See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/222066589 A new approach for the isolation of cellulose from aquatic plant tissue and freshwater sediments for stable isotope analysis Article in Organic Geochemistry · November 2008 DOI: 10.1016/j.orggeochem.2008.07.014 CITATIONS READS 40 264 3 authors, including: Holger Wissel Christoph Mayr Forschungszentrum Jülich Friedrich-Alexander-University of Erlangen-Nürnberg 23 PUBLICATIONS 326 CITATIONS 111 PUBLICATIONS 2,143 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: SALSA (South Argentinean Lake Sediment Archives and Modelling) View project ALPWÜRM View project All content following this page was uploaded by Holger Wissel on 08 November 2017. The user has requested enhancement of the downloaded file. SEE PROFILE This article appeared in a journal published by Elsevier. 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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Organic Geochemistry 39 (2008) 1545–1561 Contents lists available at ScienceDirect Organic Geochemistry journal homepage: www.elsevier.com/locate/orggeochem A new approach for the isolation of cellulose from aquatic plant tissue and freshwater sediments for stable isotope analysis Holger Wissel, Christoph Mayr 1, Andreas Lücke * Institute of Chemistry and Dynamics of the Geosphere, Sedimentary Systems (ICG 5), Energy & Environment Research Center Jülich, D-52425 Jülich, Germany a r t i c l e i n f o Article history: Received 18 February 2008 Received in revised form 24 June 2008 Accepted 25 July 2008 Available online 8 August 2008 a b s t r a c t Isotope studies of the cellulose of plant tissue and sediments are increasingly important in ecological and palaeoenvironmental studies. The reliability of analytical results in these investigations depends on the ability to separate pure cellulose from the organic and inorganic matrix. This study evaluates the performance of a new approach for the isolation of cellulose based on wet oxidation and cellulose dissolution in cuprammonium solution (CUAM) with respect to conventional techniques using solely wet oxidation or wet oxidation in combination with density separation. All the methods led to identical changes in isotopic composition of standard cellulose powder samples, providing evidence that CUAM-treated cellulose is isotopically indistinguishable from cellulose treated with conventional methods. The performance of CUAM with aquatic plant tissue was at least equal to those of the other methods used and in numerous cases achieved better results in terms of cellulose purification and precision of the isotope signal. Compared to conventional methods, contamination of several samples with small amounts of minerogenic matter and biogenic opal could be completely removed from the extraction residue with CUAM. While conventional methods failed to result in isolation of cellulose from two typical fine-grained lacustrine sediments, extraction residues from CUAM treatments revealed infrared (IR) spectra resembling those of standard cellulose powder, without evidence of minerogenic matter, biogenic opal, chitin or refractory organic matter. A series of sedimentary materials ranging from soil, to sediment trap and sediment core material were extracted with CUAM. IR spectra of all materials tested correspond to cellulose, and carbon and oxygen isotope compositions of the extracted cellulose fractions agreed with previous knowledge about the samples. Our results prove that CUAM is a reliable method that yields clean and pure cellulose for high quality isotope analysis from plant tissue and different types of sediments alike. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Investigation of the stable isotope composition of light elements in cellulose (H, C, O) represents an increasingly important approach for palaeoecological and palaeoclimatological studies. Applications have focussed on the study * Corresponding author. Tel.: +49 2461 614590; fax: +49 2461 612484. E-mail address: a.luecke@fz-juelich.de (A. Lücke). 1 Present address: GeoBio-CenterLMU and Department of Earth and Environmental Sciences, Ludwig-Maximilians-Universität, Richard Wagner-Strasse 10, D-80333 Munich, Germany. 0146-6380/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2008.07.014 of the C and O isotope composition of cellulose in tree rings (e.g. Treydte et al., 2006) and, to a lesser extent, in aquatic plants (e.g. Sternberg et al., 1984) and peat mosses (e.g. Ménot-Combes et al., 2002). Accordingly, various methods for the chemical extraction of cellulose from wood have been developed (Macfarlane et al., 1999; Brendel et al., 2000; Boettger et al., 2007) that are, in the majority, derivatives of the Jayme–Wise method (Green, 1963). The use of sedimentary cellulose in palaeohydrology was first proposed by Edwards and McAndrews (1989). As a result of technical and methodological advances, isotope studies of cellulose in organic matter (OM) Author's personal copy 1546 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 incorporated into lacustrine sediments has attracted increasing interest during the last decade (Wolfe and Edwards, 1997; Wolfe et al., 2003; Sauer et al., 2001). Compared to wood, the extraction of cellulose from sediments is, however, exceedingly more laborious and error prone. Freshwater or marine sediments have a complex composition of abiogenic and biogenic constituents. Sedimentary OM consists of a wide variety of terrestrial and aquatic plant tissue and organic compounds such as lipids, pectin, proteins, cellulose and hemicellulose, as well as refractory OM and humic substances. To our knowledge, only one method, developed at the University of Waterloo, was specially designed to solve the challenge posed by the extraction of sedimentary cellulose (Wolfe et al., 2001, 2007). Since the reliability of oxygen isotope measurements depends primarily on the extraction quality, the isolation of pure cellulose is crucial for sedimentary studies. However, in a number of applications on fine-grained lacustrine sediments we detected contamination in the cellulose extraction residues, i.e. minerogenic components, diatom frustules, chitinous remains or refractory OM with the Wolfe et al. (2007) method. Similar problems of cellulose extraction also only partly solved by density separation were described by Beuning et al. (2002). In principle, any technique applied to cellulose extraction from sediments should fulfil several fundamental criteria for facilitating optimal accuracy and precision of subsequent isotope analysis. A physically clean cellulose preparation devoid of minerogenic residues avoids bias from matrix–analyte interactions and release of traces of inorganically bound O during pyrolysis. A chemically pure cellulose preparation, often conventionally termed a-cellulose, circumvents any bias induced by impurities in the analyte from other polysaccharides such as, e.g. hemicellulose and chitin. Quantitative and reproducible yields of cellulose evidence the quality of the extraction process and prevent bias by conceivable isotopic inhomogeneity of the target cellulose. Furthermore, efficiency in terms of raw material and sample throughput as well as universality in terms of applicability to OM of diverse origin would foster the technique. This study evaluates the performance of a novel approach for the isolation of cellulose from sedimentary matter. The protocol is based on classical approaches of cellulose extraction with sodium chlorite (NaClO2) and of cellulose dissolution in cuprammonium solution ([Cu(NH3)4](OH)2; Gupta and Sowden, 1964; Green, 1963; Klemm et al., 1998). Here, we report a number of experiments with various materials, including cellulose powders, aquatic plant tissue and fine-grained sediments, using the developed method (CUAM) in comparison with the Wolfe et al. (2007) method for sediments (UWEIL, University of Waterloo Environmental Isotope Laboratory) and a modified Jayme–Wise method used in our laboratory for woody material (JUEL). Unlike other methods, the new approach achieves a clean and pure fraction of cellulose, not by removing contaminants, but by separating the target cellulose from the contaminants by way of cellulose dissolution and subsequent precipitation. The performance of the different treatments is first compared for standard cellulose materials, followed by experiments with different plant tissues and finally by a discussion of results from fine-grained freshwater sediments. 2. Experimental 2.1. Materials Commercially available cellulose powders were used as standards without further treatment. Fresh plant material from aquatic macrophytes, water mosses and algae was harvested in the field and brought directly to the laboratory. Further details of the samples are summarized in Table 1. As far as necessary, plant tissue was cleaned macroscopically through washing in demineralized water, ultrasonication and hand-picking. Aquatic macrophyte samples Myriophyllum II and Elodea II were only washed in demineralized water. The wet material was freeze dried, homogenized using ultra-centrifugal milling (Retsch, 0.75 mm sieve) at room temperature for 30–60 s and separated into aliquots for replicate chemical extractions. Sedimentary materials were sampled either directly in the field or, in the case of cores, in the laboratory. Sediments were treated with aqueous sodium hexametaphosphate [(NaPO3)6] solution (5%) at room temperature overnight (16 h) to ensure dispersion. Following dispersion, sediments were sieved at 200 lm to remove coarse organic debris, washed clean with deionized hot water ( 70 °C) and freeze dried. The fraction < 200 lm was divided into aliquots. From each material and each extraction protocol (UWEIL, JUEL, CUAM) at least three separate extractions were performed. For UWEIL preparations including the density separation step, aliquots of HZM surface and AZUL were additionally sieved at 20 lm to provide a 20–200 lm fraction. 2.2. Cellulose extraction protocols We used three different extraction methods. A protocol developed at UWEIL especially for lacustrine sediments, a modified Jayme–Wise protocol used in our laboratory for woody materials (JUEL) not adapted for sedimentary samples and the new protocol as described here (CUAM). All protocols consist of several steps. 2.2.1. UWEIL protocol For practical and safety reasons it was necessary to slightly modify single steps of the method as described by Wolfe et al. (2001, 2007). (i) Initially, carbonate was removed by dissolution with HCl (5%) for 2 h at 50 °C in a water bath. The residue was repeatedly washed with deionized water. (ii) The removal of lipids, tannins and resins was achieved by solvent extraction in a toluol/ethanol mixture (2:1) for 24 h at room temperature (3–4 until completion). Following the last solvent treatment, the mixture was centrifuged and decanted. Acetone was added to the residue and left for 24 h before the sample was again decanted and air dried in a fume hood. (iii) Chemical bleaching of the sample was accomplished by treatment with sodium chlorite (7%) acidified to pH 4–5 with concen- Author's personal copy 1547 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 Table 1 Materials used for cellulose extraction experiments and abbreviations used in the text (n.d., not detected) Abbreviation Description Corg (%) O (%) Cellulose Avicel FlukaCell IAEA-C3 Cellulose powder, Merck (for column chromatography) Cellulose powder, Fluka (cellulose from spruce) Cellulose powder, IAEA Vienna (reference material, milled plates, charge number 337) 43.3 42.2 43.7 47.3 46.8 47.2 Macrophyte Elodea I Potamogeton Callitriche Myriophyllum I Myriophyllum II Elodea II Whole Whole Whole Whole Whole Whole plant plant plant plant plant plant tissue, tissue, tissue, tissue, tissue, tissue, 40.7 37.6 41.3 42.2 35.8 36.4 40.6 36.4 37.6 41.9 39.3 36.4 Aquatic mosses Fontinalis I Drepanocladus ‘Aquatic moss’ Fontinalis II Whole Whole Whole Whole plant plant plant plant tissue, stream, Kirnitzschtal, Germany (Fontinalis antipyretica) tissue, Laguna Potrok Aike, Patagonia, Argentina (Drepanocladus perplicatus) tissue, Laguna Vizcachas, Patagonia, Argentina, undetermined tissue (>500 lm), brook, Wennigsen, Germany (Fontinalis antipyretica) 46.6 41.6 41.4 37.3 35.8 39.8 37.6 35.4 Algae Cladophora Rhizoclonium Vaucheria Whole plant tissue, Lake Holzmaar, Germany (Cladophora glomerata (L.) Kütz.) Whole plant tissue, Laguna Azul, Patagonia, Argentina (Rhizoclonium spec.) Whole plant tissue, pond, Wennigsen, Germany (Vaucheria spec.) 39.4 34.4 39.6 37.2 n.d. n.d. Sediments HZM surface HZM trap HZM 6500 SAC surface SAC 1700 POND wen SOIL for AZUL Surface sediment, Lake Holzmaar, Germany, clam-shell bottom sampler Sediment trap, epilimnion, Lake Holzmaar, Germany Sediment core, age 6500 cal BP, Lake Holzmaar, Germany, piston core Lake surface sediment, Sacrower See, Germany, gravity core Lake sediment core, age AD 1700, Sacrower See, Germany Surface mud, pond, Wennigsen, Germany Forest soil, top horizon (A), Wennigsen, Germany Surface sediment, Laguna Azul, Patagonia, Argentina, gravity core 5.2 7.5 8.3 13.1 7.0 4.2 5.8 12.0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. pond, Wennigsen, Germany (Elodea canadensis) stream, Kirnitzschtal, Germany (Potamogeton pectinatus) stream, Kirnitzschtal, Germany (Callitriche palustris) Lake Holzmaar, Germany (Myriophyllum spec.) pond FZ Jülich, Germany (Myriophyllum spec.) brook, Jülich, Germany (Elodea canadensis) trated acetic acid (96%) at 60 °C. After 10 h bleaching, the solution was exchanged and the process continued for another 10 h. Subsequently, the sample was washed (2–3) with hot deionized water ( 70 °C) to displace the bleach solution. (iv) Other polysaccharides remaining besides cellulose were removed by addition of NaOH (17%) and reaction at room temperature for 45 min. The residue was washed with cold deionized water and neutralized with dilute acetic acid. (v) To remove Fe- and Mn-oxyhydrates a leach solution containing 35 g sodium dithionite, 52 g ammonium citrate and 14 g hydroxylamine hydrochloride in 1 l deionized water was used. Leaching for 2 h at 60 °C was followed by leaching for 24 h at room temperature. Leaching was repeated at room temperature with exchanged leach solution until the cellulose concentrate was permanently discoloured. The cellulose residue was then repeatedly washed with cold deionized water (5– 7) and freeze dried. (vi) Finally, minerogenic components were removed by heavy-liquid density separation in centrifuge tubes with sodium polytungstate solution adjusted to a specific gravity of 1.9–2.0. Fe- and Mn-oxyhydrate leaching and density separation were not used for standard cellulose powders and plant tissue. For alternative demineralisation, samples were treated with HCl–HF (10%) for 16 h at room temperature. 2.2.2. JUEL protocol This protocol is a modified version of the Jayme–Wise protocol after Green (1963), originally designed for wood powder and complemented with a decarbonization step. (i) Carbonate was removed by dissolution in HCl (5%) for 2 h at 50 °C in a water bath; the residue was repeatedly washed with deionized water. (ii) Solvent extraction was substituted by an initial treatment with NaOH (5%) at 60 °C, repeated twice for 1 h (Rinne et al., 2005). (iii) Chemical bleaching was performed with NaClO2 (7%) acidified to pH 4–5 with concentrated acetic acid (96%) at 60 °C. After 10 h, the solution was exchanged and the treatment was repeated for another 10 h. The residue was washed (2– 3) with deionized hot water ( 70 °C) to displace the bleach solution. (iv) Finally, remaining non-cellulose polysaccharides (hemicellulose) were removed by treatment with NaOH (17%) for 45 min at room temperature. The extraction residue was washed with cold deionized water and neutralized with diluted acetic acid. 2.2.3. CUAM protocol Cuprammonium solution (cuam, cuoxam) or Schweizer reagent is an aqueous metal complex solution well known for its capability for dissolving cellulose in pulp chemistry (Schweizer, 1857; Saalwächter et al., 2000). Even medium to highly polymerized cellulose (DPw < 5300) dissolves quickly and completely in cuprammonium solution at a copper concentration between 15 and 30 g/l (Klemm et al., 1998). To verify the copper concentration necessary for the dissolution of cellulose from natural samples, experiments were carried out with plant tissue and sediments after conventional sodium chlorite bleaching. The copper hydroxide concentrations used were 10, 15, 20 and 30 g/l. The results showed that cellulose was dissolved at all con- Author's personal copy 1548 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 centrations tested. Reproducibility of cellulose yield and of carbon isotope values was best at a copper hydroxide concentration of 15 and 20 g/l. Based on these results, we developed a protocol merging a conventional approach for bleaching with sodium chlorite with a classical approach for cellulose dissolution in cuprammonium solution. Initial bleaching is necessary to make the cellulose in the samples readily accessible to the metal complex. In the protocol, (i) chemical bleaching of the sample was accomplished by treatment with NaClO2 (7%) acidified to pH 4–5 with concentrated acetic acid (96%) for 10 h at 60 °C. The residue was washed (2–3) in hot deionized water ( 70 °C) to remove the reactant and freeze dried. (ii) The cuprammonium solution was prepared in a 1000 ml amber glass bottle; 15 g copper hydroxide [Cu(OH)2] were dissolved in 900 ml ammonium hydroxide solution (25%) and 100 ml deionized water. The resulting blue coloured solution was stirred for 16 h at room temperature and filtered (Klemm et al., 1998, vol. 1, p. 234). The solution was stored in a cool, dark place (fridge). The dry sample ( 3 g sediment) was placed in a 50 ml stirrer jar, ca. 20–30 ml cuprammonium solution were added to each jar, which was closed with a lid. The jar was placed on a stirrer for 6 h and left for a further 10 h at room temperature to completely dissolve the cellulose. The supernatant copper complex cellulose solution was decanted into a centrifuge tube and centrifuged at 4000 rpm for 25 min. The supernatant was again decanted into another centrifuge tube loaded with 3 ml H2SO4 (20%) and subsequently filled up to 50 ml with cold deionized water. The tube was closed with a lid and the solution carefully shaken before it was left for 20 min at room temperature. The solution was again centrifuged at 4000 rpm for 25 min and decanted. Some drops of deionized water and 3 ml H2SO4 (20%) were added to the precipitate and the centrifuge pellet (blue) was destroyed. The sample was then allowed to sit (1–5 min) until the precipitate was completely ‘‘decopperised” (change of colour from blue to white) and filled up with cold deionized water. The precipitate was rinsed at least 3 with deionized water at room temperature and freeze dried. Treatment with cuprammonium solution might increase the amorphous character of cellulose and therefore probably water content. To avoid effects on isotope composition induced by additional water adsorbed to cellulose after CUAM treatment, samples of CUAM cellulose were stored in a vacuum drier at 40 °C after packing in silver capsules. Immediately prior to analysis, samples were placed overnight (16 h) in a vacuum drier at 100 °C. 2.3. IR spectroscopy IR spectra of standard materials and extracted fractions were measured using direct transmittance with the KBr pellet technique. Spectra (4000–200 cm 1, 2.4 cm resolution) were obtained using a Perkin-Elmer PE 783 spectrometer equipped with a coated thermocouple detector. Transmission spectra were converted to absorbance. The spectra were normalized relative to the difference in absorbance at 1560 and 1164 cm 1 to enable comparability of individual spectra. Comparison of band intensities can only describe qualitative differences. IR spectra are shifted relative to each other to improve visibility of single spectra in the graphs, so are given in arbitrary units (A.U.) on the ordinate. Absorbance values were smoothed by applying an 11 point moving average filter. 2.4. Mass spectrometry/isotope analysis Values of d13C were determined using 200–300 lg dry cellulose weighed into tin foil cups and combusted at 1080 °C using an elemental analyzer (EuroEA, Eurovector) interfaced on-line to an isotope ratio mass spectrometer (Isoprime, GV Instruments). Carbon content was determined through peak integration of m/z 44, 45 and 46, and calibrated against elemental standards. For measurement of d18O values, about 275 lg of cellulose was weighed in silver capsules, vacuum-dried and pyrolysed at 1450 °C in a high temperature pyrolysis analyzer (HTO, HEKAtech), and measured on-line with a coupled GV Instruments Isoprime isotope ratio mass spectrometer. Oxygen content was determined by peak integration of m/z 28, 29 and 30, and calibrated against elemental standards. Isotope results are reported as d values in per mil using conventional notation d = (Rs/Rst 1) 1000 with Rs and Rst as isotope ratios (13C/12C, 18O/16O) of samples and international standards (VPDB for carbon, VSMOW for oxygen), respectively. The overall precision of replicate analyses is estimated to be better than 0.1‰ for d13C, 0.3‰ for d18O and 5% (rel.) for C and O content. 3. Results and discussion 3.1. Variation in extract yield The effect of the extraction procedure on the amount of original sample mass remaining after treatment is shown in Fig. 1a for different standard cellulose powders. Independent of method, all treatments caused a loss of standard material and quantitative yield (100%) of cellulose was never accomplished. Highest yields were found for all materials with CUAM, in which the maximum recovery reached 92.8% (av. of three replicates). Variations between replicate treatments were found to be rather high, partly exceeding differences of 10% in yield, although extractions were conducted with great care. The range in yield for the same standard material between the methods could be due to differences in the chemical treatments and the number of processing steps involved in the protocols. Moreover, standard cellulose powders differ with respect to physical and chemical cleaning steps in their preparation as well as in degree of polymerization, so can be expected to perform differently with different protocols. Hence, these results should not be overemphasized and may not be representative for natural samples. Because of its different functionality, cellulose content in vascular and non-vascular plants can vary largely from about 42% to 50% in wood (Sjostrom, 1993) and from 2% to 39% or even complete absence in green algae (Rho and Litsky, 1979). The actual cellulose yields found for plant tissue of aquatic macrophytes, aquatic mosses and green algae fall in this range, with mean values of replicate treatments from about 7% to 23% (Fig. 1b–d). Highest amounts Author's personal copy 1549 100 80 60 40 20 0 30 25 20 15 10 5 0 30 25 20 15 10 5 0 30 25 20 15 10 5 0 0.20 (a) Avicel FlukaCell IAEA-C3 (b) Yield (%) Yield (%) H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 Elodea I Potamogeton Callitriche Myriophyll. I Myriophyll. II Elodea II Yield (%) (c) Fontinalis I Drepanocladus Aquatic moss Rhizoclonium Vaucheria Fontinalis II Yield (%) Yield (%) (d) Cladophora (e) 0.15 0.10 0.05 0.00 HZM surf HZM trap HZM 6500 SAC surf SAC 1700 POND wen SOIL for Materials Fig. 1. Extraction yield of standard cellulose powders (a), macrophyte tissue (b), water moss tissue (c), green algae (d) and sediments (e) using UWEIL (blue bars), JUEL (green bars) and CUAM (brown bars) methods. Height of bars represents arithmetic mean values of replicate treatments and thin lines the respective standard deviation (1s). of cellulose were obtained from aquatic mosses, while yields for macrophytes were lowest, with cellulose con- tents of green algae being intermediate. The differences in yield for the same tissue with the different methods are not as distinct as those found for the standard cellulose materials but may reach comparable orders of magnitude on the basis of absolute cellulose content. None of the methods stands out from the others in terms of the amount of cellulose that can be gained from a certain material. The reproducibility in replicate treatments is considerably improved vs. standard cellulose powders. In the majority of cases one standard deviation does not greatly exceed a value of 1%. Thus, within the methodological uncertainties, cellulose yields from plant tissue can comprise a good estimate of relative differences in cellulose content between plant taxa. Yields for sediments could not be compared between the different methods because of methodological problems (i.e. impurities) with UWEIL and JUEL that are discussed below. The yields with CUAM for diverse sedimentary materials from seston to soil (Fig. 1e) are about two orders of magnitude lower than those found for plant tissue. These rather low cellulose contents agree well with results from studies on natural sediments and soil with other methods of cellulose quantification (Uzaki and Ishiwatari, 1983; Ogier et al., 2001; Martens and Loeffelmann, 2002). The yields were low, independent of the carbon content of the sample. Despite rather low cellulose content in the samples, the reproducibility within the replicates is quite satisfactory (1s 6 0.01%). The small amounts of cellulose in these materials illustrate the demanding methodological requirements that have to be met for the reproducible and quantitatively comparable extraction of cellulose from sediments of various kinds. 3.2. Chemical and isotopic characterisation of extraction residues from standard cellulose powders Fig. 2 shows the IR spectra of the untreated and CUAMtreated commercial cellulose powders. They exhibit sev- 1.8 1.6 Absorbance (A.U.) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 -0.4 4000 3750 3500 3250 3000 2750 1800 1600 1400 1200 1000 800 600 400 -1 Wavenumber (cm ) Fig. 2. IR absorbance spectra of unprocessed and CUAM-processed standard cellulose powders: Avicel (untreated: light blue, treated: dark blue), IAEA-C3 (untreated: orange, treated: brown) and FlukaCell (untreated: light green, treated: dark green). Author's personal copy 1550 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 eral absorption bands and peaks characteristic of cellulose (Silverstein and Webster, 1998; Pandey, 1999; Pandey and Pitman, 2003; Liu et al., 2004). At high frequencies a strong O–H stretching (H-bonded) absorption is seen around 4000–3000 cm 1, and a prominent C–H stretching absorption at 2900 cm 1. The peaks in the fingerprint region between 1800 and 800 cm 1 have been assigned to absorbed O–H and conjugated C–O at 1640 cm 1, C–H deformation in carbohydrates at 1426 cm 1, C–H deformation in cellulose at 1373 cm 1, CH2 wagging at 1317 cm 1, O–H deformation at 1200 cm 1, C–O–C asymmetric vibration at 1162 cm 1, glucose ring stretch at 1107 cm 1, C–O stretch at 1058 cm 1, C–O stretch at 1019 cm 1 and C–H deformation in cellulose at 894 cm 1. Compared with the controls, several of these characteristic bands changed in absorbance or wavenumber in the CUAM-treated spectra. Common to all standard celluloses the peaks at 1110 and 1429 cm 1 are concealed, whereas absorbance is increased at 894 cm 1 and in the band from 350 to 550 cm 1 (Fig. 2). For Avicel and IAEA-C3 maximum absorbance of H-bonded O–H stretching is shifted to higher wavenumber (from 3365 to 3444 cm 1) and the C–H stretch is slightly shifted to lower wavenumber (from 2900 to 2895 cm 1). Similar changes in absorbance characteristics of standard celluloses are also effected by UWEIL and JUEL (not shown). The peak shifts observed are attributed to the alkaline treatment, either by NaOH or NH3, that is part of all the extraction protocols (Oh et al., 2005a,b). Alteration in absorbance at 1650 and 3364/3444 cm 1, increases for Avicel and IAEA-C3 and decreases for FlukaCell, indicate changes in crystallinity and amount of adsorbed water as induced by chemical treatment (Hurtubise and C/O 1.1 Krässig, 1960; Liu et al., 2004; Oh et al. 2005a). Similarly, coeval changes in absorbance in the bands at 1426 and 894 cm 1 are referred to as index for changes in cellulose crystallinity (A1426/A894) by Oh et al. (2005a,b). In summary, the chemical treatments caused alteration of the spectra of standard celluloses in relation to the amount of adsorbed water and crystallinity. These changes were, however, not specific but common to all three methods. Further drying at higher temperature (> 100 °C) would not necessarily lead to the removal of a larger amount of adsorbed water below the thermal decomposition threshold (Carrillo et al., 2004). According to the stoichiometric C and O content of cellulose the theoretical C/O ratio is 0.90. Among the untreated standard cellulose powders only FlukaCell has exactly this theoretical value, while values for Avicel and IAEA-C3 are slightly higher (Fig. 3a). Alteration of the ratio by the treatments is generally small to moderate, with the exception of IAEA-C3, where all methods, and especially the JUEL protocol, led to lower values. This could reflect distinctive characteristics for this material, lower degree of polymerization, probably in conjunction with an increased amount of absorbed water caused by the chemical treatment. The d13C and d18O values of the treated standard cellulose powders are shown in Fig. 3b and c. All three methods cause insignificant changes in the carbon isotope compositions although a slight unidirectional move to depleted values is visible vs. untreated cellulose. A similar but stronger shift is evident for the oxygen isotopes, with offsets reaching values between 0.5‰ and 1.0‰ depending on material and method. The depleted values are possibly 1.1 (a) 1.0 1.0 0.9 0.9 0.8 0.8 -23 -24 -24 -25 -25 18 δ O (‰) 13 δ C (‰) (b) -23 33 32 31 30 29 28 27 (c) Avicel FlukaCell IAEA-C3 33 32 31 30 29 28 27 Materials Fig. 3. C/O ratio (a), carbon isotope composition (b) and oxygen isotope composition (c) of standard cellulose powders. Comparison between untreated cellulose (black) and cellulose with three different extraction methods, UWEIL (blue), JUEL (green) and CUAM (brown). Shown are mean values (closed circles) of replicate treatments of the same material together with the overall mean (open squares) and standard deviation (bars) for all measurements of the respective material. Author's personal copy 1551 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 associated with changes in water content evident from IR spectroscopy and, however less likely, exchange between cellulose water and demineralised water. Specific isotope labelling experiments would be needed to verify this hypothesis. Nevertheless, all the methods cause carbon and oxygen isotope changes indistinguishable from each other. The results prove that the use of cuprammonium solution (alkali dissolution/acid precipitation) does not result in exceptional changes in the isotopic composition of cellulose extracted with the CUAM method compared to conventional methods. 3.3. Chemical and isotopic characterisation of extraction residues from aquatic plant tissue 3.3.1. Aquatic macrophytes Fig. 4 shows an example of IR spectra for an aquatic macrophyte of the genus Myriophyllum (Myriophyllum II), comparing the plant tissue with extraction residues from the different treatments. The spectrum of the untreated tissue reveals several major bands relating, according to Kačuráková et al. (2000), Giordano et al. (2001) and Gorgulu et al. (2007) to a range of molecular components. The strong absorption in the region 3700– 3000 cm 1 arises mainly from N–H and O–H stretching modes of proteins and polysaccharides, intermolecular H bonding and adsorbed water. The peak around 2917 cm 1 is caused by CH2 and CH3 symmetric and asymmetric stretching in lipids and proteins. In the fingerprint region, the Amide I band at ca. 1650 cm 1 is assigned to C@O stretching in proteins and pectin. The strong absorbance of the Amide I band partly disguises the C@O stretching from phospholipids and hemicellulose at 1722 cm 1. Various conspicuous bands in the region 1200–950 cm 1 are assigned to C–O stretching, OH stretching and P@O stretching, indicating a combination of different cell wall polysaccharides. The spectra of the extraction residues show notably different absorbance patterns, indicating the effectiveness of the respective chemical treatments. The excellent match between Myriophyllum II treated via JUEL and CUAM and the target cellulose spectrum (cf. Fig. 2) testifies to the complete removal of contaminants and the yield of pure cellulose with these protocols. In contrast, the UWEIL-treated spectrum of Myriophyllum II shows atypical strong bands at 1093 and 465 cm 1 and reduced absorbance at 3440 and 2900 cm 1. The distinctive peak at 465 cm 1 and the higher wave number of the band at 1093 cm 1 vs. cellulose (1063 cm 1) can best be explained by biogenic opal impurity in the extraction residue. Biogenic opal, characterised by strong absorbance at 1091 and 464 cm 1 (cf. Fig. 10b), disguises the bands typical for cellulose and dominates the spectrum of the Myriophyllum II extract for UWEIL. The opal contamination is most likely caused by frustules of epiphytic diatoms that were not removed by mechanical cleaning and alkaline treatment. Thus, Myriophyllum II is an example where the UWEIL method is insufficient for complete removal of biogenic silica. Because of the opaline dominance in the spectrum we can not further evaluate whether organic components also contaminate the cellulose in such cases. The challenge of opal contamination of aquatic macrophyte tissue can not solely be solved by mechanical pre-cleaning since the IR spectrum of the thoroughly cleaned Myriophyllum I (not shown) is, similar to Myriophyllum II, also strongly influenced by opal bands. The C/O values of macrophyte tissue from different genera decrease to varying degrees with all the treatments but emerge overall at 0.85–0.95 (Fig. 5a). The difference between the untreated and the treated tissue varies from genus to genus, most likely due to variable contents of hydrocarbons and oxygen-bearing contaminants. The UWEIL method gave unexpectedly low values (av. 0.78) for Myriophyllum I for unknown reasons. The carbon iso- 2.0 1.8 Absorbance (A.U.) 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 4000 3750 3500 3250 3000 2750 1800 1600 1400 1200 1000 800 600 400 -1 Wavenumber (cm ) Fig. 4. IR absorbance spectra of whole tissue and extraction residue of an aquatic macrophyte, genus Myriophyllum retrieved from Jülich pond (Myriophyllum II). Untreated tissue (black) in relation to tissue treated using UWEIL (blue), JUEL (green) and CUAM (brown) methods. Author's personal copy 1552 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 C/O 1.1 1.0 0.9 0.9 0.8 0.8 0.7 0.7 13 δ C (‰) (b) -16 -20 -20 -24 -24 -28 -28 -32 -32 26 18 1.1 1.0 -16 δ O (‰) (a) (c) 26 23 22 21 20 19 18 23 22 21 20 19 18 Elodea I Potamogeton Callitriche Myriophyllum I Myriophyllum II Elodea II Materials Fig. 5. C/O ratio (a), carbon isotope composition (b) and oxygen isotope composition (c) of macrophyte cellulose. Comparison between the untreated bulk material (black) and cellulose for different extraction methods, UWEIL (blue), JUEL (green) and CUAM (brown). Shown are mean values (closed circles) of replicate treatments of the same material together with the overall mean (open squares) and standard deviation (bars) for all measurements of the respective material. tope composition of macrophyte tissue showed large variations between genera (Fig. 5b). Independently, the different treatments caused only small deviations from the untreated control, with negligible differences between the single variants. The largest deviation is for Potamogeton, with a 13C enrichment of 1.0‰ after all treatments. Removal of non-cellulose compounds from plant tissue should, in general, increase the d18O value of the residue because, e.g. lipids are isotopically lighter than cellulose (Boutton, 1996; van Dongen et al., 2002). As expected, ‘‘cellulose” fractions from the extraction treatment consistently show an 18O enrichment of 3–5‰ vs. bulk tissue (Fig. 5c). Enrichment seems to be independent of the bulk value and is greatest for Elodea I. In general, observed enrichments are similar to those reported for wood cellulose (Barbour et al., 2001). The variations between different treatments are small and generally of the order of 0.5‰. The exception, the UWEIL-treated Myriophyllum II sample, shows 1‰ less enrichment than the other variants. As indicated from IR spectroscopy, the most likely reason is contamination of the cellulose by biogenic opal. At temperatures of 1450 °C, the typical temperature for organic matter pyrolysis, biogenic opal is dehydrated and partly decomposed, thereby contributing opal-bound oxygen to the oxygen bound in cellulose (Lücke et al., 2005). 3.3.2. Aquatic mosses IR spectra of extraction residues from the different treatments of Fontinalis II are shown as examples for aquatic mosses in Fig. 6. The spectra of residues generally resemble that of standard Fluka cellulose at high frequencies. Below 1200 cm 1 and especially in the band from 750 to 350 cm 1, discrepancies arise between the spectra of UWEIL and JUEL on one hand and CUAM on the other. In this region a peak at 471 cm 1, accompanied by a smaller one around 519 cm 1, indicates minerogenic impurities in both the UWEIL and JUEL fractions (cf. Fig. 10a). Moreover, absorbance at 1360 cm 1 is reduced vs. 1063 cm 1 for both the UWEIL and JUEL fractions. The argument for minerogenic contamination is also supported by a shift in maximum absorbance in the 1060 cm 1 region from 1063 cm 1 in Fluka cellulose to 1022 cm 1 in the UWEIL extract. Such contamination is absent from the CUAMtreated extraction residue of Fontinalis II. Extracts from aquatic mosses have C/O values around 0.9 (Fig. 7). Only for Fontinalis II treated with UWEIL and JUEL do C/O values decline towards 0.8. Fontinalis I is characterised by a rather high initial ratio of almost 1.3. This might be the cause of the rather high offset of 2.5‰ between the carbon isotope composition of bulk and cellulose fraction of Fontinalis I. Extracted cellulose from every other moss tissue is enriched by 0.8–1.3‰. Differences in the carbon isotope ratios of cellulose extracted with the different methods are small and are within the analytical uncertainty. Like macrophyte tissue, the cellulose fractions of aquatic mosses are enriched by 1.3–4.8‰ in oxygen isotope composition compared to bulk tissue. Although oxygen isotope differences between the treatments are generally small and are well within the analytical uncertainty, the Fontinalis II residue from the CUAM method shows a significant enrichment vs. both the other variants. As the oxygen isotopic composition of minerogenic matter is depleted relative to organic matter this has to be interpreted as inorganic contamination of the UWEIL and JUEL Author's personal copy 1553 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 1.4 1.2 Absorbance (A.U.) 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 4000 3750 3500 3250 3000 2750 1800 1600 1400 1200 1000 800 600 400 -1 Wavenumber (cm ) Fig. 6. IR absorbance spectra of extraction residues of an aquatic moss, genus Fontinalis (Fontinalis II). Shown are spectra for tissue treated using UWEIL (blue), JUEL (green) and CUAM (brown) methods in relation to standard cellulose FlukaCell (grey). 1.3 1.3 (a) 1.2 1.1 1.1 1.0 1.0 0.9 0.9 0.8 0.8 -28 -30 -28 -30 (b) -36 -36 -38 -38 -40 -40 18 δ O (‰) 13 δ C (‰) C/O 1.2 -42 26 24 22 20 18 16 14 -42 26 24 22 20 18 16 14 (c) Fontinalis antipyretica I Drepanocladus Aquatic moss Fontinalis antipyretica II Materials Fig. 7. C/O ratio (a), carbon isotope composition (b) and oxygen isotope composition (c) of cellulose from aquatic mosses. Comparison between the untreated bulk material (black) and cellulose from different extraction methods, UWEIL (blue), JUEL (green) and CUAM (brown). Shown are mean values (closed circles) of replicate treatments of the same material together with the overall mean (open squares) and standard deviation (bars) for all measurements of the respective material. treated tissue, as also indicated by IR spectroscopy (Fig. 6). A smaller, but still apparent, depleted oxygen isotope value appears for CUAM-treated ‘aquatic moss’ vs. UWEIL and JUEL (0.5–0.7‰). The spectrum of CUAM ‘aquatic moss’ reveals a comparatively strong absorption at 1635 cm 1 and an absorbance ratio between 3348 and 1060 cm 1 (A3348/ A1060) that is atypically high for cellulose (spectrum not shown). Increased absorbance at these values can be caused by adsorbed water, but could also indicate trace amounts of proteins and polysaccharides (3348, 1635 cm 1) in the cellulose fraction. As this effect is only observed in one case and not evident for tissue of other moss genera, we presume contamination by undefined organic matter containing proteins and polysaccharides that were probably removed in the NaOH step of UWEIL and JUEL. Author's personal copy 1554 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 3.3.3. Freshwater algae Typical IR spectra for whole tissue and extraction residues are exemplified by the freshwater chrysophyte Vaucheria (Fig. 8). The whole plant tissue is characterised by several bands similar to macrophyte tissue. Strong absorbance in the region 3700–3000 cm 1 is due to O–H stretching and N–H stretching modes of water, carbohydrates and proteins, CH2 and CH3 stretching bands with strong absorbance at 2959, 2924 and 2856 cm 1 indicating lipids; strong C@O and C@N stretching (Amide I and Amide II) at 1645 and 1537cm 1 are attributed to proteins; the peak at 1247 cm 1 is assigned to P@O stretching in nucleic acid and peaks in the region 1200–900 cm 1 are attributed to C–O–C stretching of cell wall polysaccharides (Giordano et al., 2001; Sigee et al., 2002; Dean et al., 2007; Gorgulu et al., 2007). The IR spectra of the treated material reveal clear differences between the methods in as much as the spectra of the UWEIL and JUEL extracts indicate organic impurities, while only the CUAM extract reveals a pure cellulose spectrum for Vaucheria (Fig. 8). The pattern of multiple absorbance peaks around 2900 cm 1 for the UWEIL and JUEL material is indicative of traces of lipids in the cellulose fraction. The spectrum of the JUEL material additionally shows maximum absorbance in the region 3700– 3000 cm 1 at 3342 cm 1. Together with comparably reduced absorbance around 3250 cm 1, including a secondary maximum at 3270 cm 1, this could indicate unknown impurities in the JUEL residue (Bahmed et al., 2003; Ramírez-Coutiño et al., 2006). In the fingerprint region peaks at 1426, 1370 and 1336 cm 1, in combination with a relative reduction in absorbance at 1650 cm 1 for the UWEIL and JUEL extract, might be explained by hemicellulose impurities (Proniewicz et al., 2001; Pandey and Pitman, 2003; Olsson and Salmén, 2004). However, this can also be caused by differences in the crystallinity of cellulose extracted from Vaucheria. This is also indicated by the in- creased peak ratio of 1425 cm 1 over 896 cm 1 (A1425/ A896) pointing towards a higher crystallinity for UWEIL and JUEL extracted cellulose vs. that from CUAM (Wada and Okano, 2001; Oh et al., 2005a,b). C/O values of cellulose extracted from green algae tissue vary between 0.85% and 0.96% (Fig. 9). Reference values for untreated Rhizoclonium and Vaucheria are missing due to lack of material after a failure of the analytical system in the analysis of oxygen isotope ratios. Results of earlier low temperature pyrolysis (1080 °C) measurements are given instead as an indicator of the general tendency between raw material and cellulose. The carbon isotope composition of extraction residues of the different treatments are indistinguishable for Rhizoclonium and Vaucheria, while CUAM cellulose from Cladophora is considerably less enriched in 13C (0.9‰). A similar picture emerges for the oxygen isotope compositions. CUAM cellulose from Cladophora is 2‰ more enriched than UWEIL and JUEL cellulose. The impurities in the cellulose fraction of Vaucheria visible in the IR spectra (Fig. 9) for UWEIL and JUEL treatments did not apparently impact on the isotopic compositions. Either they did not differ in isotopic signature from the cellulose or their contributions to the total extract were tiny. IR spectra of Cladophora reveal evidence of either impurities in the fingerprint region or increased crystallinity (A1425/A896) for UWEIL and JUEL and an additional opal component in the UWEIL extract (spectra not shown). The latter is clearly not visible in the JUEL extract spectrum despite the comparably low C/O values for both UWEIL and JUEL extracts indicating excess oxygen, potentially from biogenic opal. Overall, reasons for the isotopic similarity between UWEIL and JUEL and the dissimilarity to CUAM, respectively, observed for Cladophora and Vaucheria remain ambiguous. In summary, it appears that the performance of the CUAM method with aquatic plant tissue is in most cases at least equal to conventional cellulose extraction meth- 2.0 1.8 1.6 Absorbance (A.U.) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 4000 3750 3500 3250 3000 2750 1800 1600 1400 1200 1000 800 600 400 -1 Wavenumber (cm ) Fig. 8. IR absorbance spectra of whole tissue and extraction residue of a yellow-green alga, genus Vaucheria. The spectrum of untreated tissue (black) is shown in relation to tissue treated obtained using UWEIL (blue), JUEL (green) and CUAM (brown). Author's personal copy 1555 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 1.10 C/O 1.05 1.10 (a) 1.05 1.00 1.00 0.95 0.95 0.90 0.90 0.85 0.85 0.80 13 δ C (‰) -16 0.80 (b) -16 -18 -18 -20 -20 -22 -22 -24 18 δ O (‰) 28 -24 (c) 28 26 26 * 24 24 * 22 22 20 18 20 Cladophora Rhizoclonium Vaucheria 18 Materials Fig. 9. C/O ratio (a), carbon isotope composition (b) and oxygen isotope composition (c) of cellulose from freshwater algae. Comparison between the untreated bulk material (black) and cellulose obtained using three different extraction methods UWEIL (blue), JUEL (green) and CUAM (brown). Shown are mean values (closed circles) of replicate treatments of the same material together with the overall means (open squares) and standard deviations (bars) for all measurements of the respective material; * indicates that untreated oxygen isotope values for Rhizoclonium and Vaucheria were generated by pyrolysis at 1080 °C and illustrates the tendency but not the absolute value of change between raw material and cellulose. ods. Thus, no artificial fractionation effect for carbon or oxygen is introduced by the CUAM treatment. Moreover, the CUAM method provided reliable cellulose d18O values even for samples for which the UWEIL and the JUEL methods could not completely remove impurities from the extracted cellulose. 3.4. Chemical and isotopic characterisation of extraction residues from sediments Unlike plant tissue, sediments and soil are composed of a mixture of inorganic and organic matter of different origins. Moreover, the organic fraction is itself highly diverse since it consists of the remains of vascular and non-vascular plants, refractory organic matter and biominerals. The spectroscopic fingerprint of such common inorganic and organic constituents in sediments and soil is illustrated in Fig. 10. The IR spectrum of illite represents an example of the large group of clay minerals. Characteristic absorbance peaks appear at 3694 and 3617 cm 1 and in the fingerprint region at 1107, 1034, 1008, 912 cm 1 and at 538, 471 and 430 cm 1 (Fig. 10a). The location and intensity of these peaks may vary for other inorganic components (e.g. muscovite, montmorillonite, smectite), nevertheless, the overall pattern of spectral intensity is similar (Kovac et al., 2004). The spectrum of biogenic opal derived from diatom frustules (Fig. 10b) typically shows two prominent peaks at 1091 and 464 cm 1, distinguishing opal from clay minerals and quartz (Kovac et al., 2004; Moschen et al., 2006). In contrast to opal, the spectrum of chitin is com- plex and has typical bands allowing differentiation between chitin and cellulose (Fig. 10c and d). Characteristic for chitin are peaks in the high frequency region at 3440 cm 1 (O–H stretch, N–H stretch), 3272 cm 1 (N–H stretch) and 3108 cm 1 (Amide II band overtone) together with a broad multi-peak band around 2923 cm 1. In the fingerprint region chitin is readily distinguished from cellulose by the Amide bands I and II at 1627–1658 and 1554 cm 1, respectively, and the characteristic bands at 1381 and 1314 cm 1 (Shigemasa et al., 1996; Bahmed et al., 2003; Ramírez-Coutiño et al., 2006). We evaluated the performance of the UWEIL method including the density separation step and the CUAM method on sedimentary materials using a fine-grained surface sediment from the mesotrophic/eutrophic Lake Holzmaar. Prior to density separation the IR spectrum reveals definite evidence of impurities in the UWEIL extraction residue (Fig. 11, light blue line). Minerogenic particles are indicated by bands at 1025, 528 and 470 cm 1, while peaks at 3272 and 3108 cm 1, as well as at 1627/1658 and 1554 cm 1, represent chitinous remains. The spectrum of the sample after density separation shows, however, almost complete accordance with the former spectrum, without significant improvement (Fig. 11, dark blue line). Clearly, cleaning by density separation was not capable of further purifying the cellulose by removing embodied organic and inorganic impurities. For comparison, we tested the effect of demineralization using HF (10%), but with even less success with respect to the removal of minerogenic matter (Fig. 11, grey line). The extraction quality Author's personal copy 1556 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 6 clay mineral (illite) 4 2 0 Absorbance (A.U.) 2 biogenic opal (Fragilaria) 1 0 0.4 chitin 0.2 0.0 0.4 cellulose 0.2 0.0 4000 3500 3000 1800 1600 1400 1200 1000 800 600 400 -1 Wavenumber (cm ) Fig. 10. IR absorbance spectra of common constituents of lake sediments representing potential contaminants in the fraction of extracted cellulose. Shown are spectra of untreated illite (brown) as representative for fine clastic contaminants (clay minerals), of biogenic opal from a freshwater diatom (yellow) and of standard chitin as representative for zooplankton chitin (green) in comparison to CUAM-treated FlukaCell (blue). Mark the axis break on the abscissa. 1.0 0.8 Absorbance (A.U.) 0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 -0.8 4000 3750 3500 3250 3000 2750 1800 1600 1400 1200 1000 800 600 400 -1 Wavenumber (cm ) Fig. 11. IR absorbance spectra of the extraction residue from the Holzmaar surface sediment gained by different treatments. Shown are results for UWEIL treatment before (light blue) and after density separation (dark blue), demineralisation with HF with subsequent UWEIL treatment without density separation (grey) and CUAM treatment (brown). differs remarkably for the CUAM-treated residue from the same raw material. Here, the IR spectrum reveals the absorbance pattern of pure cellulose without any detectable impurities (Fig. 11, brown line). This is fundamental for d18O analysis, since the presence of minerogenic matter in the cellulose extract risks biasing the oxygen isotope signature of the cellulose. Recently, Gong et al. (2007) demonstrated that even preheated mineral samples can release several percent water at temperatures used for online pyrolysis (1450 °C). Moreover, in the treatment of sed- iments the skill for complete removal of chitin from cellulose is critical since zooplankton chitin (e.g. Cladocera and Chironomidae), comparable in thermal stability to cellulose, has to be expected in almost any sedimentary material. An example of a lacustrine sediment poor in minerogenic matter but rich in biogenic opal and OM is represented by the surface sediment from Laguna Azul (Fig. 12). The IR spectrum of the UWEIL extraction residue is characterised by a comparably broad and unstructured Author's personal copy 1557 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 1.6 1.4 1.2 Absorbance (A.U.) 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 4000 3750 3500 3250 3000 2750 1800 1600 1400 1200 1000 800 600 400 -1 Wavenumber (cm ) Fig. 12. IR absorbance spectra of the extraction residue from the Laguna Azul surface sediment gained by different treatments. Shown are results for UWEIL treatment before (light blue) and after density separation (dark blue), demineralisation with HF with subsequent UWEIL treatment without density separation (grey) and CUAM treatment (brown). band around 1084 cm 1 and peaks at 954, 790 and 465 cm 1 typical for biogenic opal. In this case, density separation removes opaline impurities almost completely, presumably because the majority of biogenic opal is derived from massive sponge spicules (Fig. 12, dark blue line). Only the still visible peak at 465 cm 1 might indicate remaining traces of biogenic opal or minerogenic matter. However, the spectrum of the UWEIL residue including density separation is again dominated by peaks derived from chitin and not from cellulose. Similarly, the HF treatment could remove minerogenic and opaline particles but the spectrum is still dominated by chitin (Fig. 12, grey line). Unfortunately, further evaluation of the composition of the extraction residue using IR spectroscopy with respect to other organic contaminants like hemicellulose or refractory OM is therefore impossible. In comparison to UWEIL, the IR spectrum of the CUAM aliquot shows absorbance peaks indicative neither of opal and chitin nor of minerogenic matter. Again, the CUAM treatment was capable of successfully removing contamination to give a pure cellulose extract from the Laguna Azul sediment (Fig. 12, brown line). The discrepancy in cellulose purification between UWEIL and CUAM as expressed qualitatively by the IR spectra is also apparent in the carbon isotope composition of the extracts. The offset between the Laguna Azul surface sediment extraction residue of UWEIL with density separation ( 19.0‰) and CUAM ( 17.3‰) amounts to 1.7‰ (bulk 24.4‰). We used a variety of additional sedimentary materials to further verify the performance of the CUAM extraction. This included sediment trap material, lacustrine surface sediments, a core sediment of Holocene age (6500 cal BP), a pond surface sample and a forest soil. The IR spectra provide evidence that the extraction in all cases was successful and that the CUAM treatment always isolated a pure and clean cellulose fraction (Fig. 13). The C/O ratio of the CUAM extracts from the sediments shows some variability in replicate analyses (Fig. 14). This is especially the case for samples HZM trap, SAC surface and POND wen, for no discernible reason. Nevertheless, the respective overall mean C/O ratio is close to the theoretical value of 0.90 for cellulose. The increased variability in the elemental composition is not reflected in the carbon and oxygen isotope compositions of the extraction residues, for which reproducibility is rather good. Carbon isotope enrichment in cellulose relative to bulk OM from terrestrial sediments is in the range reported for woody plants, whereas enrichment reaches 6‰ for cellulose from freshwater sediments. This difference is much larger than that observed for macrophytes and algae and reflects the diverse composition of bulk sedimentary OM. Nevertheless, absolute carbon isotope signatures of cellulose from aquatic macrophytes or algae and of cellulose from aquatic sediments are comparable and tend to be enriched relative to terrestrial OM. Since an evaluation of oxygen isotope enrichment compared to bulk OM could not be reasonably accomplished, only the oxygen isotope values of sedimentary cellulose are presented in Fig. 14. For all extracted sedimentary samples, reproducibility of oxygen isotope values within replicates was excellent (Boettger et al., 2007). Highest values are observed for cellulose from terrestrial sediments, expressing strong evaporative enrichment of 18O in these environments. In comparison, d18O values for cellulose from freshwater sediments are up to 5‰ lower. Moreover, cellulose in surface sediment of Sacrower See is enriched in 18 O vs. Lake Holzmaar surface sediment. Actual values of the oxygen isotope composition of lake water reveal that the water body of Sacrower See is indeed enriched compared to that of Lake Holzmaar by ca. 5‰ (Lücke et al., 1998; Bluszcz et al., unpublished results). About half of this isotopic difference in lake water is compensated in the cellulose, probably by admixture of enriched OM from immmersed macrophytes/shore vegetation to the surface sediments of Lake Holzmaar. Author's personal copy 1558 H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 2.4 2.2 2.0 Absorbance (A.U.) 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 4000 3750 3500 3250 3000 2750 1800 1600 1400 1200 1000 800 600 400 -1 Wavenumber (cm ) Fig. 13. IR absorbance spectra of the extraction residue of CUAM-treated sediments. Spectra are arranged from top to bottom for Holzmaar surface sediment (HZM surface), Holzmaar sediment trap (HZM trap), Holzmaar core sediment (HZM 6500), Sacrow surface sediment (SAC surface), Sacrow core sediment (SAC 1700), pond sediment (POND wenig) and forest soil (SOIL for). The spectra of CUAM-treated FlukaCell (grey) is included for reasons of comparison. 1.05 C/O 1.00 1.05 (a) 1.00 0.95 0.95 0.90 0.90 0.85 0.85 0.80 13 δ C (‰) -22 18 -22 -24 -24 -26 -26 -28 -28 -30 -30 -32 31 -32 31 30 δ O (‰) 0.80 (b) (c) 30 29 29 28 28 27 27 26 26 25 25 HZM surface HZM trap HZM 6500 SAC surface SAC 1700 POND wen SOIL for Materials Fig. 14. Carbon to oxygen ratio (a), carbon isotope composition (b) and oxygen isotope composition (c) of cellulose from sedimentary materials gained by CUAM treatment (brown). Shown are mean values (closed circles) of replicate treatments of the same material together with the overall means (open squares) and standard deviations (bars) for all measurements of the respective material. 4. Conclusions A combination of classical approaches for bleaching with sodium chlorite (NaClO2) and cellulose dissolution in cuprammonium solution ([Cu(NH3)4](OH)2) resulted in a novel approach for the quantitative extraction of recent, sub-fossil and fossil cellulose from organic matter, especially freshwater sediments. We have evaluated the perfor- mance of this novel approach (CUAM) against conventional methods for cellulose extraction (UWEIL, JUEL) used in palaeoenvironmental applications. Standard cellulose powders were used to exclude preparative artefacts with respect to isotope composition, potentially introduced by dissolution and precipitation of cellulose in cuprammonium solution employed in the CUAM protocol. Wet chemical treatment led to a reduction Author's personal copy H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561 in C/O values for Avicel and IAEA-C3 independently of the extraction method used. This might indicate an increase in the amount of water absorbed to the cellulose by the treatments. The effects on the isotopic composition of standard celluloses are, however, negligible to small for all the methods used. Carbon isotope values are slightly reduced compared to the control, while oxygen isotope compositions are systematically depleted relative to the respective control. Within the analytical uncertainties, all three methods led to identical changes in isotopic composition of standard cellulose powders, so CUAM is indistinguishable from conventional methods with respect to any operational artifacts. In accord with the isotopic results, IR investigations indicate a certain change in crystallinity and water content of standard cellulose treated using CUAM. However, these changes did again not exceed those introduced with the UWEIL and JUEL methods. To further verify possible systematic deviations in isotopic composition of cellulose due to any laboratory treatment, an in depth study would be desirable to evaluate the effects on the oxygen isotope composition of cellulose extracted from natural samples. Extractions with aquatic plant material of diverse genera from different families revealed the capacity of the CUAM method to isolate cellulose from whole plant tissue. IR spectroscopy as well as isotope composition demonstrate that the CUAM method afforded clean and pure cellulose from any sample with one exception (‘aquatic moss’). In all other cases, the CUAM performance was at least equal to those of the other methods and in numerous cases achieved better results in terms of cellulose purification. Contamination of several tissue samples with small amounts of minerogenic matter and biogenic opal could not be removed from the extraction residue with the UWEIL and JUEL methods and consequently led to partly erroneous isotope results. Underestimation in oxygen isotope composition caused by these impurities was as great as 2‰ compared to CUAM cellulose. Like results reported by Rinne et al. (2005) for woody samples, our experiments show that solvent extraction of resins and tannins is dispensible in cellulose extraction from fresh plant material and organic sediments. The main aim of the study was an evaluation of the capability of the CUAM protocol to give clean and pure cellulose from sedimentary materials for isotopic studies. First, two typical fine-grained lacustrine sediments were used in an intercomparison exercise based mainly on IR spectroscopy of the resulting extraction residues. Neither the JUEL method (as a priori expected) nor the UWEIL method, including density separation or HF treatment, was successful in isolating a pure cellulose fraction from the bulk sediments. The spectra revealed contamination of the extraction residues by minerogenic matter and biogenic opal as well as chitin. Bands characteristic of chitin dominated the spectra, especially in the fingerprint region and mainly disguised those of cellulose. Accordingly, beside other impurities, chitinous remains of zooplankton were still present in the extraction residues from JUEL and UWEIL in quantities large enough to bias the determination of the isotopic composition of the cellulose. Only extraction residues of the CUAM-treated samples revealed 1559 IR spectra resembling those of standard cellulose powders without evidence for contamination. Second, the CUAM extraction was verified with a series of sedimentary materials such as soil, sediment trap and sediment core material. The IR spectra of the extracts demonstrated that the extraction was successful in providing clean and pure cellulose from these sediments. C and O isotope compositions of the extracted celluloses are in accord with previous knowledge about the samples and substantiate the capability of the CUAM method. We have shown that CUAM is a reliable cellulose extraction method that yields clean and pure cellulose, without inorganic and organic contamination, for isotope analysis. Plant tissue and mixed sediments can be treated alike, granting full comparability of isotopic results from cellulose between different types of material. The CUAM method is potentially superior to other methods for the treatment of sedimentary material and can most likely be used not only for soils and freshwater sediments but also for marine sediments and organic rich deposits like peat and lignite. The advantage rests in the complete removal of contamination from the target through isolation of cellulose by controlled dissolution and precipitation. Nevertheless, rigorous quality controls for the extraction process is recommended since the composition of organic matter in sediments can be highly variable and age and burial depth may affect the feasibility of the method. Acknowledgements We are indebted to Mrs. A. Richter for IR spectroscopic analysis. D. Enters provided sediments from the Sacrower See. Determination of aquatic plants by B. Messyasz is gratefully acknowledged. We thank T.W.D. Edwards and P. Finch for valuable comments. Financial support was provided by the German Federal Ministry of Education and Research in the framework of the German Climate Research Programme DEKLIM (Grant 01 LD 0001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the on-line version, at doi:10.1016/ j.orggeochem.2008.07.014. Associate Editor—G. 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