-14CO2 from Free Air Carbon En-richment (FACE) experiments (Sect. 2.1.1). Second, we seekto establish a causal connection between soil C and phytCby selecting samples from plant material grown under nor-mal atmospheric CO2 conditions, but altered substrate car-bon pools (Sect. 2.1.2). In both cases phytC and an array ofsamples associated with it were selected.2.1.1 Above-ground C manipulation experimentsThe FACE experiments exposed the plants to elevated atmo-spheric CO2 concentrations by continuously releasing CO2through jets from tubes installed in the surroundings andwithin the enclosures of the cultivation plots. Target mixingratios of atmospheric and geologic CO2 were maintained onplots until leaves were senescent and/or ready for harvesting.Two grass species (Sorghum bicolor and Triticum du-rum) were grown in two FACE experiments, respectively:at the Maricopa Agricultural Center (University of Ari-zona, USA) in 1998–1999 (Ottman et al., 2001), andat the Genomics Research Centre of CREA (Consiglioper la ricerca in agricoltura e l’analisi dell’economiaagraria) in Fiorenzuola d’Arda, Italy, in 2011–2012 (Badecket al., 2012; http://centrodigenomica.entecra.it/research/durumFACE). For each experiment, a plot cultivated un-der ambient atmospheric CO2 was compared to a plot cul-tivated under atmosphere enriched by 160–200 ppm in fos-sil hydrothermal CO2, and therefore free of 14C (Leavitt,1994; Ottman et al., 2001; Badeck et al., 2012). In termsof stable isotopic labeling, at the sorghum site the enrichedCO2 had a δ13C value of −40 ‰ from 1995 to 1998. Thisstronger isotopic label was obtained from a mixture of nat-ural CO2 from the Springerville, AZ, USA geologic wellswith 15 % petroleum-derived CO2. During 1998–1999 onlyfossil hydrothermal CO2 was used (δ13C = −4.36 ‰), whilethe background air δ13C was −8 ‰ (Leavitt et al., 2001).At the durum wheat site, the commercial fossil CO2 fromthe Rapolano Terme, Poggio S. Cecilia (Tuscany) well had aδ13C of −6.07 ‰, which was slightly positive compared tothe ambient CO2 value of −8 ‰.Two samples of mixed stems and leaves (∼ 100 g) wereobtained from the sorghum site, while four separated sam-ples (300–400 g each) of stems and leaves were collected atthe durum wheat site. Eight soil samples (∼ 5 g each) col-lected from the furrows of the sorghum plots at depths of 0–15, 15–30, 30–45, and 45–60 cm were also obtained from thearchives of the Laboratory of Tree-Ring Research, Univer-sity of Arizona, USA. While two soil samples were collectedfrom the ongoing durum wheat experimental plots at a depthof 0–15 cm (∼ 15 g each) during plant biomass harvesting.To determine the precise 14C activity of the plant mate-rials, radiocarbon measurements were conducted before thephytolith extractions started. Since the commercial CO2 usedin both FACE enrichment sites was from a fossil source,its 14C signature as fraction of modern carbon (FmC orFm14C; Stuiver and Polach, 1977) was close to zero. There-fore, the 14C signature of the enriched CO2 was highly de-pleted compared to ambient air, and the plant tissues weretagged accordingly. Radiocarbon signatures of the plant tis-sue yielded Fm14C values of 0.640 (∼ 3.6 kyr BP; 14C yearsbefore present or 1950; UCIAMS53273 and 53274; Table S1in Supplement) and 0.556 (∼ 4.7 kyr BP; UCIAMS109000and 109001; Table S2 in Supplement) at the sorghum anddurum sites, respectively. Alternatively, plant tissue from am-bient CO2 plots was expected to yield the prescribed atmo-spheric 14CO2 values of the given year that the growing sea-son took place. At the sorghum site, the Fm14C value of thebulk biomass harvested at the ambient CO2 plot matchedwith the Fm14C value of the CO2 of the year of harvest (e.g.,Fm14C ≈ 1.097, equivalent to the atmospheric 14CO2 signa-ture measured from clean air in 1999 – http://calib.qub.ac.uk/CALIBomb/ database and calibration software). This 14Csignature is higher than the present-day ambient CO2 due tonuclear weapon tests carried out during the 1950s and 1960s(Levin, 1997; Levin et al., 2013). The nuclear weapon testsdoubled the 14C content in the atmosphere, which createdan isotopic chronometer (the 14C bomb peak) during the last60 years for all living organisms. At the durum wheat site,however, the 14C signature of the biomass harvested at theambient CO2 plot was slightly depleted (Fm14C ≈ 1.017), asexpected for CO2 above urban areas in Europe in the early2010s. For comparison, the 14C signature of atmospheric-clean CO2 stations in Central Europe was Fm14C = 1.040 in2012 (Levin, 1997; Levin et al., 2013).2.1.2 Below-ground C manipulation experimentThe second experiment relies on the simultaneous responseof phytC to different carbon amendment treatments ofgrasses grown under photosynthetic natural conditions (i.e.,ambient CO2 air). Sorghum bicolor plants were grown out-doors in a ventilated area at the University of California,Irvine (UCI, USA), in six well-drained 40 L planters (A,B, C, D, E and F) filled with mineral substrates. Five ofthe planters were enriched with organic nutrients character-ized by a broad range of 14C signatures (from bomb spikedto fossil – Tables 1 and 2), while the last contained an in-organic nutrient devoid of C as a control (Planter F). Al-though much concerning the direct root absorption of naturalcarbon remains unknown, beneficial responses of root andplant growth have been reported in association with the ad-dition of either inorganic carbon (Hibberd and Quick, 2002)and/or humic acids (Nardi et al., 2002). Consequently, wechose as substrate for Planter B, a natural carbonate-basedsedimentary deposit mixed with organic carbon detritus ofwww.biogeosciences.net/13/1269/2016/ Biogeosciences, 13, 1269–1286, 2016

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1272 P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particlesequal/even-age. For Planter E, fossil humic acids (extractedfrom leonardite) were chosen as the OC source.Plants were fed as needed solely with 2 L of ultra-pure wa-ter (Planter A), or with a combination of ultra-pure water andtheir respective fertilizers and SiO2 providers (Planters B–F) at a concentration of 1 % (v/v) (Table 2). Additionally,the CO2 in the air surrounding the planters was isotopicallymonitored by collecting air in evacuated 6 L cylinders forthe duration of the experiment with the purpose of charac-terizing the local atmospheric CO2 close to planters, and toserve as a reference for the 14C signatures expected fromplant tissue organs. Also, we isotopically measured commer-cial (sorghum) seeds to check if their 14C signatures were re-cent. Finally, CO2 fluxes respired from the planter substrateswere also sampled to evaluate their putative contribution tothe phytC 14C signature. After 3.5 months the Sorghum bi-color plants (stem and leaf) were harvested in preparation forphytolith extractions and isotopic analyses.2.2 Laboratory procedures2.2.1 Plant treatment and phytolith extractionStems and leaves samples (50–100 g each) were thoroughlyrinsed with warm ultrapure water to remove air-dust, dried at60 ◦C and ground using an industrial mill (IKA® M20 Uni-versal Mill). About 10 mg of each sample was kept for bulktissue 14C and δ13C analyses.Four phytolith extraction protocols with increasing aggres-siveness (via organic compound oxidation and silica dissolu-tion) were used to treat the samples from the above-groundC manipulation experiment (Fig. 2). The protocols have beenpreviously described in detail by Corbineau et al. (2013).They are based either on acid digestion and alkali or on multi-step dry ashing and acid digestion. They are summarized be-low and in Fig. 2.Protocols 1a and 1b. Plant samples were subject to strongwet-digestion steps in order to oxidize the organic matter(e.g., 1N HCl 2 h−1, hot H2SO4 24 h−1 plus 30 % H2O2 for2–3 days, and > 65 % HNO3 plus 1 g KClO3 for 24 h). Thiswas followed by 30 min of immersion in KOH solution atpH 11 (protocol 1a) or pH 13 (protocol 1b). The KOH im-mersions allowed final removal of any alkali-soluble formsof organic compounds remaining on phytolith surfaces.Protocols 2a and 2b. Plant samples were subjected to dry-ashing. Stepwise increases in temperature were used from300 to 500 ◦C and the samples were then kept at 500 ◦C for6 h (protocol 2a) or 12 h (protocol 2b). Samples were thendigested in a > 65 % HNO3 and 70 % HClO4 mixture (2 : 1).In order to assess local 14C contamination during chemi-cal extractions, four laboratories were involved in the extrac-tions. They are UCI (USA), CEREGE (CNRS, AIx-MarseilleUniversity, France), the Soils and Sediments Analysis Lab(SSAL, the University of Wisconsin-Madison, USA), andthe National Lacustrine Core Facility (LacCore, the Univer-sity of Minnesota, Twin Cities, USA). Aliquots of pre-baked(900 ◦C 3 h−1) silicon dioxide powder (SiO2; mesh# −325,Sigma Aldrich, St. Louis, MO, USA) were chemically pre-treated in parallel with the plant samples, and later analyzedas phytolith extract to provide independent blank data foreach laboratory following the procedures described in San-tos et al. (2010).Due to the limited amount of plant biomass produced bythe below-ground C manipulation experiment (Sect. 2.1.2),only two protocols were tested (1a and 2b) at only three ofthe laboratories (UCI, CEREGE, and LacCore), followed byblank sample materials as required.2.2.2 Soil extraction fractionsSoils from the above-ground C manipulation experimentwere physically cleaned of roots and stones. The bulk SOMfraction was isolated after carbonate removal in 1N HClbaths at 60 ◦C. The refractory (alkali-insoluble) fraction wasfurther isolated via multiple baths in 1M NaOH at 60 ◦C, fol-lowed by 1N HCl rinses (Santos and Ormsby, 2013). Uponchemical treatment, samples were adjusted to pH neutral anddried in a vacuum oven (Savant RT 100A refrigerated vaporvacuum pump system).Amendments from the below-ground C experiment werenot subject to any chemical pretreatment, except for the testsperformed to small aliquots of greensand (GS, Table 1), al-lowing us to isolate the organic fraction from its bulk mix-ture.2.2.3 CO2 flux measurementsIn the frame of the below-ground C manipulation experi-ment, the rate of CO2 respired from Sorghum bicolor foliage(after sprouting), root systems and substrate was measuredusing closed dynamic soil CO2 flux chambers (Czimziket al., 2006). Chamber headspace gasses were circulatedthrough an infrared gas analyzer (840, 1400, LI-COR, Lin-coln, NE, USA), for 6 min at 0.5 L per minute, and the CO2concentration was recorded every second. Once headspaceCO2 concentrations reached twice that of ambient-air, theCO2 was collected in a molecular sieve trap for isotopic anal-ysis, followed by ambient-air samples to serve as references.2.3 Analytical procedures2.3.1 Phytolith concentrate purity analysisSmall particulate organic contamination of phytolith concen-trates may considerably bias isotopic and quantitative anal-yses of phytC. The purity of the phytolith concentrates wasthus verified by Scanning Electron Microscopy with Energy-dispersive X-ray spectroscopy (SEM-EDS) (Corbineau et al.,2013). Extracted phytoliths, mounted directly on pre-cleanedaluminum stubs, were analyzed with a Schottky ThermalField Emission FEI/Philips XL-30 SEM with back-scatteringBiogeosciences, 13, 1269–1286, 2016 www.biogeosciences.net/13/1269/2016/

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P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particles 1273Figure 2. Above-ground C manipulation procedure. (a) Averaged Fm14C values versus averaged phytC yields (or concentration in % ofphytoliths). Constant solid lines correspond to the averaged Fm14C values obtained for stems and leaves (SL) of origin and the oldestextracted SOM fraction. (b) Oldest SOM-derived C contribution to phytC calculated using the mixing equation (Eq. 1) presented in the textexpressing the 14C signature of phytC as the result of mixing between the C derived from plant photosynthesis and the C derived from theoldest extracted SOM fraction. Phytolith samples are labeled according to the extraction protocol (1a, 1b, 2a, 2b described in caption and inthe text) used and the laboratory of extraction (UCI, CEREGE, LacCore and SSAL).electron detector. EDS semi-quantitative analyses of C andSi were obtained from 10 to 30 μm locations on selectedparticles. Special attention was paid to organic-like particlesshowing tissue-like or non-phytolith morphologies. A totalof ∼ 30 analyses per sample were made. Samples with allC : Si peaks area ratios < 0.1 were reported as devoid of or-ganic particles. The equal/even accuracy and precision of theEDS analyses were evaluated by multiple measurements of asilicon carbide (SiC) standard (#9441, Micro-Analyses Con-sultant Instrument Ltd, St Ives, UK): mean value of C : SiM = 1.17; standard deviation SD = 0.02 (n = 21).2.3.2 Stable isotope analysisStems/leaves, SOM fractions, nutrients/fertilizers and phy-tolith samples were analyzed for their total C content andstable C isotope ratio (δ13C) using a continuous flow stableisotope ratio mass spectrometer (Delta-Plus CFIRMS) inter-www.biogeosciences.net/13/1269/2016/ Biogeosciences, 13, 1269–1286, 2016

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1274 P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particlesTable 1. Below-ground experiment. Details of substrate amendments, their carbon content, radiocarbon values (as Fm14C and 14C age) andC isotopic signatures.Name Major contents Fm14C ±1σ 14C age ±1σ ±1σ%C (mass)a δ13C (‰)Miracle Gro® (MG) Sphagnum Moss, Perlite,Compost, NH4NO3,(NH4)3PO4,Ca3(PO4)2, K2SO449.5 (n = 2) 1.0849 0.0028 −650b 25 −26.1 0.11.0123 0.0028 −95b 25 −25 0.1Greensand (GS) Glauconite with organic and in-organic detritus, MnO2, SiO20.10c 0.1591 (n = 2) 0.0016 14765 78 −24.3(OC; n = 4) 0.1−12.6(bulk)Ionic Grow (IG) Ca(NO3)2, KNO3, H3PO4,HNO3, K2SO40.8 0.0374 (n = 2) 0.0101 26550 2192 −26.4 0.1Earth juice (EJ) Kelp meal, MgSO4borax,CoSO4,FeSO4, MnSO4, Na2MoO4,ZnSO415.44 (n = 2) 0.4991 (n = 3) 0.0013 5583 24 −24.1 (n = 2) 0.2Fossil Fuel (FF) Humic acids (from leonarditeor lignite coal)33.04 (n = 2) 0.0055 0.0003 43340 1700 −26.2 (n = 2) 0.2Inorganic in-house fertilizer (IF)d NaH2PO4, MgSO4, Ca(NO3)2,KNO3– – – – – – –Silica Blast (SB)d Na2SiO3, K2SiO3 – – – – – – ——a Total percentage of carbon was determined by manometric measurements of CO2 after combustion of solids. Those values are estimates only, as they do not take in account volatile organic C losses during thedrying procedure of the amendments as solutions; b negative 14C ages are associated with material that fixed C during the post-nuclear testing period (e.g., 1950 to the present); c GS %C is based on its total Camount by d wt, with 0.06 % of it constituted of organic matter detritus with the remaining C pool from marine carbonates. %C estimates of independent fractions were based on stable isotopic measurements of bulkand HCl treated (OC fraction) subsamples (Sect. 2.2.2). Nevertheless, the FmC 14C values of the organic C and bulk fractions are similar, and are shown here as an average value. The δ13C values of both fractionsare shown as reference; d attempts to produce CO2 from solids (upon freeze-drying) confirmed the absence of C in those amendments, and therefore those are not shown.Table 2. Below-ground experiment. Planters’ major features: substrates and amendments, living plant appearance, biomass by d wt andphytolith yields. All nutrients and fertilizers were administered in aqueous solutions, except for MG. In bold: main amendment.PlantersA B C D E FSubstrate MG GS Baked Sand Baked Sand Baked Sand Baked SandAmendments In MG In GS, IGa IGa EJ, IFb FF, IFb IFbSilica Provider In MG In GS SB SB SB SBAppearance Dark green Dark green Dark green Green Yellowish green GreenBiomass (g) 98.57 79.09 89.24 86.67 54.78 53.37Phytolith yieldc 0.12 0.78 0.83 0.83 1.77 1.35a IG has a very low %C. Therefore, its C contribution to planters B and C after dilution into solution (e.g., ∼ 0.02 g of C per feeding) was found tobe very small, a conclusion supported by isotopic analyses (Table S3); b IF (which does not contain measurable amounts of C) was added to thoseplanters to supply micronutrients to support plant growth;c as % of dry leaf and stem biomass combined.faced with a Fisons NA-1500NC (for solid materials) and aGasbench II (for CO2 input).About 10 mg of phytoliths and 25 mg of soil were weighedout into pre-baked (100 ◦C per 2 h) tin capsules (5 × 9 mmcapsules from Costech Analytical Technologies Inc., Valen-cia, CA, USA) using a pre-calibrated microbalance (Sarto-rius AG, Göttingen, Germany). For accurate integration andcalibration of carbon peaks of phytolith samples (∼ 0.1 % C),measurements were obtained by decreasing the heliumcarrier flow rate, and by measuring several size-matchedaliquots of standards from the National Institute of StandardsTechnology. Aliquots of SiO2 blanks and fossil phytoliths(MSG70) used as an internal standard at CEREGE (Alexan-dre et al., 2015; Crespin et al., 2008) were included forbackground corrections and accuracy (Santos et al., 2010),respectively. For the bulk tissue samples, aliquots of CO2gas were recovered after combustion, and sent to CFIRMS,which has a typical precision of 0.1 ‰. Stable isotope resultsare reported as δ values in ‰ relative to the Vienna Pee DeeBelemnite (vPDB).2.3.3 Radiocarbon analysisStems/leaves, SOM fractions, nutrients/fertilizers, CO2 andphytolith samples were processed for 14C accelerator massspectrometry (AMS) analyses. About 2 mg of plant tis-Biogeosciences, 13, 1269–1286, 2016 www.biogeosciences.net/13/1269/2016/

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P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particles 1275sue, 20–100 mg of SOM and 15–300 mg of phytoliths wereloaded for tube-sealed combustion (Santos et al., 2004). Toavoid CO2 adsorption on phytolith surfaces, the loaded sam-ples were kept and transferred warm (at 160 ◦C) to the evac-uated line for sealing (Santos et al., 2010). Liquid solutionswere freeze-dried directly into tubes prior to combustion. At-mospheric CO2 was extracted from 6 L collection flasks ofwhole air, by attaching the flasks to an evacuated line. Asimilar procedure was used to recover the CO2 collected inmolecular sieve traps (from flux chambers). Once the CO2was cryogenically separated from other gases, it was thentransferred to a Pyrex tube at a flame-off port and sealed(Santos et al., 2010). Samples of CO2 from tube-sealed com-bustions, flasks and traps were cryogenically isolated, andreduced to graphite (Santos et al., 2007; Xu et al., 2007), ortransferred to Gasbench II CFIRMS for isotopic analysis.The 14C measurements were performed at the Keck-CCAMS Facility (UCI). Precision and accuracy in measure-ments on > 0.7 mg of near-modern carbon samples are typ-ically 0.2–0.3 % (Beverly et al., 2010), and 1 % on samplesin the 0.01 mg C range (Santos et al., 2007). The instrumentprovides the isotopic ratio 13C / 12C, allowing for fractiona-tion effects (either spectrometer induced or arising from bio-chemical processes) to be corrected for all targets measured.Blanks from SiO2 aliquots were also measured to pro-vide background corrections. All labs and extraction proto-cols showed similar procedural blanks (∼ 0.003 mg of mod-ern C and ∼ 0.002 mg of 14C free1. Those values were sub-tracted from the 14C data, including the results obtained fromthe MSG70 reference material, for accuracy. Details on suchbackground subtractions can be found elsewhere (Santos etal., 2010). Radiocarbon results were expressed as Fm14C andwhen appropriate were discussed as ages.2.3.4 Thermal analysisWe performed thermal analysis of phytoliths on a modifiedThermal-Optical Carbon Aerosol Analyzer (RT 3080, SunsetLaboratory Inc.) (Bae et al., 2004). Phytolith concentrates of7–10 mg were loaded onto a customized spoon (Jelight Com-pany, Inc. USA), placed into the instrument and kept at 50 ◦Cfor ∼ 10 min for surface cleansing. The stepwise temperatureramp started at 50 ◦C and ended at 850 ◦C 50 min later. Pureoxygen (65 mL min−1) was used to avoid refractory carbon(char) formation. The CO2 evolved was injected into a man-ganese dioxide oven at 870 ◦C, and later quantified by a non-1The term14C free is used in association with materials fromwhich the original 14C radioisotope content has been reduced tozero or close to zero. However, those materials obviously continueto maintain their stable amounts of 12C and 13C. Consequently, theaddition of 14C free (or organic carbon from subfossil 14C signa-tures) to pools of C containing present-day atmospheric CO2 signalswill lower the overall 14C signature of the pool. For each 1 % fossilC present, an offset of 80 years is expected (Santos et al., 2016, andreference therein).dispersive infrared detector. Typical multi-point calibrationcurves, when analyzing known quantities of C ranging from2–120 μg, yielded correlation coefficients greater than 0.998.Two phytolith samples were analyzed. Durum wheat leafphytoliths extracted using protocol 1a, and MSG70, made ofhighly weathered fossil phytoliths (Alexandre et al., 2015;Crespin et al., 2008).3 Results and discussions3.1 Isotopic results from above-ground C manipulationexperimentsA total of 21 individual phytolith concentrates were pro-duced for the above-ground experiments by all laboratoriesinvolved in this project. Those samples are tabulated in theSupplement (Tables S1 and S2), followed by details on thesample processing (protocols, laboratory and measurementidentifiers). Note that when sufficient plant material wasavailable (which was the case for the durum wheat samples)some labs could replicate the extraction (i.e., processing thesame pool of biomass following the same protocol).From those 21 phytolith concentrates, 51 14C results wereproduced to determine the phytC 14C signatures (number oftargets includes duplicates and/or replicates, as specified inTables S1 and S2). Two phytC 14C targets from MSG70, afossil phytolith internal standard at CEREGE, were also pro-duced to evaluate measurement reproducibility. Overall, theprecision and accuracy of the phytC 14C data were better than0.3 %, based on duplicates and triplicates of graphite sam-ples > 0.5 mg C. For the smaller sized samples, 1 % or bet-ter were recorded in most cases, even after background cor-rections based on measurements of multiple SiO2 aliquotswere propagated into individual uncertainties (Tables S1 andS2). We have not identified significant differences in inter-laboratory analyses when using the same protocol on sub-samples of the same biomass sample, and/or when evaluatingprocedural blank materials (added to every batch analyzed –details in Sect. 2.3.3). To help with determining the phytCcarbon sources, other 14C results shown in Tables S1 and S2are from the stems/leaves and SOM fractions (e.g., the car-bon pools associated with the labile-accessible and recalci-trant (alkali-insoluble)).PhytC concentrations were consistent for a given extrac-tion method but showed a clear decreasing trend with in-creasing protocol aggressiveness. The phytC yields (phytC %relative to the d.wt of phytoliths) averages ranged from 0.24–0.06 % for the less aggressive protocols 1a and 1b and from0.05 and 0.002 % for the more aggressive protocols 2a and2b (Fig. 2a, and Tables S1, S2).Phytoliths extracted from either sorghum or durum wheatusing protocol 1a produced phytC 14C signatures closest tothe values of the stems and leaves of origin regardless ofair CO2 concentration (ambient vs. enriched CO2) and grasswww.biogeosciences.net/13/1269/2016/ Biogeosciences, 13, 1269–1286, 2016

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1276 P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particlesspecies (Fig. 2b). However, phytC 14C offsets were still ev-ident when compared to the expected values given the yearof harvest or artificial tagging (Table S1). For sorghum, ab-solute offsets varied from 85 (UCIAMS123579 and 123580)to 610 years (UCIAMS123577 and 123578) when using pro-tocol 1a. The maximum offset increased when using proto-cols 1b (2633 years; UCIAMS95338), 2a (1920 years; UCI-AMS130339), and 2b (1990 14C years; UCIAMS95335 to95337). Durum wheat ambient phytC 14C absolute offsetsvaried from 105 (UCIAMS123572) to 1925 years (UCI-AMS125986), while phytC offsets from enriched plots var-ied from 310 (UCIAMS123570 and 123571) to 2885 years(UCIAMS125983).The hypothesis that there is a contribution of SOM-derivedC to phytC was tested estimating phytC as a mixture of (i) Cderived from plant photosynthesis and (ii) C derived from theoldest SOM fraction measured. The mixing equation isOldest SOM-derived C contribution =(Fm14CphytC − Fm14CSL)/(Fm14CSOM − Fm14CSL) (1)where the 14C signatures of the oldest SOM, stems and leaves(SL) and phytC are expressed as Fm14CSOM, Fm14CSLand Fm14CphytC. Fm14CphytC was expressed relative toFm14CSOM (assigned a contribution value of 1) and Fm14CSL(assigned a contribution value of 0). The average Fm14Cvalue of the oldest SOM-C fractions measured in each exper-iment (i.e., the Fm14C average value of the SOM 45–60 cmfraction for S. bicolor plots – Table S1 in Supplement, andthe refractory 0–15 cm fraction for T. durum plots – Table S2)were used for Fm14CSOM.The mixing curves associated with the SOM-derived C tophytC hypothesis are presented in Fig. 2b. The Fm14C valuesof two phytC samples from the Sorghum Ambient CO2 ex-periment obtained using protocol 1a (UCIAMS123579 and123580) and one phytC sample from the Durum Wheat En-riched CO2 experiment obtained using protocol 1b (UCI-AMS130339) were higher than Fm14C values of the stemsand leaves of origin, indicating that the soil pool still hasremnants of 14C-labeled OC from the 1950s thermonucleartests (Levin, 1997; Levin et al., 2013). In this case the SOM-derived C was assigned a contribution value of 0, and thestems and leaves a contribution value of 1 in Fig. 2b. Con-versely, some of the phytC Fm14C values from the Du-rum Wheat Enriched CO2 experiment, obtained using pro-tocols 1a, 2a and 2b (UCIAMS123566, 123567, 125985,130334 and 130335), were lower (14C age older) than theFm14C value of the oldest SOM fraction or 1 in Fig. 2b.This pattern suggests that the so-called oldest SOM fraction,which is a mixture of old and young SOM (Schrumpf et al.,2013) may still be “younger” than present-day in terms ofits 14C signatures, if the soil C pool is still bearing somebomb-produced 14C OC, or much older if aromatic com-plexes are dominant (Telles et al., 2003; Torn et al., 2009).For the sorghum experiment this trend was particularly ob-vious, as the ambient CO2 and the upper soil layers wereclearly imprinted with bomb 14C (Levin, 1997). Therefore,Fig. 2b clearly showed that the phytC Fm14C values unam-biguously trend toward the Fm14C value (or 14C age) of theoldest SOM fraction. Overall, the crucial point to be no-ticed is that the phytC 14C offsets shifted linearly towardsnegative values if the oldest SOM fraction was older thanthe biomass of origin (Sorghum Ambient and Durum WheatAmbient, Fig. 2a), and towards positive values when the old-est SOM fraction was younger (Sorghum Enriched, Fig. 2a).Thus, phytC 14C differences were clearly linked to the oldestSOM 14C ages. Moreover, the agreement in phytC 14C val-ues obtained from stems and leaves indicated that the offsetswere not linked to plant anatomy.Regarding δ13C values, the phytC offsets relative to thetissue of origin did not systematically trend towards SOMδ13C values, except for the Sorghum Ambient phytC under-going the 2b protocol (−21.6 ± 0.1 ‰ (n = 2) as indicated inFig. 3; UCIAMS95335 and 95336). As described earlier, thisprotocol tends to isolate the most recalcitrant phytC fraction.The difference between phytC δ13C values of durum wheatand sorghum was higher (∼ 15.7 ‰) than the difference be-tween δ13C values of the stems and leaves of origin (e.g.,∼ 5.6 vs. ∼ 7.2 ‰ for wheat and sorghum, respectively), aspreviously reported for grasses with C3 and C4 photosyn-thetic pathways (Webb and Longstaffe, 2000, 2010). Withoutfurther discrimination of the molecular composition of SOM-derived C absorbed by the plant roots, in-depth discussion ofthe δ13C differences between phytC and plant biomass is dif-ficult. Nevertheless, the observed differences between phytCand stems and leaves δ13C values were consistent with pre-vious calibration studies, and were explained by preferen-tial occlusions of plant molecular 13C-depleted compoundsin phytoliths (Webb and Longstaffe, 2010).3.2 Isotopic results from below-ground C manipulationexperimentsA total of 12 individual phytolith concentrates and phytC14C targets were produced for the below-ground experi-ments, two of those from the same biomass samples (fromPlanters A and C), but subjected to different degrees ofoxidation (e.g., protocol 2b). Other 14C results shown arefrom the stems/leaves, nutrients/fertilizers, and CO2 ex-tracted from 6 L flasks and flux chambers (Fig. 4). The com-plete set of isotopic results and sample processing details aretabulated in the Supplement (Tables S3).Phytoliths produced phytC yields ranged from 0.08to 0.1 % d wt when using the less aggressive protocol 1a andfrom 0.01–0.04 % d wt when using the more aggressive pro-tocol 2b (Table S3).Significant offsets of the phytC 14C values relative tothe stem and leaf Fm14C values were again found in as-sociation with the carbon sources in the soils (e.g., sub-strates/amendments). The highest phytC 14C offset of 3610Biogeosciences, 13, 1269–1286, 2016 www.biogeosciences.net/13/1269/2016/

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P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particles 1277Figure 3. Above-ground C manipulation experiment. δ13C values of stems and leaves, phytC, and soil SOM fractions obtained for (a)sorghum and (b) durum wheat experiments. To facilitate comparisons between groups, samples from ambient and enriched CO2 plots areplotted next to each other. Values are reported as per mil (‰) related to PDB. Results of the bulk and refractory SOM fractions were averaged;consequently results and uncertainties indicate multiple data points. Individual results are shown in Tables S1 and S2.years (UCIAMS104366) was obtained from the phytC 14Cfrom Planter B when using protocol 2b, showing again thatthe increased age discrepancies were due to protocol aggres-siveness (e.g., from 1a to 2b). The effect is also observed inthe phytoliths associated with Planter C, which received verylow amounts of below-ground organic carbon relative to allother treatments (Tables 1 and 2). Specifically, the PlanterC phytC 14C offsets increased from 160 (UCIAMS130346;protocol 1a) to 1150 (UCIAMS104362; protocol 2b), and to1760 years (UCIAMS104900; protocol 2b).Even when we processed biomass samples from allPlanters following the same protocol (such as the less ag-gressive 1a protocol), 14C age discrepancies between phytCand the plant of origin were highly evident, and correlatedto the 14C signatures of amendments (UCIAMS130344 to130348). PhytC 14C offsets were greater for amendmentscontaining sufficient amounts of C of extreme 14C-signatures(e.g., positive 320 years to Planter A, and negative 680 yearsto Planter E in Table S3). Note that the Planter A substratewas composed of rich bulk-complex OC imprinted with 14C-bomb values (or Fm14C signatures higher than present-dayvalues), while the Planter E substrate received a solution offossil OC (Fm14C = 0; close to ∼ 43 kyr BP; n = 3) (Tables 1and 2). As in the above C manipulation experiment, in Fig. 4we assigned values of 0 and 1 to the Fm14C associated withstems and leaves of origin and amendments, respectively (Ta-ble S3), and used the same mixing equation (Eq. 1).The bulk stems and leaves produced Fm14C signatures thatwere very similar to the local ambient air 14CO2 values col-lected in the 6 L cylinders during the growing season, exclud-ing any possibility that the phytC 14C depletions are a prod-uct of urban fossil atmospheric CO2 fixation. The small dis-crepancies between the stem and leaf 14C values (e.g., from25 to 65 years) (Table S3) are attributed to heterogeneitiesin C distribution within plant cells during C fixation (Pauschand Kuzyakov, 2011; Wichern et al., 2011). The commercialseeds of sorghum were also measured by 14C-AMS (Fig. 4)to verify their recent radiocarbon activity (UCIAMS83120and 83121; Table S3). As expected, once early-fixed pho-tosynthetic CO2 became dominant, remobilized 14C fromseeds made little contribution to mature biomass tissue.Although Fm14C values of substrate CO2 fluxes weredepleted towards amendment 14C bulk signatures (UCI-AMS83842 to 83845, Table S3), soil CO2 plant tissue re-fixation via photosynthesis (and its influence on phytC) wasfound to be negligible, and cannot be invoked to explain theanomalous phytC 14C results. CO2 fluxes from the planters’substrates upon sprouting varied from 0.34 to 1.72 ppm s−1(≈ 10−5 g m−2 yr−1) (Table S3), indicating very little micro-bial activity. For comparison, global soil CO2 fluxes varyfrom 60 to 1000 g m−2 yr−1 (Raich and Sclesinger, 1992).δ13C offsets between phytC and stems and leaves were∼ 6.5 ‰ on average, including the phytC from Planter B(which contain a mixed C pool of OM detritus of plant originand carbonate deposits – Table 1), showing that the inorganicfraction of the soil C was not a significant source of phytC(Fig. 5). Also in Fig. 5, we show the stable isotopic signa-tures of the CO2 fluxes (UCIAMS83842 to 83845; Table S3)collected using closed dynamic soil CO2 flux chambers (Cz-imzik et al., 2006). The results fell mostly between the airwww.biogeosciences.net/13/1269/2016/ Biogeosciences, 13, 1269–1286, 2016

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1278 P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particlesFigure 4. Below-ground C manipulation procedure: oldest amendment-derived C contribution to phytC calculated using the mixing equation(Eq. 1) presented in the text expressing the 14C signature of phytC as the result of mixing between C derived from plant photosynthesis(seeds, stems and leaves represented by the green squares) and C derived from the oldest amendment (MG, EJ, GS, IG, FF defined in Table1 and represented by the red squares). Phytolith samples are labeled according to the phytolith extraction protocol used (1a and 2b) and thelaboratory of extraction (UCI, CEREGE and SSAL). Selected age benchmarks from substrate amendments and soil CO2 fluxes are shownfor reference on the right axis.and bulk plant tissue averages, as expected for CO2 pro-duced from above- and below-ground biomasses, supportingour previous observations of negligible effects of soil CO2respired to phytC.This data set clearly shows that amendment-derived C, ad-sorbed through root plants, altered the phytC 14C signatures.3.3 Thermal stability of phytCChemical compositional insights on carbonaceous materi-als can be obtained via oxidation reactivity to thermal treat-ments; such treatments have been frequently used on organiccompounds from soils and sediments (Plante et al. 2011,2013; Rosenheim et al., 2013). For instance, single-bondedcarbon structures usually show a lower thermal stability thanthose dominated by double bonds, such as conjugated andaromatic structures (Harvey et al., 2012). Here, we make useof the same chemical-thermal stability concept to evaluatethe heterogeneity of phytC in reacting to heat treatments.Thermograms obtained from phytoliths of the durumwheat leaves and fossil phytoliths (MSG70) indicated a con-tinuum of phytC CO2 with different degrees of resistanceBiogeosciences, 13, 1269–1286, 2016 www.biogeosciences.net/13/1269/2016/

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P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particles 1279Figure 5. Below-ground C manipulation experiment. δ13C valuesof the respired CO2, stems and leaves, amendments and phytC forthe five planters enriched in organic carbon nutrients (A–E). Valuesare reported as per mil (‰) related to PDB, and individual symbolsrepresent single results as reported in Table S3. For Planter B wereport two values, its OC fraction (−24.3 ‰) and its bulk fraction(−12.1 ‰ – a mixture of OC and inorganic carbon) (Table 1). Con-stant solid lines correspond to the average δ13C values of ambient-air CO2 and bulk plant tissues.or accessibility (Fig. 6). Although the overall productionof CO2 was lower for MSG70, the continuum temperature-dependency pattern of phytC was preserved. For example, at250 ◦C both phytolith extracts produced CO2, however theleaf phytoliths show lesser amounts of CO2 evolved than soilphytoliths. At 500 ◦C half of the phytC CO2 in both sampleshas been evolved, and at 800 ◦C all of the phytC has beencompletely removed.Phytoliths typically melt at ∼ 573 ◦C (Deer et al., 1992),but embedded metals (e.g., Al, Fe) within their structurescould lead to a decrease in temperature stability (Wu et al.,2014). Nevertheless, phytC that required much higher tem-peratures to fully oxidize (e.g., beyond of the phytolith melt-ing point of 573 ◦C) would be placed at the upper end of thecarbon recalcitrance continuum. (Cheng et al., 2013; Harveyet al., 2012; Plante et al., 2005, 2011, 2013). Furthermore,even if char occurred during combustion leading to someelemental carbon formation, it does not explain the Fm14CphytC discrepancies obtained here (Figs. 2 and 4) or else-where (Santos et al., 2010, 2012a, b; Sullivan and Parr, 2013;Yin et al., 2014).Santos et al. (2012a) and Yin et al. (2014) also heated phy-tolith aliquots from a single extract, and observed shifts in14C ages towards older values. This effect is similar to thatobserved in total carbon or SOM distributions in soils andsediments when subject to thermal decomposability (Planteet al., 2011, 2013). Thus, phytolith extractions that employheat treatments would better isolate the oldest soil C frac-tion within phytoliths, as previously found (in Sects. 3.1. and3.2). Basically, if the C pool in phytoliths is supposedly ho-Figure 6. Thermograms (n = 2; blue and red lines) of phytolithsobtained from (a) durum wheat leaves, phytoliths extracted follow-ing protocol 1a (Table S2), and (b) soil phytoliths MSG70 extractedusing a conventional protocol adapted to soil and sediment materi-als. Peaks are artifacts of the 100 ◦C temperature-step increments.Vertical lines indicate main temperature thresholds, as explained inthe text.mogeneous and from a single source (atmospheric CO2), the14C results from all CO2 temperature-fractions should be inabsolute agreement, as Fernandez et al. (2015) demonstratedby subjecting carbonaceous materials to ramp pyrolysis andsubsequently measuring them by 14C-AMS.3.4 The SOM-derived C to phytC hypothesis set ofevidenceResults from both above- and below-ground experimentsshowed that the 14C offsets between phytC and stems andleaves pointed toward the oldest SOM 14C values (Figs. 2 and4). This confirmed that a fraction of the old SOM-derived Coccluded in phytoliths was more resistant (or less accessible)to oxidation than the occluded C derived from recent photo-synthesis or from recent SOM. Once the most labile (or moreaccessible) C had been removed, the older and more resistantcarbon fraction became dominant. This behavior mirrors thatin a recent study showing an increase in 14C age offsets ofphytoliths with increasing combustion temperature (Yin etal., 2014), and also the thermal decomposability pattern il-lustrated in the phytC thermograms (Fig. 6).Our findings also imply that a portion of SOM-derived Cis absorbed by the roots, transferred to the stems and leaves,and finally occluded into phytoliths. In the bulk plant organs,www.biogeosciences.net/13/1269/2016/ Biogeosciences, 13, 1269–1286, 2016

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1280 P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particlesthe old SOM-derived C amount is far too small to be 14C de-tected in tissue clippings, as it is masked by the large amountsof photosynthetic atmospheric carbon tissue (bulk stems andleaves averaged ∼ 41 % carbon; Tables S1–S3). On the otherhand, in phytoliths, the old SOM-derived C becomes over-represented when the most labile-accessible phytC starts tobe oxidized. It should be noted that the 14C ages of the old-est SOM fraction are averaged bulk values that do not yieldany precise assessment of the fine-scale 14C age of the Cthat may have been absorbed. These drawbacks prevent pre-cise quantification of the old SOM (probably diluted by theyoung SOM)-derived C contribution to phytC. The impos-sibility of quantifying precisely the amounts of soil C andassociated 14C signatures in phytC precludes application ofany correction that would allow phytC to be used as a reli-able dating material. As in any other heterogeneous carbonpool, the phytC continuum can be similarly partitioned dif-ferently by distinctive chemical extractions. For instance, inPiperno (2016) the entire data set of post-bomb Neotropi-cal plant phytolith extracts were neither accurate nor precise.While 14C offsets reached discrepancies as high as 4.4 kyrbetween expected calendar ages and phytC, two pairs of phy-tolith extracts obtained by distinct chemical treatments (sul-furic vs. nitric) yielded a 50 % reproducibility rate (Table 1,Santos et al., 2016).Recent 3-D x-ray microscopy and NanoSIMS measure-ments of a phytolith sample from the Durum Wheat EnrichedCO2 experiment (TD-F-L/1a-CEREGE, Table S1) (Alexan-dre et al., 2015) suggested two locations for phytC: in micro-metric internal cavities and within the silica network. Rapidopening of internal cavities during the dissolution processresulted in losses of phytC found in these locations, whichis expected when phytoliths are subject to rapid oxidation.Conversely, phytC in the silica network is homogeneouslydistributed at the micrometric scale, and is less accessible tooxidation. These two pools of phytC may account for the het-erogeneity of phytC accessibility to oxidation.3.5 Rebuttals to possible arguments against theSOM-derived C contribution to phytC hypothesisOur experiments and data set allow the rejection of severalhypotheses for the “anomalously” old 14C ages for phytC.First, bias due to exogenous C contamination during the phy-tolith extractions performed simultaneously by several labo-ratories and artifacts of errors in background corrections arehighly unlikely. In these cases the 14C offsets would trend ina single direction, rather than being both positive and neg-ative (Figs. 2b and 4). In addition, aliquots of SiO2 blankyielded 14C values in close agreement with the expected re-sults, giving no indication of the presence of unusual contam-inants. Second, natural- or spectrometer-produced anoma-lous δ13C shifts of phytC were not observed here (Figs. 3 and5) nor elsewhere (Santos et al., 2010, 2012b; Sullivan andParr, 2013). Third, contributions of soil-respired CO2 to ma-Figure 7. Conceptualization of the impact of phytolith extractionaggressiveness and C removal on 14C age of phytoliths. Incompletedigestion leads to an accumulation of old residual OM in phytolithconcentrates. Protocol 1 removes all OM residues and better pre-serves the dual source phytC signature. Protocol 2 removes all resid-ual OM and labile (intrinsically young) phytC. For illustration pur-poses, young and old C are represented by black and orange dots,respectively (see Fig. 1b).ture plant tissue (and phytC) were also negligible (Sect. 3.2).Fourth, phytC 14C results were not biased by organic matterresidues, as the efficiency of the phytolith extraction proto-cols was fully checked by SEM-EDS analyses (e.g., accep-tance threshold of C : Si ≤ 0.1 of 30 frames or more) (Cor-bineau et al., 2013), a method superior to microscopic eval-uation alone (Figs. S1 and S2 in Supplement) (Kameník etal., 2013; Santos et al., 2012a). Moreover, our extracts wereconsistently reproducible regarding phytC yields across alllabs involved (Tables S1–S3) and thermal decomposabilityproperties (Fig. 6). Since it has been established that plantsdo not photosynthesize all carbon found within their tissues(details in Sect. 3.8), the uptake of SOM-derived C via theroot system and its allocation to phytC is the only plausibleexplanation for the phytC 14C offsets.3.6 Implications for the use of phytC as a proxy ofplant CSince plants (and more specifically phytoliths) contain abroad continuum of C with a complex mixture of chemi-cal compounds of different turnover times as evidenced here(Figs. 2, 4, and 6), we believe that insufficient to exces-sive oxidations can result in wild moves in phytC 14C datesfrom thousands, to hundreds, to back to thousands years old(Fig. 7).While pure phytoliths produced from a less aggressiveprotocol (e.g., 1a) may minimize 14C offsets to some de-Biogeosciences, 13, 1269–1286, 2016 www.biogeosciences.net/13/1269/2016/

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P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particles 1281gree, two factors remain that may explain the anomalousthousands of years old age of phytC indicated in the lit-erature (Wilding, 1967; Kelly et al., 1991; McClaran andUmlauf, 2000; Santos et al., 2010, 2012a, 2016; Sullivanand Parr, 2013; Piperno, 2016). The first factor is the in-complete removal from phytolith concentrates of refractorySOM residues, either extraneous in the case of litter andsoil samples or from the plant tissue itself. The accumula-tion effect of small quantities of residual recalcitrant (andsomewhat older) SOM derived-C from concentrates due toincomplete digestion (Fig. 7), which can be detected viaC:Si peaks with SEM-EDS (Corbineau et al., 2013), maybe undetected under natural light microscopy. For instance,Santos et al. (2010) reported phytC 14C age offsets of 2.3to 8.5 kyr BP on phytolith concentrates extracted from liv-ing grasses using conventional digestion protocols, such asKelly et al. (1991). Later, OM remnants in association withthose anomalous 14C results were detected by SEM-EDS inphytolith concentrates (Fig. 2 in Santos et al., 2012a), thusdemonstrating that even very small amounts of particulate Cwere enough to bias the phytC 14C results. Attempts to re-produce the atmospheric 14CO2 bomb-peak in phytC frombamboo litter and mature leaves subjected to microwave di-gestions, also yielded offsets of several hundreds to 3.5 kyr(Santos et al., 2012b; Sullivan and Parr, 2013). Similarly, aset of post-bomb Neotropical plant phytolith extracts pro-duced by two protocols yielded phytC 14C ages that werehighly inaccurate, e.g., phytC 14C offsets ranging from sev-eral decades to 4.4 kyr (Santos et al., 2016). In those cases,preferential bias due to post-depositional occlusion of SOMwas unlikely. All phytolith extracts analyzed were obtainedfrom living or close to living vegetation, undergoing differ-ent extraction procedures coupled with optical microscopeanalyses (for purity evaluations). Cumulative effects of OMremnants on phytoliths would also explain the higher phytCyields (Kelly et al., 1991; Li et al., 2014; Parr and Sullivan,2005; Santos et al., 2010; Song et al., 2014). The second fac-tor is the increasing relative proportion of old SOM-derivedC in phytC when phytolith extraction aggressiveness is highenough to remove the phytC fraction most sensitive to oxi-dation (e.g., the labile-accessible C fraction termed “proto-col 2” in Fig. 7). Once carbon partitioning takes place viaeither further chemical extractions or increased combustiontemperatures, phytC concentrations tend to drop followed byincreased 14C offsets to thousands of years old (Yin et al.,2014 and the present work).Since the range of old SOM-derived C content in phytCleft by a given protocol can be large (Fig. 2), and can varyin association to the abundances of C fractions within thesubstrates and their respective 14C signatures (Fig. 4), anyattempt to apply a systematic correction to obtain a phytCFm14C signature derived solely from photosynthesis is likelyto fail. We can also assume that when grasses are forcedto reach greater rooting depths (Sivandran and Bras, 2012)than the ones sampled here, where the proportion of intrinsic-older organic compounds is likely to rise (Telles et al., 2003;Torn et al., 2009; Kleber, 2010; Petsch et al., 2001), oldSOM-derived C in phytC and its Fm14C depletions wouldalso increase. Furthermore, by themselves the 14C signa-tures of phytC pools with competing 14C ages (recent SOM-derived C vs. present-day atmospheric 14CO2) are insuffi-cient to distinguish them. Therefore, the old soil C to phytCcontributions found here in association with the 14C signa-tures of phytoliths extracted from living grasses are likely tobe only a very small fraction of the total SOM contributionto phytC, as discussed earlier.Further work is still needed to assess the full impact ofSOM (e.g., the different fractions of labile vs. recalcitrantcarbon; Han et al., 2007) to the phytC pool. At natural condi-tions the presence of SOM-derived C in phytC may bias theδ13C signature to a lesser extent if the SOM and the plants oforigin have similar photosynthetic pathways (C3 or C4). Thebias may however be significant if they are not. The δ13C sig-nature of SOM can be hard to assess, especially in the caseof phytoliths extracted from sedimentary archives. Thus, wesuggest that the use of 14C and δ13C signatures of phytC asa dating tool or as a proxy of plant or atmospheric CO2 sig-natures should be reappraised in the light of the present find-ings.3.7 Implications for long-term atmospheric CO2biosequestrationThe evidence for a SOM-derived C contribution to phytCdecreases the putative effectiveness of grasslands and cropsto sequester atmospheric CO2 for two reasons. Besides neg-atively affecting phytolith C storage capacity, our findingsmost importantly invalidate phytC accumulation rates esti-mated from direct 14C dating of soil phytoliths (Parr andSullivan, 2005). In addition, other issues may also come intoplay. For instance, the phytolith biosequestration hypothesisis based essentially on the following premises. First, highphytC concentrations are required. Values of 1.5–3 % d wthave been quantified (e.g., Li et al., 2013; Parr and Sulli-van, 2011; Parr et al., 2010). These values are more than 10times higher than the concentrations recently measured byothers (< 0.1 % d wt, Santos et al., 2010). Differences in theefficiency of phytolith extraction protocols (Kameník et al.,2013), combined with the lack of proper control (blanks) andreproducibility of results (Corbineau et al., 2013) may havecontributed to these high phytC concentrations. Second, asoil phytolith stability factor of 70 to 90 % based on a few14C measurements of soil phytoliths (e.g., Parr and Sullivan,2005) has been estimated and widely used (Li et al., 2014) re-gardless of soil type. These high percentage estimates differfrom those of biogenic Si fluxes, based on Si pool measure-ments in tropical soil–plant systems. For instance, accord-ing to Alexandre et al. (2011) investigating two soil/plantsystems in intertropical areas, only 10 % of phytoliths pro-duced annually are in fact preserved for extended periods,www.biogeosciences.net/13/1269/2016/ Biogeosciences, 13, 1269–1286, 2016

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1282 P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particlesthe remaining 90 % being rapidly dissolved due to weather-ing (Oleschko et al., 2004). These proportions would reason-ably depend on environmental conditions such as activity ofelements (Si, Al, Fe, H+) in soil solution, morphology ofphytoliths (and thus vegetation type), and elemental concen-tration of phytoliths (and thus soil type).Only as an exercise, we used the highest phytC yieldmeasured in the frame of the present study (0.3 % ofphytoliths) coupled with the 10 % phytolith stability fac-tor estimated from Alexandre et al. (2011), to recalcu-late a global grassland phytC-sink. We obtain a value of4.1 × 104 tC yr−1, which is roughly one hundred times lowerthan the 3.7 × 106 tC yr−1 value reported elsewhere (Song etal., 2014 and references therein). This amount is insignificantwhen compared to the 2.6 × 109 tC yr−1 estimate for the landC sink (IPCC, 2007), or to the 0.4 × 109 tC yr−1 global meanlong-term soil C accumulation rate (Schlesinger, 1990). Thissuggests that previous conclusions on the importance of de-veloping silica accumulator crops for increasing atmosphericC sequestration should be reconsidered.3.8 Implications for our understanding of soil C poolsmobilizationOur findings have important implications for our understand-ing of the mobilization of soil C pools. Several studies haveshown that terrestrial plant roots can uptake soil dissolvedinorganic carbon (DIC). DIC can be transported directly bythe transpiration stream or fixed in mycorrhizal and root tis-sues and subsequently translocated in the form of amino acid(Gioseffi et al., 2012; Rasmussen et al., 2010; Talbot andTreseder, 2010). DIC can represent 1–3 % of total leaf-fixedCO2 (Ford et al., 2007; Ubierna et al., 2009). However, asDIC is expected to be in equilibrium with soil CO2 respiredfrom autotrophic and heterotrophic sources, its 14C signatureshould reflect an average of SOM 14C signatures, close tocontemporary. Assuming soil DIC as the soil end-memberin Fig. 2, the phytC samples from ambient CO2 experimentswould plot along mixing lines with lower slopes than the ac-tual ones. The 14C age of several thousand years systemati-cally measured for the most resistant phytC, rather suggeststhat an older SOM fraction supplies the SOM-derived C ab-sorbed by the roots, up-taken and transported to the stem andleaves tissues.The fact that roots can also acquire soil C in a molecularform has been previously inferred from the detection in roots,stems and shoots of polycyclic aromatic hydrocarbons (PAH)(Gao et al., 2010; Yu et al., 2013), and soil amino acids (AA)(Paungfoo-Lonhienne et al., 2008; Warren, 2012; White-side et al., 2009, 2012). Although reported PAH concentra-tions were three orders of magnitude below phytC concentra-tions (e.g., 10−9 g g−1 vs. 10−6 g g−1, assuming 0.1 % d wtfor both phytolith concentration in plants and phytC contentin phytoliths), AAs make up several tenths of a percent ofthe plant nitrogen requirements (Lipson and Näsholm, 2001).Arbuscular mycorrhizal fungi, which colonize 70 % of plantfamilies (Talbot and Treseder, 2010; Treseder and Turner,2007) are probably at the base of the transfer of molecular Cfrom the rhizosphere to the roots, although intact protein hasalso been shown to enter root cells without the help of my-corrhizae, most likely via endocytosis (Paungfoo-Lonhienneet al., 2008). At lower scales, AA transporters were shown toconfer the ability of plants to absorb molecular C from thesoil solution (Lipson and Näsholm, 2001; Tegeder, 2012).Root acquisition of humic substances (active and passive)and its positive effect on plant nutrient uptake has been alsoreported (Trevisan et al., 2010). The incorporation of below-ground physical, chemical and biological processes in therhizosphere (e.g., microbial priming effect or N and C cycleinteractions) have also been proposed (Heimann and Reich-stein, 2008 and references therein). The results of the presentstudy go a step further by demonstrating that part of the soilmolecular C absorbed by roots is several thousand years old.Recent studies also show that old, supposedly poorly ac-cessible SOM (Kleber, 2010; Petsch et al., 2001; Schmidtet al., 2011), can be decomposed by organisms or catalyticenzymes (Dungait et al., 2012; Marín-Spiotta et al., 2014).Common sources of dissolved Si for plants are clay miner-als and amorphous silicates (allophane, imogolite). Due totheir small size, high surface functional groups, area, andporosity, these minerals stabilize SOM either by adsorptiononto their surface or by aggregation (Basile-Doelsch et al.,2007; Jones and Singh, 2014; Kögel-Knabner et al., 2010).Further studies are needed to investigate whether dissolutionof Si-bearing forms during active uptake of Si (Ma et al.,2006) may also promote old SOM mobilization, ready to bechelated with Si, absorbed by the roots and translocated tothe stems and leaves.4 ConclusionsAlthough photosynthesis is the main source of C in planttissue, we have demonstrated here that grass biosilica (phy-toliths) occlude SOM-derived C that can be several thousandyears old, debunking the common assumption of phytC pho-tosynthetic carbon exclusivity. This finding suggests causesfor previous anomalously older phytC 14C ages found in theliterature. Moreover, the fact that phytC is not uniquely con-stituted of photosynthetic C limits the usefulness of phytCeither as a dating tool or as a significant sink of atmosphericCO2. Revised estimates of atmospheric CO2 biosequestra-tion by phytoliths led to values that are insignificant com-pared to the total land C or soil C sinks. All in all, by demon-strating that old SOM-derived C is accessible to roots andbuilds up in plant biosilica, this study constitutes a basis tofurther investigate the mechanism and amplitude of old SOMrecycling by roots for a better understanding of the C cycleat the soil/plant interface.Biogeosciences, 13, 1269–1286, 2016 www.biogeosciences.net/13/1269/2016/

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P. E. Reyerson et al.: Unambiguous evidence of old soil carbon in grass biosilica particles 1283The Supplement related to this article is available onlineat doi:10.5194/bg-13-1269-2016-supplement.Author contributions. G. M. Santos conceived the study.G. M. Santos, A. Alexandre, P. E. Reyerson, and R. Cor-bineau designed the experiments and conceived the strategiesfor phytolith extraction and purity analyses. G. M. Santos,P. E. Reyerson, A. Alexandre, A. Harutyunyan, R. Corbineauand H. Martinez De La Torre performed the experiments andcontributed to analysis tools. F. Badeck and L. Cattivelli providedbulk tissue and soil samples from T. Durum FACE. G. M. Santos,A. Alexandre, and P. E. Reyerson interpreted the data and wrotethe paper. All authors discussed the results and implications, andcommented on the paper.Acknowledgements. The authors gratefully acknowledge thesupport of the US National Science Foundation (DEB-1144888to GMS), the French FIR 2010 (Aix-Marseille Université),ECCOREV 2011, AIR Archéométrie 2011(CNRS) and LabexOT-Med 2013. PER wishes to thank J. A. Mason (University ofWisconsin-Madison) and the National Lacustrine Core Facility(University of Minnesota) for lab space usage. GMS thanksM. J. Ottman, B. Kimball, S. W. Leavitt, E. Pendall, P. Pinter,G. Hendrey, H. L. Cho and R. Rauschkolb for providing thearchived Maricopa FACE samples. Financial support provided bythe Durum experiment via the “Fondazione in rete per la ricercaagroalimentare” with the AGER program: agroalimentare e ricercais gratefully acknowledged. We thank J. Southon for help with the14C-AMS analyses, and C. Czimczik and M. Lupasco for technicalsupport with the CO2 flux measurements, help in interpreting thedata, and suggestions and comments on an early version of thispaper. We would like to thank X. Xu for the stable isotope analysisand technical support for CO2-air cryogenic extraction, and Q. Linand the Laboratory for Electron and X-ray Instrumentation (LEXI)at UC Irvine for access to lab space and assistance with X-rayanalytical techniques. We also thank S. Fahrni for providing theinorganic fertilizer used in the control planter, and K. Gallagher forher efforts on the estimates of percent carbon of some amendments.A. Alexandre thanks J. Balesdent (CEREGE) for helpful discussionon root absorption of molecular C. The authors also wish to extendtheir thanks to the Editor Roland Bol and the three anonymousreviewers for the constructive comments.Edited by: R. BolReferencesAlexandre, A., Bouvet, M., and Abbadie, L.: The role of savannasin the terrestrial Si cycle: A case-study from Lamto, Ivory Coast,Glob. Planet. Change, 78, 162–169, 2011.Alexandre, A., Basile-Doelsch, I., Delhaye, T., Borshneck, D.,Mazur, J. C., Reyerson, P., and Santos, G. 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$Above ground C manipulation procedure. (a) Averaged Fm 14 C values vs. average C yields obtained for phytC. Averaged Fm 14 C values obtained for stems and leaves (SL) of origin and the oldest extracted SOM fraction are indicated. (b) PhytC expressed as a mixture between photosynthetically-derived C and the oldest extracted SOM fraction-derived C (oldest SOM-derived C contribution = (Fm 14 C SOM − Fm 14 C SL )/(Fm 14 C phytC − Fm 14 C SL )). Phytolith samples are labeled according to the extraction protocol (1a, 1b, 2a, 2b described in caption) used and the laboratory of extraction.$

$Above-ground C manipulation experiment. δ 13 C values of stems and leaves, phytC, and soil SOM fractions obtained for a) sorghum and b) durum wheat experiments. To facilitate comparisons between groups, samples from ambient and enriched CO 2 plots are plotted next to each other. Values are reported as per mil (‰) related to PDB. Results of the bulk and refractory SOM fractions were averaged; consequently results and uncertainties indicate multiple data points. Individual results are shown in Tables S1 and S2.$

Evidence of old soil carbon in grass biosilica particles

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Full-text available

September 2015

Biogeosciences Discussions

Plant biosilica particles (phytoliths) contain small amounts of carbon called phytC. Based on the assumptions that phytC is of photosynthetic origin and a closed system, claims were recently made that phytoliths from grasslands play a significant role in atmospheric CO2 sequestration. However, anomalous phytC radiocarbon (14C) dates suggested contributions from a non-photosynthetic source to phytC. Here we address this non-photosynthetic source hypothesis using comparative isotopic measurements (14C and 13C) of phytC, plant tissues, atmospheric CO2, and soil organic matter. State-of-the-art methods assured phytolith purity, while sequential stepwise-combustion revealed complex chemical-thermal decomposability properties of phytC. Although photosynthesis is the main source of carbon in plant tissue, it is found that phytC is partially derived from soil carbon that can be several thousand years old. The accumulation of old soil organic matter derived carbon in plant biosilica suggests that Si absorption and phytolith production promote old soil organic carbon mobilization. Although the magnitude of this mechanism still needs to be properly assessed at plant and ecosystem scales, its confirmation alone argues against attempts to use phytC as a proxy of plant carbon and call for the reexamination of phytolith atmospheric CO2 biosequestration estimates.

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From radiocarbon analysis to interpretation: A comment on ” Phytolith

Article

Full-text available

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Journal of Archaeological Science

The paper “Phytolith Radiocarbon Dating in Archaeological and Paleoecological Research: A Case Study of Phytoliths from Modern Neotropical Plants and a Review of the Previous Dating Evidence” by Dolores R. Piperno presents radiocarbon analysis of phytoliths from modern Neotropical plants collected between 1964 and 2013. The analyses presented were intended to rebut the emerging hypothesis that invokes root-plant uptake, transport and reallocation of soil organic carbon into phytoliths that has been recently put forward as an explanation for the anomalous radiocarbon (14C) ages (of hundreds to thousands of years old) reported for modern grass phytoliths in Santos et al. (2010a, 2012a,b). We believe that the results presented in Piperno (2015) lack methodological rigor, mostly due to the absence of any procedural blank assessment, and that the attempts to disprove the hypothesis of uptake of soil organic matter (SOM) by phytoliths in Santos et al. (2012a) are not supported by a careful analysis. Rather than supporting the position that 100% of the carbon in phytoliths is of photosynthetic origin, which allows the use of phytolith carbon (or phytC) as a dating tool, the analysis of 14C in phytoliths from modern Neotropical plants presented in the study shows that the 14C ages are strongly affected by other sources of carbon. In this comment, we carefully reassess the 14C results in phytoliths from modern Neotropical plants presented in Piperno (2015) in the context of the 14C bomb-pulse methodology, SOM ages and turnover rates, and offer an alternative interpretation of the experimental results.

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SEM images of TD-F-L wheat phytolith assemblage including silica sheets (a, b), stellate type from intercellular space (c); GSC phytolith: Rondel types (d, e) and Polylobate type (f).

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From radiocarbon analysis to interpretation: A comment on “Phytolith Radiocarbon Dating in Archaeological and Paleoecological Research: A Case Study of Phytoliths from Modern Neotropical Plants and a Review of the Previous Dating Evidence”, Journal of Archaeological Science (2015), doi: 10.1016/j.jas.2015.06.002.” by Dolores R. Piperno

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DownloadRecommendFollowShare

New highlights of phytolith structure and occluded carbon location: 3-D X-ray microscopy and NanoSIMS results

Article

Full-text available

February 2015

Biogeosciences

Phytoliths contain occluded organic compounds called phytC. Recently, phytC content, nature, origin, paleoenvironmental meaning and impact in the global C cycle have been the subject of increasing debate. Inconsistencies were fed by the scarcity of in situ characterizations of phytC in phytoliths. Here we reconstructed at high spatial resolution the 3-D structure of harvested grass short cell (GSC) phytoliths using 3-D X-ray microscopy. While this technique has been widely used for 3-D reconstruction of biological systems it has never been applied in high-resolution mode to silica particles. Simultaneously, we investigated the location of phytC using nanoscale secondary ion mass spectrometry (NanoSIMS). Our data evidenced that the silica structure contains micrometric internal cavities. These internal cavities were sometimes observed isolated from the outside. Their opening may be an original feature or may result from a beginning of dissolution of silica during the chemical extraction procedure, mimicking the progressive dissolution process that can happen in natural environments. The phytC that may originally occupy the cavities is thus susceptible to rapid oxidation. It was not detected by the NanoSIMS technique. However, another pool of phytC, continuously distributed in and protected by the silica structure, was observed. Its N/C ratio (0.27) is in agreement with the presence of amino acids. These findings constitute a basis to further characterize the origin, occlusion process, nature and accessibility of phytC, as a prerequisite for assessing its significance in the global C cycle.

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