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FEMS Microbiology Ecology 46 (2003) 121^128
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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
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£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
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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
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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.
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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.
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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.
(1997) Metal selectivity of in situ microcolonies in bio¢lms of the
Elbe river. J. Bacteriol. 179, 31^40.
[2] Pelz, O., Tesar, M., Wittich, R.-M., Moore, E.R.B., Timmis, K.N.
and Abraham, W.-R. (1999) Towards elucidation of microbial community metabolic pathways: Unravelling the network of carbon sharing in a pollutant degrading bacterial consortium by immunocapture
and isotopic ratio mass spectrometry. Environ. Microbiol. 1, 167^
174.
[3] Boschker, H.T.S. and Middelburg, J.J. (2002) Stable isotopes and
biomarkers in microbial ecology (Minireview). FEMS Microb.
Ecol. 40, 85^95.
[4] Abraham, W.-R., Hesse, C., Pelz, O., Hermann, S., Tesar, M.,
Moore, E.R.B. and Timmis, K.N. (2001) Sharing of nutritional resources in bacterial communities determined by isotopic ratio mass
spectrometry of biomarkers. In: Focus on Biotechnology (Hofman,
M. and Anne¤, J., Eds.), Vol. 2: Biotechnology for the Environment
(Durieux, A. and Simon, J.P., Eds.), pp. 143^154. Kluwer, Dordrecht.
[5] Boschker, H.T.S., de Brouwer, J.F.C. and Cappenberg, T.E. (1999)
The contribution of macrophyte-derived organic matter to microbial
biomass in salt-marsh sediments: Stable isotope analysis of microbial
biomarkers. Limnol. Oceanogr. 44, 309^319.
[6] Pelz, O., Chatzinotas, A., Andersen, N., Bernasconi, S.M., Hesse, C.,
Abraham, W.-R. and Zeyer, J. (2001) Use of isotope and molecular
techniques to identify the toluene degrading population in anaerobic
microcosms. Arch. Microbiol. 175, 270^281.
[7] Lechavelier, M.P. (1982) Lipids in bacterial taxonomy. In: CRC
Handbook of Microbiology (Laskin, A.I. and Lechavelier, H.A.,
Eds.), pp. 435^541. CRC Press, Boca Raton, FL.
127
[8] Abraham, W.-R., Meyer, H., Lindholst, S., Vancanneyt, M. and
Smit, J. (1997) Phospho- and sulfolipids as biomarkers of Caulobacter, Brevundimonas and Hyphomonas. Syst. Appl. Microbiol. 20,
522^539.
[9] Fredrickson, H.L., Cappenberg, T.E. and de Leeuw, J. (1986) Polar
lipid fatty acid compositions of Lake Vechten seston ^ an ecological
application of lipid analysis. FEMS Microb. Ecol. 38, 381^396.
[10] Vestal, J.R. and White, D.C. (1989) Lipid analysis in microbial ecology. Bioscience 39, 535^541.
[11] Zelles, L., Bai, Q.Y., Beck, T. and Beese, F. (1992) Signature of fatty
acids in phospholipids and lipopolysaccharides as indicators of microbial biomass and community structure in agricultural soils. Soil
Biol. Biochem. 24, 317^323.
[12] Tesar, M., Hoch, C., Moore, E.R.B. and Timmis, K.N. (1996) Westprinting : Development of a rapid immunochemical identi¢cation for
species within the genus Pseudomonas sensu stricto. Syst. Appl. Microbiol. 19, 577^588.
[13] Co⁄n, R., Fry, B., Peterson, B. and Wright, R. (1989) Identi¢cation
of bacterial carbon sources with stable isotope analysis. Limnol. Oceanogr. 34, 1306^1310.
[14] Cifuentes, L.A., Co⁄n, R.B., Solorzano, L., Cardenas, W., Espinozas, J. and Twilley, R. (1996) Isotopic and elemental variations of
carbon and nitrogen in a mangrove estuary. Coastal Shelf Sci. 43,
781^800.
[15] Abraham, W.-R., Hesse, C. and Pelz, O. (1998) Ratios of carbon
isotopes in microbial lipids as indicator of substrate usage. Appl.
Environ. Microbiol. 64, 4202^4209.
[16] Bligh, E.G. and Dyer, W.J. (1959) A rapid method for total lipid
extraction and puri¢cation. Can. J. Biochem. Physiol. 37, 911^
917.
[17] Vancanneyt, M., Witt, S., Abraham, W.-R., Kersters, K. and Fredrickson, H.L. (1996) Fatty acid content in whole-cell hydrolysates
and phospholipid fractions of pseudomonads : a taxonomic evaluation. Syst. Appl. Microbiol. 19, 528^540.
[18] Craig, H. (1957) Isotopic standard for carbon and oxygen and correction factors for mass-spectrometric analysis of carbon dioxide.
Geochim. Cosmochim. Acta 12, 133^149.
[19] Silfer, J., Engel, M., Macko, S. and Jumeau, E. (1991) Stable carbon
isotope analysis of amino acid enantiomers by conventional isotope
ratio mass spectrometry and combined gas chromatography/isotope
ratio mass spectrometry. Anal. Chem. 63, 370^374.
[20] Demmelmaier, H. and Schmidt, H.-L. (1993) Precise N13 C-determination in the range of natural abundance on amino acids from protein
hydrolysates by gas chromatography-isotope ratio mass spectrometry. Isotopes Environ. Health Stud. 29, 237^350.
[21] Abrajano Jr., T.A., Murphy, D.E., Fang, J., Comet, P. and Brooks,
J.M. (1994) 13 C/12 C ratios in individual fatty acids of marine mytilids
with and without bacterial symbionts. Org. Geochem. 12, 611^617.
[22] Goodman, K.J. and Brenna, J.T. (1992) High sensitivity tracer detection using high-precision gas chromatography-combustion isotope
ratio mass spectrometry and highly enriched [U-13 C]-labeled precursors. Anal. Chem. 64, 1088^1095.
[23] Docherty, G., Jones, V. and Evershed, R.P. (2001) Practical and
theoretical considerations in the gas chromatography/combustion/isotope ratio mass spectrometry delta C-13 analysis of small polyfunctional compounds. Rapid Commun. Mass Spectrom. 15, 730^738.
[24] Engel, M.H., Macko, S.A., Qian, Y. and Silfer, J.A. (1994) Stable
isotope analysis at the molecular level: a new approach for determining the origins of amino acids in the Murchison meteorite. Adv.
Space Res. 15, 99^106.
[25] Silfer, J.A., Qian, Y., Macko, S.A. and Engel, M.H. (1994) Stable
carbon isotope compositions of individual amino acid enantiomers in
mollusc shell by GC/C/IRMS. Org. Geochem. 21, 603^609.
[26] Huberty, C.J. (1975) Discriminant analysis. Rev. Educ. Res. 45, 543^
598.
[27] Rawn, J.D. (1989) Biochemistry, Ch. 16: Fatty Acid Metabolism.
Nril Patterson Publishers, Burlington, NC.
FEMSEC 1564 17-9-03
128
W.-R. Abraham, C. Hesse / FEMS Microbiology Ecology 46 (2003) 121^128
[28] Monson, K.D. and Hayes, J.M. (1982) Biosynthetic control of the
natural abundance of carbon 13 at speci¢c positions within fatty
acids in Saccharomyces cerevisiae. J. Biol. Chem. 257, 5568^5575.
[29] Lund, L.J., Horne, A.J. and Williams, A.E. (2000) Estimating denitri¢cation in a large constructed wetland using stable nitrogen isotope
ratios. Ecol. Eng. 14, 67^76.
[30] Robinson, D., Handley, L.L. and Scrimgeour, C.M. (1998) A theory
for 15N/14N fractionation in nitrate-grown vascular plants. Planta
205, 397^406.
[31] Hayes, J.M. (1993) Factors controlling 13C contents of sedimentary
organic compounds: Principles and evidence. Mar. Geol. 113, 111^
125.
[32] Hayes, J.M. (2001) Fractionation of carbon and hydrogen isotopes in
biosynthetic processes. Stable Isotope Geochem. 43, 225^277.
[33] Blair, N.A., Leu, A., Mun‹oz, E., Olsen, J., Kwong, E. and Des
Marais, D. (1985) Carbon isotopic fractionation in heterotrophic
microbial metabolism. Appl. Environ. Microbiol. 50, 996^1001.
[34] Macko, S.A., Fogel, M.L., Hare, P.E. and Hoering, T.C. (1987) Isotopic fractionation of nitrogen and carbon in the synthesis of amino
acids by microorganisms. Chem. Geol. 65, 79^92.
[35] Phillips, R.L., Zak, D.R., Holmes, W.E. and White, D.C. (2002)
Microbial community composition and function beneath temperate
trees exposed to elevated atmospheric carbon dioxide and ozone.
Oecologia 131, 236^244.
[36] Abelson, P.H. and Hoering, T.C. (1961) Carbon isotope fractionation
in formation of amino acids by photosynthetic organisms. Proc. Natl.
Acad. Sci. USA 47, 623^632.
[37] Henn, M.R. and Chapela, I.H. (2000) Di¡erential C isotope discrimination by fungi during decomposition of C3- and C4-derived sucrose. Appl. Environ. Microbiol. 66, 4180^4186.
[38] Hobbie, E.A., Macko, S.A. and Shugart, H.H. (1999) Insights into
nitrogen and carbon dynamics of ectomycorrhizal and saprotrophic
fungi from isotopic evidence. Oecologia 118, 353^360.
[39] Ho«gberg, P., Plamboeck, A.H., Taylor, A.F.S. and Fransson, P.M.A.
(1999) Natural 13C abundance reveals trophic status of fungi and
host-origin of carbon in mycorrhizal fungi in mixed forests. Proc.
Natl. Acad. Sci. USA 96, 8534^8539.
FEMSEC 1564 17-9-03