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FEMS Microbiology Ecology 46 (2003) 121^128 www.fems-microbiology.org Isotope fractionations in the biosynthesis of cell components by di¡erent fungi: a basis for environmental carbon £ux studies Wolf-Rainer Abraham , Christian Hesse GBF ^ German Research Center for Biotechnology, Research Group Chemical Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany Received 5 September 2002; received in revised form 23 July 2003 ; accepted 25 July 2003 First published online 27 August 2003 Abstract The isotope fractionation of carbon from substrates possessing different isotope ratios into fatty acids of polar lipids and amino acids was determined for four different fungi (Rhizopus arrhizus, Mortierella isabellina, Fusarium solani, Aspergillus niger). Carbon isotope ratios of fungi closely followed that of the substrates. Palmitic acid (C16:0), derived from phospholipids, did not display a large carbon isotope fractionation against the substrate. Stearic acid (C18:0), however, was depleted in 13 C against C16:0 in all strains. The desaturation of C18:0 to oleic acid (C18:1g9) had little effect on the carbon isotope ratio. The subsequent desaturation of C18:1g9 to linolic acid (C18:2g6,9) enriched the resulting C18:2g6,9 by +3.9x and varied little among strains. This result is important because C18:2g6,9 is often used as a biomarker in environmental studies. Most amino acids were enriched in 13 C compared to the substrates, but isoleucine and lysine were close to the isotope ratio of the substrate and phenylalanine and leucine were depleted. Interestingly, the carbon isotope ratios of many amino acids differed significantly among different species. A discriminant analysis based on the isotope ratio of four amino acids (Thr, Ile, Phe, Val) resolved the two phyla in the first discriminant function and all four strains in the first two discriminant functions and confirmed a taxon-specific manner of isotope fractionation. The derived rules provide the basis for the use of stable isotopes in environmental studies to elucidate the role of fungi in the carbon flow in the environment. 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Amino acid; Fatty acid; Isotope ratio mass spectrometry ; Phospholipid; Ascomycotina; Zygomycotina 1. Introduction Microorganisms in the biosphere are responsible for the degradation and recycling of nearly all organic compounds including man-made pollutants. To achieve the degradation of chemical compounds they usually do not act independently from their neighbours but work in microbial consortia. These consortia show a multitude of interactions to ensure their functioning, e.g. they possess organisms protecting the microbial community from toxic metals [1], detoxifying intermediates, which are dangerous for some of their members [2] or forming aggregates to protect the community from grazers. One important characteristic * Corresponding author. Tel. : +49 (531) 6181 419 ; Fax : +49 (531) 6181 411. E-mail address : [email protected] (W.-R. Abraham). Abbreviations : GC/C/IRMS, gas chromatography/combustion/isotope ratio mass spectrometry ; C16:0, palmitic acid; C18:0, stearic acid; C18:1g9, oleic acid; C18:2g6,9, linolic acid of microbial communities is their substrate sharing, which allows them to degrade complex or not easily accessible compounds [2]. Furthermore, it enables them to compensate better for environmental changes like shifts in temperature, salinity, pH or shock loadings with pollutants. Almost nothing is known about metabolic networks in microbial community and, furthermore, currently e¡orts are focused on interactions among bacteria [3], ignoring the impact of fungi in many ecosystems. The elucidation of this carbon sharing between di¡erent members of microbial communities is a prerequisite for the understanding of many aspects of carbon £ux in the environment and the use of microbial consortia for biotechnological applications. To resolve the activities within microbial communities and to understand the role of their individual members are the main challenges in microbial ecology. This requires methods for determination of carbon £ux within these consortia with a minimal disturbance of the communities and without cultivating their individual members. One of the most promising techniques to directly monitor carbon 0168-6496 / 03 / $22.00 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0168-6496(03)00203-4 FEMSEC 1564 17-9-03 122 W.-R. Abraham, C. Hesse / FEMS Microbiology Ecology 46 (2003) 121^128 £uxes in microbial consortia is the determination of the isotope ratios of the substrates and their comparison to those of taxon-speci¢c biomarkers of the consortium [4^6]. These biomarkers can be any kind of biomolecules. Currently, phospholipids [7,8], their fatty acids [9^11] and outer membrane proteins [12] are the most promising candidates. For isotope analyses these molecules are separated in a gas chromatograph and then oxidised to carbon dioxide, which is ¢nally analysed in an isotope ratio mass spectrometer. Alternatively, the compounds are oxidised in an elemental analyser (EA) coupled to an isotope ratio mass spectrometer [13]. A similar routine is applied to the isotope analysis of nitrogen [14]. To get valuable results in microbial communities, the isotope fractionation in pure cultures needs to be determined. Such a study has been done for the fatty acids of bacterial glyco- and phospholipids [15], but none has been reported for fungi. In the present study we determined the isotope fractionation of carbon from substrates with di¡erent isotope ratios into fatty acids of polar lipids and amino acids. For this study we chose Rhizopus arrhizus ATCC 11145 and Mortierella isabellina DSM 1414 as members of the Zygomycotina and Fusarium solani DSM 62416 and Aspergillus niger DSM 2182 belonging to the Ascomycotina. The aim of this work was to evaluate di¡erent cell compounds of fungi for carbon £ux studies in the environment by means of determination of the isotope fractionations for di¡erent amino acids and fatty acids and comparing the observed isotope fractionations with di¡erent fungi and di¡erent substrates. cedure [16] as described [17]. The total lipid fraction was fractionated and the fatty acid methyl esters prepared as described previously [9]. Individual fatty acid methyl esters were identi¢ed by their retention time compared to standards. 2. Materials and methods 2.4. Notation R. arrhizus ATCC 11145, M. isabellina DSM 1414, F. solani DSM 62416 and A. niger DSM 2182 were grown in minimal media with three di¡erent carbon sources. For comparison all strains were also grown in the same media, but with sodium nitrate of N15 N 6.02x instead of N15 N 9.05x. Minimal medium for fungi contained: 3 g sodium nitrate, 0.25 g yeast extract (N13 C 322.88x, N15 N 32.64x), 1.3 g dipotassiumhydrogen phosphate, 0.5 g potassium chloride, 0.5 g magnesium sulphate heptahydrate, 50 mg iron(II)sulphate heptahydrate and 0.7 g citric acid monohydrate (N13 C 324.9x) in 1 l of distilled water. To this minimal medium 10 g l31 of glucose (N13 C 310.10x), mannose (N13 C 323.71x) or mannose (N13 C 315.55x) were added. All strains were grown at 30‡C and 100 rpm in 2-l £asks ¢lled with 200 ml medium and the biomass was harvested after 72 h. The ratio of inoculum to harvested biomass (w/w) was between 1:200 and 1:500 depending on substrate and strain. All strains were grown and analysed twice in all media. The standard notation for expression of high-precision gas IRMS results in the N notation de¢ned as 2.1. Polar lipid fatty acid analysis Lipids were extracted using a modi¢ed Bligh^Dyer pro- 2.2. Determination of isotope ratios by EA/combustion/isotope ratio mass spectrometry (EA/C/IRMS) Biomass (200 mg wet weight) was washed three times with phosphate bu¡er (20 ml) and then freeze-dried. A portion (1^0.25 mg) of carbon substrate or dried biomass was combusted in an EA Fisons 1108 with CHN packing and analysed with the isotope ratio mass spectrometer. The analyses were run ¢ve times. 2.3. Gas chromatography (GC)/C/IRMS Measurements were performed on a Finnigan MAT 252 isotope ratio mass spectrometer in triplicate. It was coupled with a HP 5890 gas chromatograph via a combustion interface. The fatty acid methyl esters were separated with a Restek Rtx-2 column (60 m, 0.32 mm inner diameter, 0.25 Wm ¢lm thickness). The column e¥uent was combusted on-line in an oxidation oven (copper, nickel, platinum catalyst, 980‡C), passed through a reactor with elemental copper (600‡C) for reducing NOx and removing surplus O2 . Combustion gas was dried by a water-permeable membrane (Na¢on). N ðxÞ ¼ ððRFAME =RPDB Þ31ÞU103 ð1Þ where RFAME and RPDB are the 13 C/12 C isotope ratios corresponding to the sample and to the international standard PeeDee Belemnite, a South Carolinian carbonate rich in 13 C (RPDB = 0.0112372 N 0.0000090), respectively [18]. The isotope ratio of nitrogen is measured against air (N15 N = 0x) where the 15 N ratio is RAir = 0.003669. 2.5. Calculation of the isotope ratios of the fatty acids The derivatisation of the fatty acids introduced one additional carbon, which was not present in the parent compound and which altered the original isotope ratio of the fatty acids. However, the derivatisation process introduced a distinct reproducible fractionation that was constant for each fatty acid [19,20]. The measured isotope ratios of the FAMEs were corrected for the isotope ratio of the methyl moiety to get the FEMSEC 1564 17-9-03 W.-R. Abraham, C. Hesse / FEMS Microbiology Ecology 46 (2003) 121^128 isotope ratios of the fatty acids. This was done by using the formula to calculate the isotope ratios of the fatty acids [21,22]. N 13 CFA ¼ ððC n þ 1ÞUN 13 C FAME 3N 13 CMeOH Þ=Cn 13 ð2Þ 13 N CFA = N C of the fatty acid ; Cn = number of carbons in the fatty acid; N13 CFAME = N13 C of the fatty acid methyl ester (FAME); and N13 CMeOH = N13 C of methanol used for the methylating reaction = 337.57x. 2.6. Amino acid analysis Wet cells (about 200 mg) were treated with 6 M HCl (100‡C, 24 h) in order to hydrolyse all proteins to amino acids. Evaporation with a gentle stream of nitrogen at 60‡C was followed. To remove water as azeotrope dichloromethane was added and then evaporated. The carboxyl groups were esteri¢ed with isopropanol/acetyl chloride (5:1, v/v, 105‡C, 45 min). The esteri¢ed samples were evaporated to dryness and the amino group was acylated with tri£uoroacetic acid anhydride at 100‡C for 15 min. After each reaction step any excess of reagents was evaporated by a gentle stream of nitrogen. Capillary gas chromatographic analyses were performed on a Hewlett Packard 5890 Series II gas chromatograph equipped with a capillary column HP Ultra 2 (50 m; i.d. 0.2 mm; ¢lm thickness 0.11 mm) and FID. The oven programme was: 60‡C for 2 min, ¢rst increased to 200‡C at 5‡C min31 , afterwards to 290‡C at 8‡C min31 , followed by an isothermal period of 10 min. 2.7. GC/C/IRMS The amino acid derivatives were separated as described for the fatty acid methyl esters, but a capillary column HP Ultra 2 (50 m; i.d. 0.32 mm; ¢lm thickness 0.25 mm) was used. 2.8. Calculation of the isotope ratios of the amino acids The measured isotope ratios of the amino acid derivatives were corrected for the isotope ratio of the tri£uoroacetate and isopropyl moieties and the controlled isotope e¡ect on the derivatisation to get the isotope ratios of the amino acids. This was done by using the formula: N 13 CAA ¼ ðN 13 CDAA ðnAA þ xniso þ ynTFA Þ 3N 13 Ckorr ðxniso þ ynsTFA ÞÞ=nAA ð3Þ N13 CAA = N13 C of amino acid; nAA = number of carbons in the amino acid; niso = number of carbons in isopropyl = 3; nTFA = number of carbons in tri£uoroacetic acid = 2; x = number of isopropyls in amino acid derivative ; y = number of tri£uoroacetates in amino acid derivative ; and N13 CDAA = N13 C of amino acid derivative. 123 Eq. 3 gives 13 N Ckorr ¼ ðN 13 CDAA ðnAA þ xniso þ ynTFA Þ 3N 13 CAA nAA Þ=ðxniso þ ynTFA Þ ð4Þ The isotope ratio N13 CAA of amino acids was determined using amino acid standards for the EA connected to the isotope ratio mass spectrometer. The same amino acids were derivatised to give the isopropyl ester tri£uoroacetates for which the isotope ratios N13 CDAA were determined using GC/C/IRMS [23]. Eq. 4 was then applied to determine N13 Ckorr from these two isotope ratios. Finally, N13 Ckorr was used in Eq. 3 to calculate the isotope ratios of the amino acids obtained from the di¡erent fungi [24,25]. The fatty acid and amino acid data were analysed using Statistica 5.0 software (Statsoft, Tulsa, USA). Discriminant analysis was used to test for a phylogenetic component in isotope fractionations in cell compounds. A review on the various uses of discriminant analysis is given in Huberty [26]. To discern between the four fungi a canonical analysis with three orthogonal, i.e. independent, functions was computed using the isotope ratios of 12 amino acids from the fungi grown on the three carbon substrates with the two nitrates. A Chi-square with successive removal of discriminant functions test gave for all discriminant functions P-levels 6 0.003 and Wilks’ lambda 6 0.25. 3. Results 3.1. Carbon and nitrogen isotope fractionations of fungi When grown on glucose the fungi were depleted in 13 C (by 31.37 N 0.07x) compared to the substrate, but grown on mannose (N13 C 315.55x) they were enriched (+1.46 N 0.40x). With the isotopically more depleted mannose (N13 C 323.71x) the isotope ratio of the fungi was not signi¢cantly di¡erent from that of the substrate. With glucose as the carbon source the Ascomycotina (Fusarium 30.39 N 0.05x, Aspergillus 30.56 N 0.22x) were less depleted in 13 C than the Zygomycotina (Rhizopus 32.44 N 0.18x, Mortierella 32.11 N 0.09x). Grown on mannose (N13 C 315.55x) the fungi were all enriched in 13 C (+0.88 to +2.43x) and with the lighter mannose (N13 C 323.71x) M. isabellina was depleted (31.38x) but the other three species were enriched (+0.13 to +0.57x). The nitrogen isotope ratios of the fungi were more variable than those for carbon but always depleted in 15 N relative to the source. Surprisingly, F. solani showed a distinct overall reduced depletion of 15 N against nitrate relative to the other three strains (32.9x compared to 39.2x for Rhizopus, 38.7x for Mortierella, and 37.9x for Aspergillus). The di¡erence between the nitrogen isotope ratios of the two sodium nitrates used were also seen in the di¡erences in N15 N of the biomasses of FEMSEC 1564 17-9-03 124 W.-R. Abraham, C. Hesse / FEMS Microbiology Ecology 46 (2003) 121^128 M. isabellina (2.3x), F. solani (6.6x) and A. niger (11.0x). The exception was R. arrhizus, which did not show such a clear tendency (Fig. 1). The Zygomycotina species (R. arrhizus and M. isabellina) re£ected the isotope ratios of the di¡erent nitrates more clearly than those of the Ascomycotina (A. niger and F. solani). 3.2. Carbon isotope fractionation of fatty acids of phospholipids Biosynthesis of fatty acids is achieved in the multi-enzyme complex fatty acid synthase and ends with palmitic acid (C16:0). C16 :0 is then the starting material for dehydro-fatty acids and longer fatty acids giving this speci¢c fatty acid a central role in the biosynthesis of fatty acids in Eubacteria and higher organisms [27]. C16:0 is present in all eukaryotes [7] and we found it in the phospholipids of all of the strains we used. It is used here as a standard to determine the depletion or enrichment in 13 C from the substrate into fatty acids. Averaged across all fungi and substrates, a 13 C enrichment of less than +1.0x of C16:0 against the substrate and +1.1x against the biomass was found. The next step in the biosynthesis of fatty acid [28], the formation of stearic acid (C18:0) from C16 :0, had a pronounced isotope fractionation of 34.0x compared to the substrate and 34.7x compared to C16:0 (Table 1). Here Zygomycotina and Ascomycotina revealed a di¡erent behaviour. Strains of the Zygomycotina were depleted in 13 C in C18 :0 by 31.4x compared to C16:0 whereas the depletion for the Ascomycotina was up to 35.5x. A rather low isotope fractionation of 30.7x resulted from the desaturation of C18:0 to oleic acid (C18:1g9). The amount of C18 :0 in M. isabellina was too low for useful N13 C data Fig. 1. 15 N isotope ratios of biomass obtained from di¡erent nitrates. (Filled symbols: N15 N 9.05x nitrate ; open symbols N15 N 6.02x nitrate; square: N13 C 23.71x mannose ; rhombus : N13 C 15.55x mannose ; triangle : glucose.) but from the comparison of isotope ratios of C16:0 and C18:1g9 we concluded that this species showed a carbon isotope fractionation similar to R. arrhizus. The next desaturation from C18:1g9 to linolic acid (C18:2g6,9), however, displayed 13 C enrichment of +3.9x which was not much di¡erent between the two groups (+3.6x for Zygomycotina and +4.4x for Ascomycotina). The isotope enrichment in F. solani was considerably larger than for the other species (+7.6x vs. +3.1 to +4.1x). Table 1 Mean carbon isotope fractionation of amino acids and fatty acids (vN13 C) compared to biomass obtained for four substrates for four fungi with standard deviations vN13 C/vN15 N R. arrhizus ATCC 11145 M. isabellina DSM 1414 F. solani DSM 62416 A. niger DSM 2182 vN15 N biomass vN13 C biomass Ala Asx Glx Gly Ile Leu Lys Phe Pro Ser Thr Val C16:0 C18:0 C18:1g9 C18:2g6,9 39.2 N 2.5 30.5 N 1.8 20.0 N 4.8 5.4 N 2.8 10.5 N 3.0 34.3 N 10.6 1.4 N 5.4 36.9 N 5.8 32.6 N 7.4 38.3 N 5.3 4.2 N 4.3 14.2 N 9.1 21.7 N 10.5 19.6 N 5.1 31.0 N 1.8 31.8 N 1.9 32.3 N 1.9 2.0 N 2.0 38.7 N 1.5 30.7 N 1.7 13.9 N 1.2 6.0 N 2.0 10.7 N 2.8 19.0 N 11.2 5.4 N 8.1 38.4 N 2.7 0.8 N 9.6 36.3 N 3.9 3.7 N 2.8 1.6 N 6.5 13.2 N 3.4 10.4 N 11.2 30.3 N 1.8 n.d. 31.6 N 1.9 2.5 N 1.8 32.9 N 1.0 0.8 N 1.3 12.6 N 2.2 9.9 N 1.5 8.6 N 1.2 14.8 N 5.6 2.2 N 2.9 37.7 N 2.1 2.6 N 1.9 0.6 N 3.1 8.1 N 1.8 1.4 N 8.1 2.0 N 2.0 4.8 N 2.4 31.0 N 1.6 33.2 N 1.6 35.6 N 1.5 30.2 N 1.6 37.9 N 1.5 0.4 N 0.8 11.2 N 1.5 6.9 N 1.4 4.5 N 2.1 7.6 N 4.2 33.0 N 2.5 36.4 N 2.2 30.1 N 2.6 32.4 N 2.6 2.0 N 2.6 32.7 N 4.4 30.5 N 2.3 3.2 N 3.4 2.4 N 1.3 0.6 N 2.0 31.6 N 1.2 2.0 N 1.3 For the biomass, di¡erences to the isotope ratios of the substrates are given. FEMSEC 1564 17-9-03 W.-R. Abraham, C. Hesse / FEMS Microbiology Ecology 46 (2003) 121^128 125 3.3. Carbon isotope fractionation of amino acids In all cases alanine, glycine, proline, valine, aspartate and glutamate contained more 13 C than the substrates they were derived from. In most cases threonine and serine were also 13 C enriched during their biosyntheses, while isoleucine and lysine were isotopically similar to the substrate. Phenylalanine and especially leucine were depleted in 13 C compared to the substrate. The phyla behaved differently with regard to the isotope di¡erentiation of 13 C when building the amino acids (Fig. 2). While the Zygomycotina enriched 13 C in threonine by about +15x only minor enrichments of about +2x were found with the Ascomycotina. A similar but less pronounced behaviour was found for alanine, valine and glycine. Phenylalanine was more depleted in 13 C by the Zygomycotina than by the Ascomycotina. All strains showed a very similar 13 C depletion in leucine of about 37 to 38x (Table 1). A comparison of the mean isotope fractionations observed for the di¡erent amino acids for all substrates revealed that they showed constant isotope fractionations with regard to the substrate. The observed isotope fractionation for carbon in the individual amino acids was in most cases larger than the di¡erences of N13 C of the substrates used in this study (Fig. 2). Compared to biomass, leucine, lysine and phenylalanine were depleted in 13 C whereas the other amino acids, especially alanine, glycine, threonine, and valine were enriched. The di¡erence of isotope ratios between biomass and leucine was very similar for the four fungi but it diverged considerably for glycine, threonine, serine, and valine. Again, a taxon-speci¢c in£uence was seen, where the Zygomycotinae were usually more depleted in 13 C than the Ascomycotinae. For phenylalanine, however, this trend was reversed. One of the most pronounced di¡erences in isotope fractionation was found for glutamate/glutamine Fig. 2. Di¡erence of isotope ratios of amino acids to the substrates used. The mean values (based on the three substrates and two nitrates) for di¡erent fungi are shown for each amino acid. For clarity, data points belonging to the same strain are connected with straight lines for Zygomycotina (¢lled rhombus: R. arrhizus ATCC 11145, ¢lled square: M. isabellina DSM 1414) and broken lines for Ascomycotina (¢lled triangle: F. solani DSM 62416, cross : A. niger DSM 2182). Fig. 3. Plot of N13 C of biomass versus glutamate for four fungi grown in six di¡erent media. Larger enrichments of 13 C in Ascomycotina compared to Zygomycotina are observed. where the Zygomycotina enriched 13 C in this amino acid considerably more than the Ascomycotina (+12.1x vs. +7.0x) (Fig. 3). To prove whether the phylogeny of the four species in our study is mirrored in the di¡erent isotope ratios of their amino acids multivariate statistical analyses were applied. The discriminant analysis based on the isotope ratio of the 12 amino acids resolved the phyla Ascomycotina and Zygomycotina in the ¢rst discriminant function and all four Fig. 4. Discriminant analysis based on the isotope ratios of the amino acids threonine, isoleucine, phenylalanine and valine obtained from growth in six di¡erent media (P 6 0.0001, Wilks’ lambda 0.007220). The cumulative proportions of explained variance are 72.3% for the ¢rst discriminant function (eigenvalue 15.378) and 98.7% for the second (eigenvalue 5.628). Zygomycotina (triangle : R. arrhizus, square: M. isabellina) are resolved from Ascomycotina (rhombus : F. solani, cycle: A. niger) in the ¢rst discriminant function and all strains are resolved in the ¢rst two discriminant functions. FEMSEC 1564 17-9-03 126 W.-R. Abraham, C. Hesse / FEMS Microbiology Ecology 46 (2003) 121^128 strains in the ¢rst two discriminant functions. Using the isotope ratios of the four amino acids threonine, phenylalanine, isoleucine and valine in the discriminant analysis, essentially, the same separation of the four species could be achieved (Fig. 4). The ¢rst three amino acids were su⁄cient to discern Zygomycotina and Ascomycotina. Here, threonine was most discriminative having N13 C s 310x for the Zygomycotina and N13 C 6 310x for the Ascomycotina (Table 1). 4. Discussion and conclusions The carbon isotope ratios of the fungi followed closely those of the substrates. The largest isotope fractionation was found with glucose as substrate and here the fungi belonging to the Zygomycotina depleted 13 C signi¢cantly more than those to Ascomycotina. For the two mannose substrates no such distinction between Zygomycotina and Ascomycotina could be made. This result agrees with our earlier ¢nding that biomass of bacteria was depleted in 13 C when grown on glucose but was not signi¢cantly di¡erent from the isotope ratio of the substrate when grown on mannose [15]. All isotope fractionations that we observed for di¡erent fungi were in the range of N 2.5x. For environmental studies it is important to know this margin of isotope fractionations by fungi, which allows the identi¢cation of distinct carbon sources only if they di¡er by more than 2.5x. The nitrogen isotope ratios of the biomasses, however, are not so easy to interpret. Here, a taxon-speci¢c isotope fractionation was observed and F. solani was distinct from the other strains. It has been reported for constructed wetlands that preferably the lighter nitrate was consumed [29] and the results presented here for fungi ¢t well to a theory for 15 N/14 N fractionation in nitrate-grown plants [30], with the exception of F. solani. One explanation for the deviations found in F. solani could be that this species used at least some of the nitrogen of the yeast extract instead of the nitrate. This observation indicates that application of a single trophic level shift in isotope fractionation including fungi is not justi¢ed given our current level of understanding. Compound-speci¢c analyses revealed large isotope fractionations, presumably indicating di¡erences in the division of carbon £ows at branch points [31,32] and/or the isotope discriminations of di¡erent enzymes involved in compound biosynthesis [33,34]. Some clear rules were found for the di¡erent fatty acids. C16 :0 derived from the phospholipid fraction did not display a large carbon isotope fractionation compared to the substrate. C18:0, however, was depleted in 13 C against C16:0 in all strains. The 13 C depletion was less pronounced in fungi belonging to the Zygomycotina than in those of the Ascomycotina. Such di¡erence between Zygomycotina and Ascomycotina may implicate di¡erent ratios of prolongation of the C16 :0 precursor to C18:0 and de novo synthesis of C18:0 as has been proposed by a model of Monson and Hayes [28]. The desaturation of C18:0 to C18:1g9 did not alter much the carbon isotope ratio of the product and the observed isotope fractionations fell well into the range reported by Monson and Hayes. The subsequent desaturation of C18:1g9 to C18:2g6,9 favoured the 13 C-rich substrate and the resulting C18:2g6,9 was enriched by +3.9x, an amount varying little among the di¡erent strains. This is an important result because C18:2g6,9 is a fatty acid characteristic for eukaryotes and only rarely found in larger amounts in Eubacteria. Therefore, it is often used as a biomarker for fungi in environmental studies [35]. Most amino acids were enriched in 13 C compared to the substrates. Alanine, glycine, proline, valine, aspartate and glutamate were always enriched in 13 C, isoleucine and lysine were close to the isotope ratio of the substrate and phenylalanine and leucine were depleted in 13 C compared to the substrate. Similar results were reported for Chlorella pyrenoidosa and other photosynthetic organisms [36]. It is interesting to note that, on the one hand, species-speci¢c di¡erences in the carbon isotope ratios of many amino acids were observed (Fig. 2) and, on the other hand, the mean of the carbon isotope ratios of individual amino acids from the four fungi of a given substrate showed more or less a constant o¡set for the three di¡erent substrates, re£ecting the isotope ratios of the substrates. As seen in this study di¡erent isotope fractionations, depending on the phylogenetic position of the species, were also found for some fatty acids. This observation agrees with a report that sucrose derived from C3 and C4 plants was fractionated di¡erentially by three ecologically relevant basidiomycete species under controlled laboratory conditions in a taxon-speci¢c manner [37]. This report, however, focused on the biomass and did not state which cell compounds showed di¡erences in their isotope composition and which were not in£uenced by the phylogenetic position. The observation con¢rms again the in£uence of phylogenetic position on the isotope fractionation of carbon. Having the notion that phylogeny is based on the relatedness of the genes and pathways of the organisms and that isotope fractionation is caused by the selectivity of the enzymes involved in the biosynthesis of the compounds, then similar isotope fractionations can point to similar pathways and enzymes acting in fractionation. Future studies using a much broader range of fungal species have to con¢rm the connection of phylogenetic position and isotope fractionation. One application of stable isotope analysis is in the elucidation of carbon £uxes in symbioses. If fungi are involved in symbiosis, the characteristic isotope fractionation of amino acids can be used to determine the share of the di¡erent partners in some detailed carbon £uxes [38,39]. Determination of the isotope ratios of the individual amino acids of the host and the fungal symbiont allows the quanti¢cation of amino acid sharing between the FEMSEC 1564 17-9-03 W.-R. Abraham, C. Hesse / FEMS Microbiology Ecology 46 (2003) 121^128 di¡erent partners. This is only possible by using the isotope fractionations for individual amino acids, which have been reported in this study. From our study it becomes evident that in fungi the carbon isotope ratios of fatty acids from phospholipids are easier to predict than those of the amino acids. Therefore, if amino acids are used for tracing the £ow of carbon in microbial consortia, a careful correction of the isotope fractionation taking place during their biosynthesis has to be done. This di⁄culty can be overcome if substrates that are su⁄ciently enriched in 13 C are used. On the other hand, the much larger range of isotope fractionations observed in amino acids and their dependence on fungal phylogenetic position o¡er some advantages in ecological studies. Acknowledgements We are indebted to Dagmar Wenderoth, Andrea Brinkop, and Peter Wol¡ for their excellent technical assistance. The International Atomic Energy Agency, Vienna (Austria), is acknowledged for providing free reference materials for the calibration of the IRMS. This work was supported by a grant of the German Federal Ministry for Science, Education and Research (Project No. 0319433C) and HGF strategy fund ‘Soil Functions’. References [1] Lu«nsdorf, H., Bru«mmer, I., Timmis, K.N. and Wagner-Do«bler, I. 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