Carbon isotope variability in monosaccharides and lipids of aquatic

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MARINE ECOLOGY PROGRESS SERIES Mar Ecol Prog Ser

Vol. 232: 83–92, 2002

Published May 3

Carbon isotope variability in monosaccharides and lipids of aquatic algae and terrestrial plants Bart E. van Dongen*, Stefan Schouten, Jaap S. Sinninghe Damsté Department of Marine Biogeochemistry and Toxicology, Royal Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB Den Burg, Texel, The Netherlands

ABSTRACT: The stable carbon-isotope compositions of individual monosaccharides and lipids, as well as the bulk stable carbon-isotope composition of total cell material from different aquatic and terrestrial plants were determined. With the exception of a Phaeocystis sp. bloom sample, monosaccharides were generally enriched in 13C by 0 to 9 ‰ compared to the total cell material and significantly enriched (1 to 16 ‰) in 13C compared to lipids (fatty acids, phytol, sterols and alkenones) within single organisms. The depletion of 13C in n-alkyl lipids relative to monosaccharides was larger than the depletion of 13C in isoprenoid lipids relative to monosaccharides. In addition, an isotopic enrichment was observed in the 13C content of C5 monosaccharides compared to glucose in some of the organisms studied, indicating isotopic heterogeneity within carbohydrates. The magnitude of the differences between monosaccharides, total cell material and lipids was far greater than previously reported. Thus, selective assimilation of ingested carbohydrates can lead to isotopic enrichments of heterotrophic biomass and zooplankton and subsequent trophic levels. In addition, since dissolved organic carbon (DOC) and particulate organic carbon (POC) has a significant carbohydrate fraction, the δ13CPOC and δ13CDOC signal will be significantly influenced by the relative amounts and the δ13C values of the carbohydrates present. This has significant implications for the isotopic integrity of the organic matter fractions during carbon cycling in food chains of aquatic ecosystems. KEY WORDS: Carbohydrates · 13C · Stable carbon isotope · Food web · Lipids · Aquatic · Terrestrial Resale or republication not permitted without written consent of the publisher

INTRODUCTION Carbohydrates are the most abundant biochemicals on earth (Aspinall 1983). For instance, carbohydrates comprise about 75 wt% of vascular plant tissues (Sjöström 1981) and 20 to 40 wt% of plankton (Parsons et al. 1984). The carbohydrates comprise the bulk of the biomass in marine and terrestrial environments together with proteins and lignins (Tissot & Welte 1978, Parsons et al. 1984). In addition, carbohydrates are a significant component of oceanic dissolved organic matter (DOM; Mopper et al. 1980, Benner et al. 1992, Pakulski & Benner 1994, McCarthy et al. 1996, Borch & Kichman 1997, Skoog & Benner 1997, Burdige et al. 2000). DOM represents one of the largest

*E-mail: [email protected] © Inter-Research 2002 · www.int-res.com

dynamic reservoirs of reduced carbon on earth (~1018 g carbon, i.e. the same magnitude as all living vegetation on the earth’s continents), and is larger than the atmospheric CO2 pool (Hedges 1992). Because carbohydrates, together with proteins and lignins, represent > 40% of the degraded carbon, they are important nutrients for the benthic and pelagic communities (Cowie et al. 1992). Even at low concentrations, excreted carbohydrates provide a substantial food source for heterotrophic bacteria and zooplankton (Wright & Hobbie 1966, Gocke 1975, Williams & Yentsch 1976), as indicated by their rapid turnover rates. In the assessment of food web structures in freshwater and marine ecosystems, the potential of stable carbon-isotope (δ13C) ratios as natural tracers in food chains is increasingly acknowledged (e.g. Michener & Schell 1994) and a number of studies utilizing stable

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carbon isotopes have been published (e.g. Yoshii et al. 1999, Van der Zanden et al. 2000). These studies show that the δ13C of the heterotrophic biomass is primary influenced by the δ13C of the autotrophic food source. Consequently, because carbohydrates are an important constituent of autotrophic carbon and selective assimilation can take place (e.g. Volkman et al. 1980, Grice et al. 1998), the carbon-isotope composition of carbohydrates will significantly influence the carbonisotope composition of the heterotrophs and zooplankton. Thus, information on the carbon-isotope composition of carbohydrates is of primary importance for reconstructing food web relations based on isotopic compositions. In addition, since carbohydrates are an important component of DOM their carbon-isotope composition will significantly influence the bulk DOM carbon-isotope composition. Until now, using bulk measurements, carbohydrates were generally thought to be slightly enriched in 13C. The magnitude of this enrichment was found to be up to 3 to 4 ‰ relative to total cell material and 4 to 10 ‰ relative to lipids (e.g. Deines 1980). Wong et al. (1975) reported an enrichment in 13C of bacterial carbohydrates of up to 6 ‰ relative to the total cell material and up to 16 ‰ in 13C relative to the lipids. Very little is known about the δ13C values of carbohydrates at the molecular level. In contrast, much attention has been paid to another class of biochemicals, the lipids. This is mainly because of the easy accessibility of these compounds by gas chromatography techniques due to their apolar nature. Differences between δ13C values of individual lipids within single organisms were found to be large (Schouten et al. 1998). However, the magnitude of the biosynthetic enrichment of δ13C in monosaccharides relative to total cell material and lipids is unclear and detailed molecular isotopic analyses of individual monosaccharides are lacking. Recently, we developed a new method (Van Dongen et al. 2001) to reliably measure the δ13C values

of individual monosaccharides. In this study we determined the stable carbon-isotope composition of individual monosaccharides in a range of marine and freshwater algae and terrestrial higher plants. These were compared with the 13C content of the total cell material and individual lipids to infer the implications for carbon isotopic studies of food webs and carbon cycling in aquatic systems.

MATERIALS AND METHODS Cell material. The aquatic and terrestrial plants that were analyzed in this study and their culture conditions or sampling sites are listed in Table 1. The freshwater species Tetraedron minimum, Scenedesmus communis and Pediastrum boryanum were cultured under conditions described by Blokker et al. (1998a,b). Two different cultures of T. minimum were cultured, 1 continuous culture and 1 batch culture. The continuous culture was kept at a constant pH of 7.0, while the pH of the batch culture was between 5 and 6 at the start and between 8 and 9 at the end. Cultures of the marine species Isochrysis galbana and Rhodomonas sp. were grown in Erlenmeyer flasks (3 l) with a continuous air supply. Light was kept on a 16:8h light: dark regime and dilution rates ranged between 0.13 and 0.22 d–1. The algae were grown on F/2 medium (Guillard 1975). The alga Phaeocystis sp. was collected with plankton nets (50 µm mesh) during spring blooms in the western Dutch Wadden Sea and the southern North Sea in 1991 and 1992 and stored frozen (–20°C) until use. Field specimens of Sphagnum cuspidatum and the roots of Erica tetralix were obtained from the peat bog reserve ‘Bargerveen’ (Zwartemeer, SE Drenthe, The Netherlands: Baas et al. 2000). Immediately after collection, the plants were washed repeatedly with water and any non-indigenous material was removed. The samples were stored frozen until use.

Table 1. Aquatic and terrestrial plants analyzed in this study with their culture or field condition Organism Freshwater Tetraedron minimum Scenedesmus communis Pediastrum boryanum Marine Isochrysis galbana Rhodomonas sp. Phaeocystis sp. Terrestrial Sphagnum cuspidatum Erica tetralix

Class

Culture type

Sampling site

T (°C)

CO2 source

Chlorophyceae Chlorophyceae Chlorophyceae

Batch/Continuous Batch Batch

– – –

19 19 19

Air + 2% CO2 Air + 2% CO2 Air + 2% CO2

Haptophyceae Cryptophyceae Haptophyceae

Continuous Continuous –

– – North Sea bloom

15 15 –

Air Air Air

Bryophyta Ericaceae

– –

Bargerveen peat bog Bargerveen peat bog

– –

Air Air

van Dongen et al.: 13C isotopes in algal monosaccharides and lipids

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rides are measured as their alditol acetates, the origin of some of the alditol acetates is uncertain. For instance, glucitol could originate from glucose as well as fructose (Macko et al. 1998). In these cases the results of the MBD method, which preserves the original isomeric structure, in combination with the alditol acetate method, were used to infer the origin of the different alditol acetates. The MBD method, a derivatization method to make monosaccharides GC-amenable, was used to determine the δ13C values of the different monosaccharides as described by Van Dongen et al. (2001). Typically, 0.5 ml of a solution of 10 mg methylboronic acid in 1 mL pyridine was added to the hydrolyzed material and the solution was heated at 60°C for 30 min. Fig. 1. Extraction and isolation scheme of plant cell material Subsequently, 15 µl N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was added and the solution was stirred at 60°C for 5 min. To remove any solid material, the soluMonosaccharide analysis. Cell material of the plants was collected, freeze-dried and extracted as described tion was filtered through a pipette filled with MgSO4 by Schouten et al. (1998). The extraction, separation with ethyl acetate as an eluent. The solution was concentrated using evaporation under nitrogen as necesand identification of the monosaccharides as well sary. The monosaccharide fractions were analyzed by as the lipids are schematically depicted in Fig. 1. Briefly, cell material was ultrasonically extracted with gas chromatography (GC), gas chromatography-mass methanol (3 ×), methanol/dichloromethane (1:1, 3 ×) spectro-metry (GC/MS), and isotope-ratio monitoring and hexane/dichloromethane (hex/DCM, 1:1, v/v mixGC/MS (IRM-GC/MS) as described in ‘Instumental analysis’. ture; 3 ×). The extracts were analyzed for lipids as Lipids. The separation and identification of lipids are described in the following subsection. Approximately 7 to 15 mg of dried residues left after extraction were schematically depicted in Fig. 1. Saponification, methstirred with 4 ml 12 M H2SO4 at room temperature for ylation and silylation were performed as described by 2 h. The solution was diluted to 1 M and the polysacSchouten et al. (1998). In some instances, phytol and charides were hydrolyzed for 4.5 h at 85°C. The acidic sterols were present in relatively low amounts compared solution was neutralized with BaCO3. The precipitate to fatty acids, and δ13C values could not be determined was removed by centrifugation, the residue washed accurately. In these cases, the total ex-tracts were filtered twice with bidistilled water and the combined water over an Al2O3-column using ethyl acetate as an eluent to remove the fatty acids. The total extracts of Erica Tetralix layers were freeze-dried. The hydrolyzed material was and Sphagnum cuspidatum were eluted over a SiO2analyzed for monosaccharides using 2 different methcolumn using hex/DCM (9:1, v/v mixture) as eluent to ods. To determine the relative amounts of the different collect the n-alkanes and DCM/methanol (1:1, v/v mixmonosaccharides, they were analyzed using the alditol ture) to collect the fatty acids and sterols and phytol. The acetate method as described by Klok et al. (1982, long-chain alkenones present in Isochrysis galbana were 1984). The methylboronic derivatization method (MBD measured in the saponified fraction to avoid co-elution method; described in subsection ‘Instrumental analywith the long-chain alkenoates present. The lipid fracses’) was not used to determine the relative amounts of tions were analyzed by GC, GC/MS and IRM-GC/MS as all the monosaccharides since the derivatization of a described in the following section. number of the monosaccharides is incomplete (Van Instrumental analyses. Gas chromatography (GC) Dongen et al. 2001). The alditol acetate method was performed with a Hewlett Packard 6890 instruinvolves the reduction of monosaccharides with NaBH4 ment and gas chromatography-mass spectrometry to alditols and acetylation of the alcohol groups using (GC-MS) using a Hewlett-Packard 5890 gas chromatoacetic anhydride and pyridine with the use of myograph interfaced with a VG Autospec Ultima mass inositol as internal standard. Because the monosaccha-

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spectrometer. Lipid analysis was carried out as described by Schouten et al. (1998). For analysis of alditol acetates, a fused silica capillary column (l = 25 m; i.d. = 0.32 mm) coated with CP Sil-88 (film thickness 0.25 µm) was used. Samples were dissolved in ethyl acetate and injected at 70°C. The temperature was raised to 150°C at 20°C min–1, 3°C min–1 to 240°C and held for 10 min. Since the number of added carbon atoms during the derivatization reaction is different for the different monosaccharides the relative amounts were corrected for added carbon. In order to measure The δ13 values of the monosaccharides, the MBD method was used for the derivatization as described by Van Dongen et al. (2001). The derivatized compounds were separated on a fused silica capillary column (l = 30 m; i.d. = 0.32 mm) coated with CP Sil-19 (film thickness 0.12 µm). Samples were injected at 70°C and the temperature was raised to 80°C (20°C min–1) and held for 10 min. Subsequently, the GC was heated at 4°C min–1 to 180°C followed by 10°C min–1 to 280°C and held for 1 min. The alditol acetates and methyl boronic acid derivatives were identified on the basis of known relative retention times established by the analysis of standard mixtures and on the basis of mass spectra (Reinhold et al. 1974, Klok et al. 1982, 1984, Van Dongen et al. 2001). Isotope-ratio-monitoring gas chromatography-mass spectrometry (IRM-GC-MS) was performed using a Finnigan DELTA-C. The same columns, column conditions and temperature program was used as in the case of GC analysis. The δ13C values reported were obtained by at least 2 analyses and the results were averaged to obtain a mean value. The MBD method implies the introduction of extra carbon atoms from the derivatization agent: 2 in the case of arabinose and xylose

Fig. 2. Structures of the compounds analyzed

and 5 in the case of glucose (Fig 2). To correct for the alteration in the stable carbon-isotope composition due to the introduction of these additional carbon atoms, the δ13C of the derivatization agents (methylboronic acid and BSTFA) were determined (Van Dongen et al. 2001). The corrections for the introduced carbon due to the methylation or silylation of the fatty acids, phytol and sterols were performed as described by Schouten et al. (1998). The stable carbon-isotope compositions are reported in delta notation as determined against the Vienna PDB 13C standard. The stable carbon-isotope composition of the bulk cell material (δ13Cbulk) was determined by automatic online combustion (Carlo Erba CN analyser 1502 series) followed by conventional isotope ratio-mass spectrometry (Fisons optima: Fry et al. 1992).

RESULTS AND DISCUSSION 13

C content of monosaccharides

The monosaccharide fraction of the freshwater algae and the marine alga Rhodomonas sp. mainly consisted of glucose (65 to 90%) and other C6-monosaccharides (mannose, rhamnose and galactose: Fig. 3). In Isochrysis galbana the major component was also glucose (30%), but it was relatively less abundant. Phaeocystis sp. had as major monosaccharide arabinose (46%), and the terrestrial plants Sphagnum cuspidatum and Erica tetralix xylose (24 and 47%). In addition to these monosaccharides, considerable amounts of other C5 (mainly ribose), C6 (rhamnose, mannose and galactose) and C7-monosaccharides were found. Using the MBD method, the δ13C values of the different monosaccharides (where possible) were determined (Table 2). For the cultured algae (except Isochrysis galbana) only the δ13C of glucose could be reliably determined. The contribution of other monosaccharides was too small and consequently could not been determined or, in the case of mannose, the derivatization by the MBD method was incomplete (Van Dongen et al. 2001) and the δ13C values obtained were deemed not reliable. For Isochrysis galbana, Phaeocystis sp. and both terrestrial plants, the δ13C of glucose and the C5-monosaccharides arabinose and/or xylose could be determined (Table 2). Sometimes small amounts (< 6%) of other monosaccharides were co-eluting with the C5monosaccharides, i.e. fucose co-eluted

van Dongen et al.: 13C isotopes in algal monosaccharides and lipids

Fig. 3. Percentage of monosaccharides relative to total amount of alditols in different aquatic and terrestrial organisms. Other C6-monosaccharides included rhamnose, mannose, galactose and fucose. The structures of the C7-monosaccharides are unknown. Full specific names as in Table 1

with arabinose and ribose co-eluted with xylose. In those instances an average δ13C value for co-eluting peaks was determined. Except for Phaeocystis sp., all δ13C values of the monosaccharides were enriched in 13C compared with δ13Cbulk (values of bulk cell material; Table 2, Fig. 4), i.e. 2 to 9 ‰ for glucose, 5 to 7 ‰ for xylose and 6 to 8 ‰ for arabinose. In contrast, the monosaccharides δ13C were depleted up to 4 ‰ in 13C relative to the δ13Cbulk

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value, for Phaeocystis sp. Compared with previous reports (e.g. Wong et al. 1975, Deines 1980) the magnitude of these differences is greater than has been reported on the basis of bulk determinations of monosaccharides. These differences are also far more variable than previously reported. This indicates a large heterogeneity in the relative isotopic compositions of monosaccharides in photoautotrophic organisms. The large enrichment (up to 8 ‰) in 13C of the individual monosaccharides relative to δ13Cbulk in the case of the Sphagnum species (S. cuspidatum) is surprising, since the 13C content of monosaccharides versus bulk material found by Macko et al. (1990) for a Sphagnum species were significantly different (depleted by up to 1 ‰). The reason for this could be that different Sphagnum species have different isotopic contents. Another difference between the 2 studies is the fact that Macko et al. used the alditol acetate method for isotopic analysis; this is prone to larger errors in δ13C determination (Van Dongen et al. 2001). To explain the large 13C-enrichment of monosaccharides we have to take into account that other biochemicals, such as lipids, are generally depleted in 13C compared with monosaccharides (see below). The reason why the δ13Cbulk of the Phaeocystis sp. was enriched compared with the δ13C values of the different monosaccharides could be the fact that this was a bloom sample. It is possible that isotopic fractionation within the cell during bloom conditions may be different than during non-bloom conditions. In addition, it is possible

Fig. 4. δ13C values of monosaccharides and lipids relative to δ13Cbulk in different aquatic and terrestrial organisms. The δ13C values of the fatty acids and sterols used are the average values of the C14-C18 fatty acids and C27-C29 sterols, respectively. Full specific names as in Table 1

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Table 2. δ13Cbulk and δ13C values (‰) of monosaccharides, fatty acids, phytol, sterols and alkenones in different aquatic and terrestrial organisms (with analytical error, which includes error in measurement as well as error through derivatization). Full specific names as in Table 1 Component

T. minimum S. communis P. boryanum Rhodomonas sp.

I. galbana

Phaeocystis S. cuspidatum sp.

E. tetralix

Bulk material Monosaccharides Arabinose Xylose Glucose Fatty acidsc C14 C16 C18 Phytol Sterolsc C27 C28 C29 Alkenonesc C37:nk C38:nk

–47.9 ± 0.1

–13.8 ± 0.2

–17.5 ± 0.3

–25.6 ± 0.1

–25.9 ± 0.2

–20.2 ± 0.6a –18.2 ± 0.9b –21.3 ± 0.9

–19.5 ± 0.7 –23.8 ± 0.7

–24.5 ± 0.5 –24.0 ± 0.1 –27.8 ± 0.3 –20.3 ± 0.7

–35.0 ± 0.2 –33.1 ± 0.5 –31.2 ± 0.5

–39.8 ± 0.1

–40.1 ± 0.1

–12.1 ± 0.2

a

–39.2 ± 0.8

–35.5 ± 0.4

–32.8 ± 0.4

–4.9 ± 0.4

–53.9 ± 0.1 –54.6 ± 0.4 –53.6 ± 0.7

–50.2 ± 0.6 –47.0 ± 0.4 –48.3 ± 0.2

–47.3 ± 0.3 –47.7 ± 0.5 –40.9 ± 0.7

–21.4 ± 0.4 –20.4 ± 0.5 –20.2 ± 0.2 –16.3 ± 0.2

–8.2 ± 0.6a –7.1 ± 0.9 –6.8 ± 0.3

a b

–19.1 ± 0.1 –23.0 ± 0.3 –19.0 ± 0.1 –14.8 ± 0.3 d

e

–48.7 ± 0.4e h –48.5 ± 0.4h

e

–47.0 ± 0.6e i –47.3 ± 0.4i

e

+ 38.4 ± 0.7e i + 39.5 ± 0.5i

f

–16.9 ± 0.2f

g

–21.8 ± 0.1g

–22.2 ± 0.5d –19.7 ± 0.5f

f

a

h

–18.0 ± 1.0a –21.0 ± 0.9 –23.7 ± 0.8

–28.8 ± 0.6 –29.3 ± 0.3

–35.1 ± 0.2h –34.4 ± 0.4j

j

–18.6 ± 0.1 –17.9 ± 0.4

a

Small contribution of fucose Small contribution of ribose c Subscripts indicate number of carbon atoms d Cholest-5-enol e 24-methylcholest-7-enol and 24-methylcholesta-7, 22-dienol f 24-ethylcholesta-5, 22-dienol g 24-methylcholest-5, 22-dienol h 24-methylcholest-5-enol and 24-methylcholesta-5, 22-dienol i 24-ethylcholest-7-enol j 24-ethylcholest-5-enol and 24-ethylcholesta-5, 22-dienol k Average of n = 2 to n = 4 b

that other undetermined 13C-enriched carbon material, present in the bloom but not derived from Phaeocystis sp., was influencing the δ13Cbulk. Alternatively, there may be other, as yet undetermined, 13C-enriched (compared with the δ13Cbulk) components present in Phaeocystis sp. itself. Comparison of the δ13C values of the C5-monosaccharides arabinose and xylose with those of glucose within the same organism revealed significant differences. Xylose was up to 4 ‰ enriched in 13C and arabinose up to 6 ‰ enriched in 13C compared with glucose, with the exception of Isochrysis galbana. To the best of our knowledge this is the first time that such large differences between C5 and C6-monosaccharides have been reported. It is possible that a fractionation effect occurs during the decarboxylation of glucose to form C5-monosaccharides. However, such a kinetic isotope effect is likely to result in a depletion in 13C of the product formed (C5-monosaccharide) relatively to the reactant available (C6-monosaccharide) at the reaction site (Hayes 1993, 2001). It is also possible that there are significant differences between the carbon-isotope com-

positions of the different carbon atoms in glucose, as found by Ivlev et al. (1987) and Rossmann et al. (1991), which then lead to an isotopic difference between glucose and the C5-monosaccharides. In order to differentiate between these 2 possible explanations more detailed information about the biosynthesis of C5monosaccharides and the 13C fractionation of the enzymes involved needs to be obtained.

13

C content of lipids

The most dominant lipids present in the total extracts of all the algae except Isochrysis galbana were C14-C18 saturated and unsaturated fatty acids, phytol, and C28C29 sterols, with a distribution similar to those reported by Wood (1988). Phytol (Fig. 2) and sterols were also found in I. galbana, but the most abundant compounds were C37 and C38 alkenones (Fig. 2; Marlowe et al. 1994). Only traces of fatty acids were found. The dominant compounds in the total extract of Sphagnum cuspidatum and the roots of Erica tetralix were C16-C18

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89

Fig. 5. Biosynthesis of fatty acids and isoprenoids starting from glucose. Carbon atoms are numbered according to their original position in the glucose carbon skeleton

fatty acids and C28-C29 sterols. Minor amounts of C21C23 n-alkanes, triterpenoids and wax esters were found, with a distribution similar to those reported for S. cuspidatum by Baas et al. (2000). Significant amounts of phytol were found in S. cuspidatum. Phytol is the main alkyl side chain of chlorophyll and no chlorophyll is present in roots, and hence it was not present in the roots of E. tetralix. When possible, the δ13C of the lipids were determined, for comparison with the δ13C of the different monosaccharides (Table 2). As the unsaturated and saturated C18 fatty acids were often co-eluting, an average δ13C value of the total cluster of unsaturated and saturated C18 fatty acids was determined. For the same reasons, the average δ13C value of the C37 and C38 alkenones were determined in Isochrysis galbana. The fatty acids were considerably depleted in 13C compared with glucose (Table 2, Fig. 4), i.e. the C14-C18 fatty acids were 12 to 16 ‰ depleted for freshwater and marine algal cultures, 3 to 6 ‰ for Phaeocystis sp., and 5 to 13 ‰ for terrestrial plants. Phytol and sterols were generally less depleted in 13C compared with glucose (Table 2) than the fatty acids, i.e. phytol was 8 to 14 ‰ depleted for the freshwater and marine algal cultures and about 7 ‰ depleted in 13C for Sphagnum cuspidatum. In contrast, the δ13C value of phytol from Phaeocystis sp. was similar to that of glucose. The sterols were 6 to 15 ‰ depleted compared with glucose in the freshwater and marine algal cultures and about 11 ‰ depleted in 13C in S. cuspidatum. In contrast, the C27 sterol as well as the C28 sterol measured in Phaeocystis sp. were similar in 13C compared with glucose. The alkenones in I. galbana were 11 to 12 ‰ depleted in 13C compared to glucose. The isotopic differences between the fatty acids, phytol, the sterols and the alkenones in the algal cultures are comparable to those reported previously by Schouten et al. (1998). An enrichment in 13C of monosaccharides compared to lipids has been generally assumed (Hayes 1993, 2001). However, the magnitude of the difference between monosaccharides and lipids determined here is far greater than has been anticipated. The reason for

the depletion of lipids versus monosaccharides is assumed to be a consequence of kinetic isotope effects during enzymatic reactions. Glucose is formed in the Calvin-Benson cycle during photosynthesis. Subsequently, the glucose is cleaved to 2 pyruvate molecules, which are converted to acetyl coenzyme A through decarboxylation (Fig. 5). n-alkyl lipids are synthesized starting from acetyl coenzyme A and isoprenoid lipids are synthesized from pyruvate (Hayes 1993, 2001, Rohmer et al. 1993, Schwender et al. 1996, Lichtenthaler et al. 1997, Disch et al. 1998, and present Fig 5.). The decarboxylation from pyruvate to acetyl coenzyme A is associated with a kinetic isotopic fractionation effect (e.g. Monson & Hayes 1980, 1982a,b, Melzer & Schmidt 1987), leading to a depletion in 13C in the carboxyl atom of acetyl coenzyme A. Thus, n-alkyl lipids are usually depleted in 13C relative to the isoprenoid lipids constructed from pyruvate (Schouten et al. 1998). The different pathways may explain the differences in 13C depletion between individual lipids and monosaccharides. An alternative explanation for the 13C depletion of lipids relative to monosaccharides may be the heterogeneous distribution of 13C in glucose. During the decarboxylation of pyruvate to acetyl coenzyme A the original glucose carbon atoms 3 or 4 are removed. According to Rossmann et al. (1991), these carbon positions are relatively enriched in 13C (about 2 to 6 ‰). This would mean that the acetyl coenzyme A unit formed is relatively depleted in 13C compared with the pyruvate unit and consequently compared to glucose. Because straight-chain lipids are mainly constructed from acetyl coenzyme A, they are depleted in 13C compared with glucose. However, as pointed out by Hayes (1993, 2001), this relative enrichment in 13C of carbon atoms within glucose is too small to explain the observed large depletion in δ13C values of lipids. In addition, the difference between monosaccharides and lipids are even larger (up to 16 ‰ in δ13C), and thus an even larger enrichment in 13C within glucose is required. The observed differences between C5-monosaccharides and glucose indicate that the loss of a sin-

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gle carbon atom can lead to a significant difference in δ13C values (up to 6 ‰). This means that the potential difference in 13C between the carbon atoms in glucose could be significantly larger than reported by Rossman et al. (1991). This makes the alternative explanation for the depletion of lipids, the heterogeneous distrubution of 13C in glucose, more likely, although little is known about the pathways by which these C5-monosaccharides are formed.

Influence of culture conditions on isotopic heterogeneity of biochemicals The large range in differences in 13C content between the monosaccharides, lipids and the bulk material for the different cultured algae could either be due to species-specific fractionation differences or to the culture conditions (e.g. Riebesell et al. 2000). In batch cultures, algae experience different growth conditions at the end of the log growth phase than at the beginning. This can result in changes in the culture medium, such as increasing pH. In water there is a balance between the concentration of CO2, HCO3– and CO32 –. If the pH increases, this equilibrium is shifted to CO32 –, causing a relatively lower dissolved CO2 concentration (Hayes 1993, 2001). This means that less CO2 is available and relatively more enriched CO2 is incorporated, which will result in less 13C-depletion. Thus, these conditions probably result in less 13C-depletion at the end of the end of the log growth phase. In comparison, in the continuous culture there are constant limiting nutrient conditions, which will result in different, but consistent, isotopic fraction patterns. This will probably influence the differences in isotopic composition between the compounds in a single organism. Therefore, we cultured the same algae (Tetraedron minimum) under different conditions, i.e. under continuous culture and under batch culture conditions, and compared the two (Table 3). Glucose was approximately 7 ‰ enriched compared with δ13Cbulk and generally 10 ‰ enriched compared with the fatty acids in the continuous culture. Under batch culture conditions, Table 3. Tetraedron minimum. δ13Cbulk and the δ13C values (‰) of glucose and fatty acids (weighted mean average of C14C18 fatty acids, with analytical error, which includes error in measurement as well as error through derivatization) under different culture conditions Component Bulk material Glucose Fatty acids

Batch

Continuous

–47.9 ± 0.1 –39.2 ± 0.8 –53.6 ± 0.3

–34.7 ± 0.1 –27.6 ± 0.8 –37.6 ± 0.3

glucose was approximately 9 ‰ enriched compared with δ13Cbulk and generally 15 ‰ enriched enriched compared with the fatty acids. The differences in isotopic content between the monosaccharides, lipids and the bulk material for the different cultures of Tetraedron minimum (batch versus continuous: Table 3) seem to support the assumption that culture conditions can influence the differences in 13C content between monosaccharides, lipids and bulk material. Both types of cultures showed the same isotopic differences between fatty acids, monosaccharides and bulk. However, it seems that the different culture conditions influenced the isotopic composition of the fatty acids more than glucose compared with bulk-material values. Differing growth conditions may also explain the difference between the results for cultured algae and those for the natural bloom sample.

Conclusions From our results it is clear that large isotopic differences can exist in 13C content between monosaccharides, lipids and bulk material in photoautotrophic organisms. These organisms comprise the base of the aquatic food-web chain and, depending on the (relative) assimilation of their different biochemicals components, they determine the isotopic composition of heterotrophic bacteria and zooplankton. Zooplankton can alter their bulk isotopic composition through selective assimilation of ingested compounds. Recent studies showed that components like carbohydrates are relatively easily digested and selectively assimilated (e.g. Cowie & Hedges 1996). Thus, not the δ13C values of the algae but the δ13C values of the carbohydrates (and amino acids) produced by these algae will mainly determine the δ13C values of the zooplankton. Suspended particulate organic carbon (POC) and DOM originate from various sources — primary production, resuspended sediment and terrestrial detritus discharged by rivers. The δ13CDOM and δ13CPOC signal is often used as a biomarker indicating the origin of the organic matter (e.g. Salomons & Mook 1981, Fry & Sherr 1984, Laane et al. 1990, Mook & Tan 1991). Organic matter produced by marine phytoplankton is assumed to have an average δ13C value of –21 ‰, while terrestrial C-3 plant-derived organic matter has a content of –27 ‰. DOM and POC consist, to a significant degree, of carbohydrates and, as shown in this study, monosaccharides are significantly enriched in 13C compared with the isotopic composition of the total cell material. This would mean that if the amount of carbohydrates present in the DOM and POC differ significantly, the δ13CDOM and δ13CPOC signals will also differ

van Dongen et al.: 13C isotopes in algal monosaccharides and lipids

significantly. This will make it more difficult to distinguish between the different sources of the organic matter based solely on δ13CDOM and δ13CPOC signals, and alternative methods need to be used, e.g. 14 C analysis (Megens et al. 1998).

Acknowledgements. We thank P. Blokker, W. C. M. Klein Breteler and M. Baas for supplying samples, and W. C. M. Klein Breteler and 3 anonymous referees for their useful comments on the manuscript. H. T. Kloosterhuis is acknowledged for performing bulk carbon isotope measurements. M. Kienhuis, J. P. Werne, E. Schefuss and R. D. Pancost are thanked for analytical assistance, and M. van der Meer for helpful discussions. This research was supported by the NetherlandsBremen Oceanography Program (NEBROC). This is NIOZ contribution 3655.

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Editorial responsibility: Barry & Evelyn Sherr (Contributing Editors), Corvallis, Oregon, USA

Submitted: September 17, 2000; Accepted: January 2, 2001 Proofs received from author(s): April 5, 2002

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